https://www.cazypedia.org/api.php?action=feedcontributions&user=Bobby+Lamont&feedformat=atomCAZypedia - User contributions [en-ca]2024-03-28T23:53:24ZUser contributionsMediaWiki 1.35.10https://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16108Carbohydrate Esterase Family 22020-12-01T18:42:45Z<p>Bobby Lamont: /* Family Firsts */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known, Catalytic Dyad<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO])) <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2 ([{{PDBlink}}2WAA PDB ID 2WAA]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16107Carbohydrate Esterase Family 22020-12-01T18:37:54Z<p>Bobby Lamont: /* Three-dimensional structures */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known, Catalytic Dyad<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16106Carbohydrate Esterase Family 22020-12-01T18:33:36Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known, Catalytic Dyad<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A ([{{PDBlink}}3U37 PDB ID 3U37]), ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16105Carbohydrate Esterase Family 22020-12-01T18:28:48Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known, Catalytic Dyad<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad <cite>Montanier2009</cite>. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16104Carbohydrate Esterase Family 22020-12-01T18:17:01Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known, Catalytic Dyad<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have also demonstrated preferential de-O-acetylation of xylopyranosides at positions 3 and 4, over the 2 position. In expanded substrate profiles, CE2 enzymes were also noted to deacetylate glucopyranosyl and mannopyranosyl residues at the 6-O position. The greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl compared to xylopyranosides has prompted the naming of some CE2 family members as 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]), and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in esterase triads <cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. In cases where CE2 enzymes have been noted to have a potential catalytic aspartate residue, there often exists a tryptophan that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate from completing the triad. ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue <cite>Montanier2009</cite>. Beyond the catalytic residues, CE2 enzymes have also been noted to possess an aromatic amino acid (either a tyrosine or a tryptophan) above their binding clefts that promotes greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine, a glycine, and an asparagine residue that are invariant across the CE2 family and commonly found in other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16088Carbohydrate Esterase Family 22020-11-30T14:07:57Z<p>Bobby Lamont: /* Substrate specificities */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known, Catalytic Dyad<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. CE2 family members have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical position 2, and deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position. CE2 family members may be considered as 6-de-O-acetylases because of their greater catalytic activity when deacetylating mannopyranosyl and glucopyranosyl at the 6-O position rather than the deacetylation of xylopyranosides at the 3 and 4 positions <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad<cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16086Carbohydrate Esterase Family 22020-11-27T20:34:32Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known, Catalytic Dyad<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons, CE2 family members may be considered as 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad<cite>Montanier2009 Till2013</cite>. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to this rule, as it has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and lead to the formation of a serine-substrate tetrahedral intermediate that is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate that resulted in a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl-β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed with glucomannan as the substrate for these enzymes, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes that there is a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([{{PDBlink}}3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains the α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([{{PDBlink}}4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]), ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]), and ''Cj''CE2B ([{{PDBlink}}2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16038Carbohydrate Esterase Family 22020-11-17T15:21:37Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to rule. It has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new tetrahedral intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16036Carbohydrate Esterase Family 22020-11-16T20:45:05Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to rule. It has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) ]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16035Carbohydrate Esterase Family 22020-11-16T20:34:01Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to rule. It has a functioning catalytic triad with no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B. The catalytic Ser-His dyad residues are shown as stick models in red, and α-helices and β-sheets and loops are shown in cyan, magenta, and salmon as cartoon models, respectively.]