Carbohydrate Esterase Family 2 - Revision history

Harry Brumer: Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

2021-12-18T21:18:23Z

Text replacement - "\^\^\^(.*)\^\^\^" to "$1"

Harry Brumer: Changed "Clan" to "fold" in table header

2021-04-06T15:39:21Z

Changed "Clan" to "fold" in table header

Harry Brumer at 01:25, 4 December 2020

2020-12-04T01:25:36Z

Harry Brumer: /* Three-dimensional structures */ Removed time-sensitive phrasing.

2020-12-02T01:17:23Z

Three-dimensional structures: Removed time-sensitive phrasing.

Harry Brumer: /* Kinetics and Mechanism */

2020-12-02T01:15:14Z

Kinetics and Mechanism

Harry Brumer: /* Catalytic Residues */

2020-12-02T01:14:21Z

Catalytic Residues

Harry Brumer: /* Catalytic Residues */

2020-12-02T01:13:05Z

Catalytic Residues

Harry Brumer: /* Substrate specificities */

2020-12-02T01:12:51Z

Substrate specificities

Joel Weadge: /* Three-dimensional structures */

2020-12-01T19:25:59Z

Three-dimensional structures

Joel Weadge: /* Catalytic Residues */

2020-12-01T19:24:46Z

Catalytic Residues

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 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]: ^^^Bobby Lamont^^^
+* [[Author]]: [[User:Bobby Lamont|Bobby Lamont]]
-* [[Responsible Curator]]s:  ^^^Anthony Clarke^^^ and ^^^Joel Weadge^^^
+* [[Responsible Curator]]s:  [[User:Anthony Clarke|Anthony Clarke]] and [[User:Joel Weadge|Joel Weadge]]
 ----

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 |{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE2'''
 |-
-|'''Clan'''
+|'''Fold'''
 |α/β-hydrolase
 |-

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 == Substrate specificities ==
 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>.
 == Catalytic Residues ==
 Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) from ''Clostridium thermocellum'', ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) from ''Cellvibrio japonicus'', and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) from ''Butyrivibrio proteoclasticus'' contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine 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, 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>.

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 == Three-dimensional structures ==
+All reported CE2 structures are α/β-hydrolases, e.g. ''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 its β-sheet.
 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>.

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 == Kinetics and Mechanism ==
 [[File:CJCE2B_cropped.png||thumb|300px|right|'''Figure 1.''' ''Cj''CE2B from ''C. japonicus'' ([{{PDBlink}}2W9X PDB ID 2W9X]) ]]
-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 involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic 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 then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic 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>.
+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 involves a catalytic histidine residue acting as a general base. The histidine abstracts a proton from the hydroxyl group of the catalytic 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 then acts as a general acid and donates a proton to the sugar substrate that leads to its release while the acetyl group remains attached to the catalytic 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>.
 The characterized enzymes were all tested using ''p''NP-Ac as a substrate, with ''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  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 over 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 a substrate preference for glucomannan among CE2 family members <cite>Montanier2009</cite>

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 == Catalytic Residues ==
-Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) from ''Clostridium thermocellum'', ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) from ''Cellvibrio japonicus'' and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) from ''Butyrivibrio proteoclasticus'' contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine 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>.
+Most CE2 family members contain a catalytic dyad <cite>Montanier2009</cite>. For example, the structurally characterized ''Ct''CE2 ([{{PDBlink}}2WAO PDB ID 2WAO]) from ''Clostridium thermocellum'', ''Cj''CE2A ([{{PDBlink}}2WAA PDB ID 2WAA]) from ''Cellvibrio japonicus'', and Est2A ([{{PDBlink}}3U37 PDB ID 3U37]) from ''Butyrivibrio proteoclasticus'' contain conserved serine and histidine residues that form the catalytic dyad and lack a third aspartate residue that is typically found in serine 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>.
 == Kinetics and Mechanism ==

← Older revision		Revision as of 01:12, 2 December 2020
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−	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>.	+	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>.

	== Catalytic Residues ==		== Catalytic Residues ==

← Older revision		Revision as of 19:25, 1 December 2020
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−	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.	+	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 its β-sheet.