Carbohydrate Esterase Family 7 - Revision history

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

2021-12-18T21:18:46Z

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

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

2021-04-06T15:40:45Z

Changed "Clan" to "fold" in table header

Emily Rodriguez at 19:34, 21 October 2020

2020-10-21T19:34:20Z

Emily Rodriguez at 18:02, 13 August 2020

2020-08-13T18:02:29Z

Harry Brumer: /* Three-dimensional structures */ Removed time-dependent "currently..." phrasing

2020-08-05T19:15:33Z

Three-dimensional structures: Removed time-dependent "currently..." phrasing

Joel Weadge at 14:41, 30 July 2020

2020-07-30T14:41:22Z

Joel Weadge at 20:18, 23 July 2020

2020-07-23T20:18:09Z

Joel Weadge at 20:16, 23 July 2020

2020-07-23T20:16:50Z

Joel Weadge at 20:14, 23 July 2020

2020-07-23T20:14:35Z

Michael Suits: maritima

2020-07-23T02:51:30Z

maritima

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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]]: ^^^Emily Rodriguez^^^
+* [[Author]]: [[User:Emily Rodriguez|Emily Rodriguez]]
-* [[Responsible Curator]]s:  ^^^Michael Suits^^^ and ^^^Joel Weadge^^^
+* [[Responsible Curator]]s:  [[User:Michael Suits|Michael Suits]] and [[User:Joel Weadge|Joel Weadge]]
 ----

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 |{{Hl2}} colspan="2" align="center" |'''Carbohydrate Esterase Family CE7'''
 |-
-|'''Clan'''
+|'''Fold'''
 |(α/β/α)-Sandwich
 |-

@@ Line 38: / Line 38: @@
 Several structures have been reported for the CE7 family. These include the AXE from ''B. pumilus'' ([{{PDBlink}}2XLB PDB ID 2XLB]), cephalosporin-C deacetylase (CAH) from ''B. subtilis'' strain 168 ([{{PDBlink}}1ODS PDB ID 1ODS]), the AXE from ''Paenibacillus'' sp. R4 ([{{PDBlink}}6AGQ PDB ID 6AGQ]), acetyl xylan esterase 1 (Axe1) from ''T. saccharolyticum'' strain JW/SL-YS485 ([{{PDBlink}}3FCY PDB ID 3FCY]), the AXE (TM0077) from ''T. maritima'' ([{{PDBlink}}1VLQ PDB ID 1VLQ]) and, lastly, an unclassified protein named Axe1-NaM1 ([{{PDBlink}}6FKX PDB ID 6FKX]).
-Enzymes in the CE7 family contain high levels of multimerization, with quaternary structures containing 4, 5, 6 and 8 subunits described <cite>Vincent2003</cite>. For each of the  six resolved structures from the CE7 family, individual subunits adopt a classic α/β-hydrolase fold that consists of a central eight-stranded β-sheet surrounded by α-helices on both sides (See Fig. 1A) <cite>Levisson2012</cite>. Together, the multimerized subunits adopt a donut-like shape that results in the formation of a tunnel leading to the centre of the protein (See Fig. 1B). Each of the active sites from the individual multimers are arranged so that they face into the centre of the tunnel <cite>Vincent2003</cite>.
+Enzymes in the CE7 family contain high levels of multimerization, with the quaternary structure adopting a dimer of trimer hexamer structure<cite>Vincent2003</cite>. For each of the  six resolved structures from the CE7 family, individual subunits adopt a classic α/β-hydrolase fold that consists of a central eight-stranded β-sheet surrounded by α-helices on both sides (See Fig. 1A) <cite>Levisson2012</cite>. Together, the multimerized subunits adopt a donut-like shape that results in the formation of a tunnel leading to the centre of the protein (See Fig. 1B). Each of the active sites from the individual multimers are arranged so that they face into the centre of the tunnel <cite>Vincent2003</cite>.
 == Family Firsts ==