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16034Carbohydrate Esterase Family 22020-11-16T20:32:35Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. ''Cj''CE2A is an exception to rule in which it has a functioning catalytic triad and no interrupting tryptophan residue between the catalytic histidine and aspartate <cite>Montanier2009</cite>). CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B. The catalytic Ser-His dyad residues are shown as stick models in red, and α-helices and β-sheets and loops are shown in cyan, magenta, and salmon as cartoon models, respectively.]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16033Carbohydrate Esterase Family 22020-11-16T20:04:47Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B. The catalytic Ser-His dyad residues are shown as stick models in red, and α-helices and β-sheets and loops are shown in cyan, magenta, and salmon as cartoon models, respectively.]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X])(See Fig. 1), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16030Carbohydrate Esterase Family 22020-11-16T20:01:07Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B. The catalytic Ser-His dyad residues are shown as stick models in red, and α-helices and β-sheets are shown in cyan and magenta as cartoon models, respectively.]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16029Carbohydrate Esterase Family 22020-11-16T20:00:17Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B. The catalytic Ser-His dyad residues are shown as stick models in red, and α-helices and β-sheets are shown in cyan and magenta, respectively.]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 is also unique because this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), that make up a modular protein, called ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosomes in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of ''Neocallimastix patriciarum'' <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain found in CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE later became known as ''Ct''CE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16023Carbohydrate Esterase Family 22020-11-16T19:52:47Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (''p''NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and some enzymes have preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. When CE2 enzymes contain a potential catalytic aspartate residue (like ''Cj''CE2A with a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>), often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues; thereby preventing the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of backbone atoms from the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) The individual monomer of the protein, ''Cj''CE2B.]]<br />
The possession of an α/β hydrolase fold containing a catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine; thereby rendering it nucleophilic. The serine can then attack the ester bond of the substrate and the formation of a serine-substrate tetrahedral intermediate is stabilized by the residues of the enzyme's oxyanion hole. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and deprotonates a water molecule so that it can attack the acetyl-serine ester linkage; thereby generating a new transition state intermediate that is also stabilized by the residues of the oxyanion hole. Upon collapse of this transition state, the acetyl group is released from the enzyme and the serine is re-protonated so that it is ready for another catalytic cycle <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. Est2A was also tested using ''p''-nitrophenyl butyrate which gave a ''k''<sub>cat</sub>/''K''<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-''O''-acetyl-nitrophenyl β-D-xylopyranosides that showed increased ''k''<sub>cat</sub>/''K''<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the ''k''<sub>cat</sub>/''K''<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>µM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B, respectively; thereby suggesting for these enzymes at least, a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains an α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. The common structure of the N-terminal β-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>. The α/β-hydrolase domain that is C-terminal to the jelly roll consists of a three layered α/β stack composed of five β-strands, arranged in parallel to form a central β-sheet, that is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A has 9 α-helices packing both sides of the β-sheet. <br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16005Carbohydrate Esterase Family 22020-11-16T18:41:36Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. Japonicus'' ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]).]]