@@ Line 43: / Line 43: @@
 ;First Characterized:The first cephalosporin-C deacetylase (CAH) of this family to be characterized was from ''B. subtilis'' strain 168 in 1975 <cite>Abbot1975</cite>. CAH is able to deacetylate cephalosporin-C, as well as cephalosporin-C derivatives (eg., cephalosporanic acid and cephalothin) at the C-3 position <cite>Abbot1975</cite>.  The first acetyl xylan esterase to be characterized was Axe1, from the anaerobic thermophilic bacterium ''T. saccharolyticum'' strain JW/SL-YS485 in 1995 <cite>Shao1995</cite>.  When in the presence of acetylated xylan, Axe1 showed esterase activity that liberated acetate, leading to its name <cite>Shao1995</cite>. However, Axe1 has also demonstrated activity against other acetylated sugars <cite>Shao1995</cite>.
 ;First Mechanistic Insight: The first mechanistic insight into the CE7 family came from the CAH enzyme from ''B. subtilis'' in 1994 when Takimoto and coworkers tested the effects of various chemicals (''eg.'', EDTA, PMSF, diisopropyl flurophosphate) on CAH activity and concluded that serine is likely important for catalysis <cite>Takimoto1994</cite>. Almost 10 years later in 2003, Vincent and colleagues resolved the crystal structure of CAH, confirming that serine was present in the active site and that an aspartate and histidine completed the catalytic triad <cite>Vincent2003</cite>. More recently, co-crystallization of the AXE (TM0077) from ''T. martitima'' with PMSF and paraoxan ([{{PDBlink}}3M82 PDB ID 3M82] and [{{PDBlink}}3M83 PDB ID 3M83], respectively),  two known serine protease inhibitors, led to trapping of the tetrahedral intermediate; thereby providing further support for the serine-histidine-aspartate mediated mechanism described for this family.
-;First 3-D Structure:The first resolved structure in this family was the cephalosporin-C deacetylase, CAH ([{{PDBlink}}1ODS PDB ID 1ODS]) from ''B. subtilis'' strain 168 in 2003 <cite>Vincent2003</cite>. The quaternary structure consisted of a trimer of dimers to make up a donut-shaped homohexamer with 32kDa subunits that each adopt the α/β hydrolase fold (See Fig. 1) <cite>Vincent2003</cite>. The hydrophobic core containing the six active site centers is thought to help exclude large substrates and is shielded from solvents through a lid-like domain <cite>Vincent2003</cite>. In order for the hexamer to form, there are α-helix interactions as well as a β-strand-like interface associating between monomers (See Fig. 1A) <cite>Vincent2003,Sista_Kameshwar2018</cite>.
+;First 3-D Structure:The first resolved structure in this family was the cephalosporin-C deacetylase, CAH ([{{PDBlink}}1ODS PDB ID 1ODS]) from ''B. subtilis'' strain 168 in 2003 <cite>Vincent2003</cite>. The quaternary structure consisted of a trimer of dimers to make up a donut-shaped homohexamer with 32kDa subunits that each adopt the α/β hydrolase fold (See Fig. 1) <cite>Vincent2003</cite>. The hydrophobic core containing the six active site centers is thought to help exclude large substrates<cite>Vincent2003</cite>. In order for the hexamer to form, there are α-helix interactions as well as a β-strand-like interface associating between monomers (See Fig. 1A) <cite>Vincent2003,Sista_Kameshwar2018</cite>.
 == References ==
 <biblio>