<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state which is also stabilized by the residues of the oxyanion hole. The acetyl group is released and the histidine will act as a general acid and donate a proton to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16004Carbohydrate Esterase Family 22020-11-16T17:35:40Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' Figure legend.]]<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a general base. The histidine increases the nucleophilicity of the catalytic serine through the extraction of a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine then acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state which is also stabilized by the residues of the oxyanion hole. The acetyl group is released and the histidine will act as a general acid and donate a proton to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16003Carbohydrate Esterase Family 22020-11-16T17:27:23Z<p>Bobby Lamont: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|α/β-hydrolase<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' Figure legend.]]<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16002Carbohydrate Esterase Family 22020-11-16T17:23:55Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' Figure legend.]]<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=16001Carbohydrate Esterase Family 22020-11-16T17:06:29Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
[[File:CJCE2B_cropped.png|300px|right]]<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=User:Bobby_Lamont&diff=16000User:Bobby Lamont2020-11-16T17:04:34Z<p>Bobby Lamont: </p>
<hr />
<div>[[Image:Wiki.png|200px|right]]<br />
Bobby Lamont obtained his Bachelor of Science degree in molecular biology and genetics from the University of Guelph. He is currently enrolled in the Master of Chemistry program at Wilfred Laurier University. Under the supervision of Professor Anthony Clarke, he studies the structure and function of the protein, PatA.<br />
<br />
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<br />
<br />
<br />
<br />
<br />
----<br />
<br />
<biblio><br />
<br />
<br />
</biblio><br />
<br />
<!-- Do not remove this Category tag --><br />
[[Category:Contributors|Lamont,Bobby]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=File:CJCE2B_cropped.png&diff=15999File:CJCE2B cropped.png2020-11-16T17:00:27Z<p>Bobby Lamont: </p>
<hr />
<div></div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15998Carbohydrate Esterase Family 22020-11-16T15:32:57Z<p>Bobby Lamont: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|Known<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15997Carbohydrate Esterase Family 22020-11-16T15:32:41Z<p>Bobby Lamont: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|Serine Hydrolase<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15996Carbohydrate Esterase Family 22020-11-16T15:31:41Z<p>Bobby Lamont: </p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE2.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15995Carbohydrate Esterase Family 22020-11-16T15:30:26Z<p>Bobby Lamont: /* Family Firsts */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
The first 3-D structures of CE2 family members, ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B were revealed in the same study that confirmed the catalytic mechanism of the family <cite>Montanier2009</cite>.<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15994Carbohydrate Esterase Family 22020-11-16T15:28:43Z<p>Bobby Lamont: /* Family Firsts */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
The catalytic dyad of Ser-His residues was confirmed by the arrangement of these residues in the crystal structures of CtCE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), CjCE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]), and CjCE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]) as well as sequence alignment analysis showing its invariance across the CE2 family <cite>Montanier2009</cite>.<br />
;First 3-D structure:<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15993Carbohydrate Esterase Family 22020-11-16T15:09:18Z<p>Bobby Lamont: /* Family Firsts */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
The first instance of CE2 family characterization was from the investigation of the BnaA, BnaB, and BnaC proteins that were discovered via cDNA library sequencing of Neocallimastix patriciarum <cite>Dalrymple1997</cite>. BnaA and BnaC proteins exhibited acetyl xylan esterase ability. BnaB exhibited high sequence similarity to an uncharacterized C-terminal domain of the protein, CelE <cite>Hall1988</cite>. The uncharacterized domain of CelE which would later become known as, CtCE2 <cite>Montanier2009</cite>.<br />
<br />
;First mechanistic insight: <br />
<br />
;First 3-D structure:<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15992Carbohydrate Esterase Family 22020-11-16T15:07:24Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. An example of a proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
<br />
;First mechanistic insight: <br />
<br />