@@ Line 36: / Line 36: @@
 Kinetic parameters and specific activities have been determined for some members of CE7. CAH from ''Bacillus subtilis'',  AXE from ''Paenibacillus'' sp. R4 and AXE (TM0077) from ''T. maritima'' were assayed for their activity against ''p''NP-acetate and were found to have ''K''<sub>M</sub> values of 0.29 +/- 0.01, 0.15 +/- 0.01 and 0.19 +/- 0.03 mM, respectively, and ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 260, 360 and 310 s<sup>-1</sup>mM<sup>-1</sup>, respectively <cite>Levisson2012,Vincent2003,Takimoto1994,Park2018</cite>. There has been discrepancy regarding the ability of these CE7 enzymes to utilize acetylated xylan as a substrate. While a ''Bacillus pumilus'' AXE has demonstrated a specific activity against acetylated xylan of 41 +/- 8 U/mg, ''T. maritima'''s AXE had no demonstrated activity and the acetyl xylan esterase, Axe1, from ''Thermoanerobacterium saccharolyticum'' strain JW/SL-YS485 had no activity in one study and 5.2 U/mg (8-fold lower than the ''B. pumilus'' enzyme) in another study <cite>Levisson2012,Lorenz1997,Degrassi1998,Shao1995</cite>. In light of the apparent low/no activity against acetylated xylan, the enzymes from ''T. maritima'' and ''T. saccharolyticum'' were assayed against smaller substrates, where the ''T. maritima'' AXE (TM0077) had high activity against glucose pentaacetate with a ''k''<sub>cat</sub> of 2900 s<sup>-1</sup> and Axe1 from ''T. saccharolyticum'' had high activity against xylose tetraacetate with separately reported specific activities of 210 and 740 U/mg <cite>Levisson2012,Lorenz1997,Shao1995</cite>. Lastly, CAH from ''B. subtilis'' has been kinetically characterized with regards to activity towards cephalosporin-C with a ''K''<sub>M</sub> of 24 mM and a ''k''<sub>cat</sub>/''K''<sub>M</sub> of 7.0 s<sup>-1</sup>mM<sup>-1</sup>, which has supported its naming as a cephalosporin-C deacetylase <cite>Takimoto1994</cite>.
 == Three-dimensional structures ==
-There are six resolved structures currently reported for the CE7 family. These include the AXE from ''B. pumilus'' ([{{PDBlink}}2XLB PDB ID 2XLB]), cephalosporin-C deacetylase (CAH) from ''B. subtilis'' strain 168 ([{{PDBlink}}1ODS PDB ID 1ODS]), the AXE from ''Paenibacillus'' sp. R4 ([{{PDBlink}}6AGQ PDB ID 6AGQ]), acetyl xylan esterase 1 (Axe1) from ''T. saccharolyticum'' strain JW/SL-YS485 ([{{PDBlink}}3FCY PDB ID 3FCY]), the AXE (TM0077) from ''T. maritima'' ([{{PDBlink}}1VLQ PDB ID 1VLQ]) and, lastly, an unclassified protein named Axe1-NaM1 ([{{PDBlink}}6FKX PDB ID 6FKX]).
+Several structures have been reported for the CE7 family. These include the AXE from ''B. pumilus'' ([{{PDBlink}}2XLB PDB ID 2XLB]), cephalosporin-C deacetylase (CAH) from ''B. subtilis'' strain 168 ([{{PDBlink}}1ODS PDB ID 1ODS]), the AXE from ''Paenibacillus'' sp. R4 ([{{PDBlink}}6AGQ PDB ID 6AGQ]), acetyl xylan esterase 1 (Axe1) from ''T. saccharolyticum'' strain JW/SL-YS485 ([{{PDBlink}}3FCY PDB ID 3FCY]), the AXE (TM0077) from ''T. maritima'' ([{{PDBlink}}1VLQ PDB ID 1VLQ]) and, lastly, an unclassified protein named Axe1-NaM1 ([{{PDBlink}}6FKX PDB ID 6FKX]).
 Enzymes in the CE7 family contain high levels of multimerization, with quaternary structures containing 4, 5, 6 and 8 subunits described <cite>Vincent2003</cite>. For each of the  six resolved structures from the CE7 family, individual subunits adopt a classic α/β-hydrolase fold that consists of a central eight-stranded β-sheet surrounded by α-helices on both sides (See Fig. 1A) <cite>Levisson2012</cite>. Together, the multimerized subunits adopt a donut-like shape that results in the formation of a tunnel leading to the centre of the protein (See Fig. 1B). Each of the active sites from the individual multimers are arranged so that they face into the centre of the tunnel <cite>Vincent2003</cite>.