;First 3-D structure:<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15991Carbohydrate Esterase Family 22020-11-16T15:06:43Z<p>Bobby Lamont: /* Family Firsts */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. The proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: <br />
<br />
;First mechanistic insight: <br />
<br />
;First 3-D structure:<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15990Carbohydrate Esterase Family 22020-11-16T15:03:08Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
The possession of an α/β hydrolase fold containing the catalytic serine nucleophile suggests that the reaction mechanism may proceed similar to other enzymes in the SGNH family. The proposed reaction mechanism associated with the SGNH family of enzymes begins with the catalytic histidine residue acting as a base and increasing the nucleophilicity of the catalytic serine by extracting a proton from the hydroxyl group of the serine and leaving a negatively charged oxygen sidechain. The serine will attack the ester bond of the substrate, and stabilized by the residues of the oxyanion hole, a serine-substrate tetrahedral intermediate will form. The histidine acts as a general acid and donates a proton to the substrate causing its release and leaving an acetyl group attached to the serine. The histidine acts as a general base and influences a water molecule to attack the ester of the acetyl group attached to the catalytic serine generating a new tetrahedral intermediate state. The acetyl groups is released and the histidine will act as a general acid and donate a proton to back to the serine and restore the catalytic site to it’s original state <cite>Alalouf2011</cite>.<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15989Carbohydrate Esterase Family 22020-11-16T14:57:36Z<p>Bobby Lamont: /* References */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
#Alalouf2011 pmid=21994937<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15988Carbohydrate Esterase Family 22020-11-16T14:56:22Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15987Carbohydrate Esterase Family 22020-11-16T14:55:37Z<p>Bobby Lamont: /* Kinetics and Mechanism */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
The characterized enzymes were all tested using ''p''NP-Ac, which showed k<sub>cat</sub>/K<sub>M</sub> of 2.01, 0.71, 0.38 and 3.13 s<sup>-1</sup>×µM<sup>-1</sup> for Est2A, ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. Est2A was also tested using p-nitrophenyl butyrate which gave a k<sub>cat</sub>/K<sub>M</sub> value of 2.33 x 10<sup>-3</sup> s<sup>-1</sup>×µM<sup>-1</sup> showing the significant decrease in catalytic efficiency as substrate size increased <cite>Till2013</cite>. In order to test for positional specificity, the enzyme kinetics of ''Ct''CE2, ''Cj''CE2B, and ''Cj''CE2C were tested using 2-, 3-, and 4-O-acetyl ''p''4-nitrophenyl β-D-xylopyranosides that showed increased k<sub>cat</sub>/K<sub>M</sub> values for the hydrolysis of the substrate at position 3 and 4 as opposed to position 2 <cite>Topakas2010</cite>. Enzyme kinetic assays on birchwood xylan showed k<sub>cat</sub>/K<sub>M</sub> values of 7.33 x 10<sup>-5</sup>, 7.67 x 10<sup>-4</sup>, and 2.33 x 10<sup>-4</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B respectively. When the assay was performed on these same enzymes but with the use of glucomannan, the k<sub>cat</sub>/K<sub>M</sub> values were 9.67 x 10<sup>-4</sup>, 6.5 x 10<sup>-4</sup>, and 2.68 x 10<sup>-2</sup> s<sup>-1</sup>xµM<sup>-1</sup> for ''Ct''CE2, ''Cj''CE2A and ''Cj''CE2B respectively; thereby suggesting for these enzymes at least a clear substrate preference for glucomannan <cite>Montanier2009</cite><br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15973Carbohydrate Esterase Family 22020-11-14T20:48:34Z<p>Bobby Lamont: /* Three-dimensional structures */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009 Bayer2004</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15972Carbohydrate Esterase Family 22020-11-14T20:48:06Z<p>Bobby Lamont: /* References */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
#Bayer2004 pmid=15487947<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15971Carbohydrate Esterase Family 22020-11-14T20:44:41Z<p>Bobby Lamont: /* Three-dimensional structures */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of ''Ct''CE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15970Carbohydrate Esterase Family 22020-11-14T20:44:08Z<p>Bobby Lamont: /* Three-dimensional structures */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include ''Clostridium thermocellum''’s ''Ct''CE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), ''Cellvibrio japonicus''’ ''Cj''CE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and ''Cj''CE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and ''Butyrivibrio proteoclasticus''’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of ''Ct''CE2, ''Cj''CE2A, and ''Cj''CE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, ''Ct''Cel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, ''Ct''Cel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in ''C. thermocellum'' <cite>Montanier2009</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15969Carbohydrate Esterase Family 22020-11-14T20:42:11Z<p>Bobby