@@ Line 28: / Line 28: @@
 == Substrate specificities ==
-Carbohydrate Esterase Family 7 currently contains enzymes classified as acetyl xylan esterases (AXEs) and cephalosporin-C deacetylases<cite>Nakamura2017</cite>. One exception is the AXE from ''Thermotoga martitima'' that lacks detectable activity towards acetylated xylan, but rather, shorter acetylated xylo-oligomers<cite>Levisson2012</cite>. A common feature of the oligomeric enzymes belonging to this family is that they usually serve as multifunctional deacetylases and are active on a broad range of small substrates with relatively low acetyl-positional specificity<cite>Levisson2012,Krastanova2005,Lorenz1997,Vincent2003</cite>. Some examples include xylose tetraacetate, glucose pentaacetate, α-napthyl acetate, 4-methylumbelliferyl acetate and ''p''-nitrophenyl acetate (''p''NP-acetate)<cite>Degrassi2000</cite>. Substrate binding is proposed to be largely mediated through non-specific hydrophobic interactions with surface amino acid residues<cite>Vincent2003</cite>.
+Carbohydrate Esterase Family 7 currently contains enzymes classified as acetyl xylan esterases (AXEs) and cephalosporin-C deacetylases <cite>Nakamura2017</cite>. One exception is the AXE from ''Thermotoga martitima'' that lacks detectable activity towards acetylated xylan, but rather, shorter acetylated xylo-oligomers <cite>Levisson2012</cite>. A common feature of the oligomeric enzymes belonging to this family is that they usually serve as multifunctional deacetylases and are active on a broad range of small substrates with relatively low acetyl-positional specificity <cite>Levisson2012,Krastanova2005,Lorenz1997,Vincent2003</cite>. Some examples include xylose tetraacetate, glucose pentaacetate, α-napthyl acetate, 4-methylumbelliferyl acetate and ''p''-nitrophenyl acetate (''p''NP-acetate) <cite>Degrassi2000</cite>. Substrate binding is proposed to be largely mediated through non-specific hydrophobic interactions with surface amino acid residues <cite>Vincent2003</cite>.
 == Catalytic Residues ==
-Members of this family use the catalytic triad of serine, histidine and aspartate<cite>Biely2012</cite>. The aspartate promotes the amphoteric nature of the histidine residue so that it can abstract a proton from the serine residue to render the serine nucleophilic<cite>Levisson2012</cite>. The Gly-X-Ser-X-Gly motif, containing the nucleophilic serine, is common amongst the broad α/β-hydrolase family to which the CE7 enzymes belong<cite>Mitsushima1995</cite>. The glycine residues near the catalytic serine are important to prevent any steric hindrance that may occur from amino acids with larger side chains and form the basis for the nucleophilic “elbow” turn motif that is present in these enzymes<cite>Levisson2012</cite>. [[Image:CAH Bsubtilis CE7.jpg|thumb|300px|Figure 1: CAH from B. subtilis ([{{PDBlink}}1ODS PDB ID 1ODS]).   A) An individual monomer from the overall CAH structure showing the α/β-hydrolase fold formed by each subunit and the β-strand-like interface domain needed for association of the monomers. The catalytic triad of Ser181, Asp 269 and His 298 are highlighted in cyan. B) Characteristic donut-shaped quaternary structure of the CAH hexamer. Each monomer is represented in a different colour.]]
+Members of this family use the catalytic triad of serine, histidine and aspartate <cite>Biely2012</cite>. The aspartate promotes the amphoteric nature of the histidine residue so that it can abstract a proton from the serine residue to render the serine nucleophilic <cite>Levisson2012</cite>. The Gly-X-Ser-X-Gly motif, containing the nucleophilic serine, is common amongst the broad α/β-hydrolase family to which the CE7 enzymes belong <cite>Mitsushima1995</cite>. The glycine residues near the catalytic serine are important to prevent any steric hindrance that may occur from amino acids with larger side chains and form the basis for the nucleophilic “elbow” turn motif that is present in these enzymes <cite>Levisson2012</cite>. [[Image:CAH Bsubtilis CE7.jpg|thumb|300px|Figure 1: CAH from B. subtilis ([{{PDBlink}}1ODS PDB ID 1ODS]).   A) An individual monomer from the overall CAH structure showing the α/β-hydrolase fold formed by each subunit and the β-strand-like interface domain needed for association of the monomers. The catalytic triad of Ser181, Asp 269 and His 298 are highlighted in cyan. B) Characteristic donut-shaped quaternary structure of the CAH hexamer. Each monomer is represented in a different colour.]]
 == Kinetics and Mechanism ==
-The CE7 family of esterases catalyze the removal of O-acetyl groups from cephalosporin-C, xylan and xylo-oligosaccharides<cite>Vincent2003,Biely2012</cite>. Members of this family present their active site residues in typical positions observed for α/β-hydrolases<cite>Sista_Kameshwar2018</cite>. The catalytic triad residues responsible for de-O-acetylation are found on loops near the C-terminus of the β-strands 5, 7 and 8 for the catalytic serine, aspartate and histidine, respectively<cite>Ollis1992</cite>. Similar to other α/β-hydrolases, the CE7 enzymes follow a mechanism where the serine is deprotonated by the histidine imidazole side chain; thereby allowing for nucleophilic attack of the serine on the incoming carbonyl of the substrate’s acetyl group<cite>Sista_Kameshwar2018</cite>. The result is a transient tetrahedral oxyanion intermediate, which is stabilized by the backbone amide group of a highly conserved glutamate residue (found in a signature RGQ motif adjacent to the nucleophilic serine at the end of β-strand 4) and a less conserved tyrosine that together form the oxyanion hole of the enzyme<cite>Levisson2012,<cite>Vincent2003</cite>. The tetrahedral intermediate then collapses resulting in an acetyl-enzyme intermediate and release of the de-O-acetylated product. A water molecule entering the active site is deprotonated by histidine and then nucleophilic attack by water on the acetyl-enzyme takes place to liberate acetate and the free enzyme<cite>Sista_Kameshwar2018</cite>. A recent study on the CE7 AXE (TM0077) from ''T. martitima'' provided further support into the catalytic mechanism<cite>Levisson2012</cite>. TM0077 was co-crystallized with PMSF and paraoxan ([{{PDBlink}}3M82 PDB ID 3M82] and [{{PDBlink}}3M83 PDB ID 3M83], respectively), two known serine protease inhibitors, that led to a 110° rotation of the catalytic serine and the formation of a covalent tetrahedral intermediate that was unable to collapse back to the starting materials (TM0077 and PMSF/paraoxan)<cite>Levisson2012</cite>. Trapping the tetrahedral intermediate is strong evidence that CE7 enzymes proceed by the proposed mechanism.
+The CE7 family of esterases catalyze the removal of O-acetyl groups from cephalosporin-C, xylan and xylo-oligosaccharides <cite>Vincent2003,Biely2012</cite>. Members of this family present their active site residues in typical positions observed for α/β-hydrolases <cite>Sista_Kameshwar2018</cite>. The catalytic triad residues responsible for de-O-acetylation are found on loops near the C-terminus of the β-strands 5, 7 and 8 for the catalytic serine, aspartate and histidine, respectively <cite>Ollis1992</cite>. Similar to other α/β-hydrolases, the CE7 enzymes follow a mechanism where the serine is deprotonated by the histidine imidazole side chain; thereby allowing for nucleophilic attack of the serine on the incoming carbonyl of the substrate’s acetyl group <cite>Sista_Kameshwar2018</cite>. The result is a transient tetrahedral oxyanion intermediate, which is stabilized by the backbone amide group of a highly conserved glutamate residue (found in a signature RGQ motif adjacent to the nucleophilic serine at the end of β-strand 4) and a less conserved tyrosine that together form the oxyanion hole of the enzyme <cite>Levisson2012,Vincent2003</cite>. The tetrahedral intermediate then collapses resulting in an acetyl-enzyme intermediate and release of the de-O-acetylated product. A water molecule entering the active site is deprotonated by histidine and then nucleophilic attack by water on the acetyl-enzyme takes place to liberate acetate and the free enzyme <cite>Sista_Kameshwar2018</cite>. A recent study on the CE7 AXE (TM0077) from ''T. martitima'' provided further support into the catalytic mechanism <cite>Levisson2012</cite>. TM0077 was co-crystallized with PMSF and paraoxan ([{{PDBlink}}3M82 PDB ID 3M82] and [{{PDBlink}}3M83 PDB ID 3M83], respectively), two known serine protease inhibitors, that led to a 110° rotation of the catalytic serine and the formation of a covalent tetrahedral intermediate that was unable to collapse back to the starting materials (TM0077 and PMSF/paraoxan) <cite>Levisson2012</cite>. Trapping the tetrahedral intermediate is strong evidence that CE7 enzymes proceed by the proposed mechanism.