Lamont: /* Substrate specificities */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac<sub>c</sub>) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), Cellvibrio japonicus’ CjCE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and CjCE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and Butyrivibrio proteoclasticus’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum <cite>Montanier2009</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15968Carbohydrate Esterase Family 22020-11-14T20:41:30Z<p>Bobby Lamont: /* Substrate specificities */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-Ac) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), Cellvibrio japonicus’ CjCE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and CjCE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and Butyrivibrio proteoclasticus’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum <cite>Montanier2009</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15967Carbohydrate Esterase Family 22020-11-14T20:39:31Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. ''Cj''CE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, ''Ct''CE2, ''Cj''CE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), Cellvibrio japonicus’ CjCE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and CjCE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and Butyrivibrio proteoclasticus’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum <cite>Montanier2009</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15966Carbohydrate Esterase Family 22020-11-14T20:37:15Z<p>Bobby Lamont: /* Three-dimensional structures */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 ([https://www.rcsb.org/3d-view/ngl/2WAO PDB ID 2WAO]), Cellvibrio japonicus’ CjCE2A ([https://www.rcsb.org/3d-view/ngl/2WAA PDB ID 2WAA]) and CjCE2B ([https://www.rcsb.org/3d-view/ngl/2W9X PDB ID 2W9X]), and Butyrivibrio proteoclasticus’ Est2A ([https://www.rcsb.org/3d-view/ngl/3U37 PDB ID 3U37]). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) <cite>Montanier2009 Till2013</cite>. This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side <cite>Montanier2009</cite>, but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively <cite>Till2013</cite>.<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure <cite>Montanier2009 Till2013</cite>. The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C ([https://www.rcsb.org/3d-view/ngl/4IM4 PDB ID 4IM4]), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum <cite>Montanier2009</cite>.<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15965Carbohydrate Esterase Family 22020-11-14T20:07:39Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad <cite>Montanier2009</cite>. The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity <cite>Montanier2009 Till2013</cite>. Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases <cite>Montanier2009 Till2013</cite>.<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 (PDB ID 2WAO), Cellvibrio japonicus’ CjCE2A (PDB ID 2WAA) and CjCE2B (PDB ID 2W9X), and Butyrivibrio proteoclasticus’ Est2A (PDB ID 3U37). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) (Montanier et al. 2009; Till et al. 2013). This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side (Montanier et al. 2009), but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively (Till et al. 2013).<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure (Montanier et al. 2009; Till et al. 2013). The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C (PDB ID 4IM4), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum (Montanier et al. 2009).<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15964Carbohydrate Esterase Family 22020-11-14T20:05:49Z<p>Bobby Lamont: /* Substrate specificities */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) <cite>Montanier2009 Till2013</cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad (Montanier et al. 2009). The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity (Montanier et al. 2009; Till et al. 2013). Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases (Montanier et al. 2009; Till et al. 2013).<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 (PDB ID 2WAO), Cellvibrio japonicus’ CjCE2A (PDB ID 2WAA) and CjCE2B (PDB ID 2W9X), and Butyrivibrio proteoclasticus’ Est2A (PDB ID 3U37). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) (Montanier et al. 2009; Till et al. 2013). This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side (Montanier et al. 2009), but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively (Till et al. 2013).<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure (Montanier et al. 2009; Till et al. 2013). The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C (PDB ID 4IM4), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum (Montanier et al. 2009).<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15963Carbohydrate Esterase Family 22020-11-14T20:04:48Z<p>Bobby Lamont: /* Substrate specificities */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) <cite>Montanier2009 Till2013<cite>. Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases <cite>Topakas2010</cite>.<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad (Montanier et al. 2009). The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity (Montanier et al. 2009; Till et al. 2013). Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases (Montanier et al. 2009; Till et al. 2013).<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 (PDB ID 2WAO), Cellvibrio japonicus’ CjCE2A (PDB ID 2WAA) and CjCE2B (PDB ID 2W9X), and Butyrivibrio proteoclasticus’ Est2A (PDB ID 3U37). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) (Montanier et al. 2009; Till et al. 2013). This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side (Montanier et al. 2009), but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively (Till et al. 2013).<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure (Montanier et al. 2009; Till et al. 2013). The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C (PDB ID 4IM4), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum (Montanier et al. 2009).<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15962Carbohydrate Esterase Family 22020-11-14T19:59:16Z<p>Bobby Lamont: /* References */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) (Montanier et al. 2009; Till et al. 2013). Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases (Topakas et al. 2010).<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad (Montanier et al. 2009). The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity (Montanier et al. 2009; Till et al. 2013). Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases (Montanier et al. 2009; Till et al. 2013).<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 (PDB ID 2WAO), Cellvibrio japonicus’ CjCE2A (PDB ID 2WAA) and CjCE2B (PDB ID 2W9X), and Butyrivibrio proteoclasticus’ Est2A (PDB ID 3U37). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) (Montanier et al. 2009; Till et al. 2013). This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side (Montanier et al. 2009), but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively (Till et al. 2013).<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure (Montanier et al. 2009; Till et al. 2013). The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C (PDB ID 4IM4), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum (Montanier et al. 2009).<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
#Hall1988 pmid=3066698<br />
#Till2013 pmid=23345031<br />
#Topakas2010 pmid=19968989<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15961Carbohydrate Esterase Family 22020-11-14T19:54:48Z<p>Bobby Lamont: /* References */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) (Montanier et al. 2009; Till et al. 2013). Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases (Topakas et al. 2010).<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad (Montanier et al. 2009). The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity (Montanier et al. 2009; Till et al. 2013). Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases (Montanier et al. 2009; Till et al. 2013).<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 (PDB ID 2WAO), Cellvibrio japonicus’ CjCE2A (PDB ID 2WAA) and CjCE2B (PDB ID 2W9X), and Butyrivibrio proteoclasticus’ Est2A (PDB ID 3U37). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) (Montanier et al. 2009; Till et al. 2013). This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side (Montanier et al. 2009), but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively (Till et al. 2013).<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure (Montanier et al. 2009; Till et al. 2013). The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C (PDB ID 4IM4), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum (Montanier et al. 2009).<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Faik2010 pmid=20375115<br />
#Montanier2009 pmid=19338387<br />
#Dalrymple1997 pmid=9274014<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15960Carbohydrate Esterase Family 22020-11-14T18:42:26Z<p>Bobby Lamont: /* Three-dimensional structures */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) (Montanier et al. 2009; Till et al. 2013). Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases (Topakas et al. 2010).<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad (Montanier et al. 2009). The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity (Montanier et al. 2009; Till et al. 2013). Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases (Montanier et al. 2009; Till et al. 2013).<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
<br />
There are four reported structures for the CE2 family. These structures are all reported to be α/β-hydrolases and include Clostridium thermocellum’s CtCE2 (PDB ID 2WAO), Cellvibrio japonicus’ CjCE2A (PDB ID 2WAA) and CjCE2B (PDB ID 2W9X), and Butyrivibrio proteoclasticus’ Est2A (PDB ID 3U37). They contain an N-terminal β-sheet “jelly-roll” domain that acts as a carbohydrate binding domain (CBM) and is linked to a C-terminal domain that contains a α/β-hydrolase fold (SGNH-hydrolase motif) (Montanier et al. 2009; Till et al. 2013). This α/β-hydrolase fold consists of a three layered α/β stack composed of five beta strands arranged in parallel that form a central β-sheet, which is packed between α-helicies. In the case of CtCE2, CjCE2A, and CjCE2B, the sheet has 5 α-helices in total packed on each side (Montanier et al. 2009), but Est2A, has 9 α-helices packing both sides of the β-sheet. The common structure of the N-terminal B-sheet “jelly-roll” domain across CE2 enzymes is comprised of two opposing β-sheets that have 4 and 5 β-strands, respectively (Till et al. 2013).<br />
<br />
<br />
The CE2 family members are typically monomeric, but there are some exceptions. Specifically, Est2A has been found to form tetramers that combine to make an overall octameric structure (Montanier et al. 2009; Till et al. 2013). The overall structure of CtCE2 also displayed that this domain is connected to the C-terminal end of a GH5 family cellulase protein, CtCel5C (PDB ID 4IM4), which make up a modular protein, called, CtCel5C-CE2. This protein is incorporated into cell-wall degrading cellulosome in C. thermocellum (Montanier et al. 2009).<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Faik2010 pmid=20375115<br />
<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15959Carbohydrate Esterase Family 22020-11-14T18:41:17Z<p>Bobby Lamont: /* Catalytic Residues */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) (Montanier et al. 2009; Till et al. 2013). Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases (Topakas et al. 2010).<br />
<br />
== Catalytic Residues ==<br />
<br />
Most CE2 family members contain a catalytic dyad (Ser-His) as opposed to a catalytic triad that is typically found in esterases. CjCE2A is an exception and contains a Ser-His-Asp catalytic triad (Montanier et al. 2009). The structurally characterized, CtCE2, CjCE2B, and Est2A are examples of CE2 enzymes that contain catalytic dyads of conserved serine and histidine residues and lack the aspartate residue found in the triad. Without the support of an aspartate residue, the histidine residue of the catalytic dyads are supported by main-chain carbonyl groups provided by a backbone amino acid. The catalytic aspartate residue that would commonly complete the catalytic triad simply does not exist in many CE2 members. When CE2 enzymes contain a potential catalytic aspartate residue, often enough, there also exists a tryptophan residue that sits between the catalytic histidine and aspartate residues thereby prevent the aspartate residue from completing the triad. CE2 enzymes also possess an aromatic residue (either a tyrosine or a tryptophan residue) above their binding clefts that promote greater substrate specificity (Montanier et al. 2009; Till et al. 2013). Lastly, the oxyanion hole is comprised of the catalytic serine residue, a glycine, and an asparagine residue that appears to be invariant across the CE2 family and that of other related acetyl-esterases (Montanier et al. 2009; Till et al. 2013).<br />
<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Faik2010 pmid=20375115<br />
<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamonthttps://www.cazypedia.org/index.php?title=Carbohydrate_Esterase_Family_2&diff=15958Carbohydrate Esterase Family 22020-11-14T18:40:36Z<p>Bobby Lamont: /* Substrate specificities */</p>
<hr />
<div><!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption --><br />
{{CuratorApproved}}<br />
* [[Author]]: ^^^Bobby Lamont^^^<br />
* [[Responsible Curator]]s: ^^^Joel Weadge^^^ and ^^^Anthony Clarke^^^<br />
----<br />
<br />
<!-- The data in the table below should be updated by the Author/Curator according to current information on the family --><br />
<div style="float:right"><br />
{| {{Prettytable}} <br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''<br />
|-<br />
|'''Clan''' <br />
|<br />
|-<br />
|'''Mechanism'''<br />
|<br />
|-<br />
|'''Active site residues'''<br />
|<br />
|-<br />
|{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''<br />
|-<br />
| colspan="2" |{{CAZyDBlink}}CE3.html<br />
|}<br />
</div><br />
<!-- This is the end of the table --><br />
<br />
== Substrate specificities ==<br />
<br />
All of the well characterized carbohydrate esterase family 2 enzymes have been shown to remove acetate groups from the synthetic molecule, 4-nitrophenyl acetate (4p-NP-AcC) (Montanier et al. 2009; Till et al. 2013). Contrary to typical acetyl xylan esterases, CE2 family members are shown to have a strong preference for the deacetylation of xylopyranosides at positions 3 and 4 instead of the typical deacetylation at position 2. CE2 family members were also shown to have significant preference for deacetylation of glucopyranosyl and mannopyranosyl residues at the 6-O position and significantly preferred deacetylation of glucopyranosyl and mannopyranosyl residues relative to the deacetylation of xylopyranosides. For these reasons CE2 family members are considered to be 6-de-O-acetylases (Topakas et al. 2010).<br />
<br />
== Catalytic Residues ==<br />
== Kinetics and Mechanism ==<br />
<br />
== Three-dimensional structures ==<br />
<br />
== Family Firsts ==<br />
;First characterized: .<br />
;First mechanistic insight: .<br />
;First 3-D structure: .<br />
<br />
== References ==<br />
<biblio><br />
#Faik2010 pmid=20375115<br />
<br />
</biblio><br />
<br />
[[Category:Carbohydrate Esterase Families|CE002]]</div>Bobby Lamont