-Kinetic parameters and specific activities have been determined for some members of CE7. CAH from ''Bacillus subtilis'',  AXE from ''Paenibacillus'' sp. R4 and AXE (TM0077) from ''T. maritima'' were assayed for their activity against ''p''NP-acetate and were found to have ''K''<sub>M</sub> values of 0.29 +/- 0.01, 0.15 +/- 0.01 and 0.19 +/- 0.03 mM, respectively, and ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 260, 360 and 310 s<sup>-1</sup>mM<sup>-1</sup>, respectively<cite>Levisson2012,Vincent2003,Takimoto1994,Park2018</cite>. There has been discrepancy regarding the ability of these CE7 enzymes to utilize acetylated xylan as a substrate. While a ''Bacillus pumilus'' AXE has demonstrated a specific activity against acetylated xylan of 41 +/- 8 U/mg, ''T. maritima'''s AXE had no demonstrated activity and the acetyl xylan esterase, Axe1, from ''Thermoanerobacterium saccharolyticum'' strain JW/SL-YS485 had no activity in one study and 5.2 U/mg (8-fold lower than the ''B. pumilus'' enzyme) in another study<cite>Levisson2012,Lorenz1997,Degrassi1998,Shao1995</cite>. In light of the apparent low/no activity against acetylated xylan, the enzymes from ''T. maritima'' and ''T. saccharolyticum'' were assayed against smaller substrates, where the ''T. maritima'' AXE (TM0077) had high activity against glucose pentaacetate with a ''k''<sub>cat</sub> of 2900 s<sup>-1</sup> and Axe1 from ''T. saccharolyticum'' had high activity against xylose tetraacetate with separately reported specific activities of 210 and 740 U/mg<cite>Levisson2012,Lorenz1997,Shao1995</cite>. Lastly, CAH from ''B. subtilis'' has been kinetically characterized with regards to activity towards cephalosporin-C with a ''K''<sub>M</sub> of 24 mM and a ''k''<sub>cat</sub>/''K''<sub>M</sub> of 7.0 s<sup>-1</sup>mM<sup>-1</sup>, which has supported its naming as a cephalosporin-C deacetylase<cite>Takimoto1994</cite>.
+Kinetic parameters and specific activities have been determined for some members of CE7. CAH from ''Bacillus subtilis'',  AXE from ''Paenibacillus'' sp. R4 and AXE (TM0077) from ''T. maritima'' were assayed for their activity against ''p''NP-acetate and were found to have ''K''<sub>M</sub> values of 0.29 +/- 0.01, 0.15 +/- 0.01 and 0.19 +/- 0.03 mM, respectively, and ''k''<sub>cat</sub>/''K''<sub>M</sub> values of 260, 360 and 310 s<sup>-1</sup>mM<sup>-1</sup>, respectively <cite>Levisson2012,Vincent2003,Takimoto1994,Park2018</cite>. There has been discrepancy regarding the ability of these CE7 enzymes to utilize acetylated xylan as a substrate. While a ''Bacillus pumilus'' AXE has demonstrated a specific activity against acetylated xylan of 41 +/- 8 U/mg, ''T. maritima'''s AXE had no demonstrated activity and the acetyl xylan esterase, Axe1, from ''Thermoanerobacterium saccharolyticum'' strain JW/SL-YS485 had no activity in one study and 5.2 U/mg (8-fold lower than the ''B. pumilus'' enzyme) in another study <cite>Levisson2012,Lorenz1997,Degrassi1998,Shao1995</cite>. In light of the apparent low/no activity against acetylated xylan, the enzymes from ''T. maritima'' and ''T. saccharolyticum'' were assayed against smaller substrates, where the ''T. maritima'' AXE (TM0077) had high activity against glucose pentaacetate with a ''k''<sub>cat</sub> of 2900 s<sup>-1</sup> and Axe1 from ''T. saccharolyticum'' had high activity against xylose tetraacetate with separately reported specific activities of 210 and 740 U/mg <cite>Levisson2012,Lorenz1997,Shao1995</cite>. Lastly, CAH from ''B. subtilis'' has been kinetically characterized with regards to activity towards cephalosporin-C with a ''K''<sub>M</sub> of 24 mM and a ''k''<sub>cat</sub>/''K''<sub>M</sub> of 7.0 s<sup>-1</sup>mM<sup>-1</sup>, which has supported its naming as a cephalosporin-C deacetylase <cite>Takimoto1994</cite>.
 == Three-dimensional structures ==
 There are six resolved structures currently reported for the CE7 family. These include the AXE from ''B. pumilus'' ([{{PDBlink}}2XLB PDB ID 2XLB]), cephalosporin-C deacetylase (CAH) from ''B. subtilis'' strain 168 ([{{PDBlink}}1ODS PDB ID 1ODS]), the AXE from ''Paenibacillus'' sp. R4 ([{{PDBlink}}6AGQ PDB ID 6AGQ]), acetyl xylan esterase 1 (Axe1) from ''T. saccharolyticum'' strain JW/SL-YS485 ([{{PDBlink}}3FCY PDB ID 3FCY]), the AXE (TM0077) from ''T. maritima'' ([{{PDBlink}}1VLQ PDB ID 1VLQ]) and, lastly, an unclassified protein named Axe1-NaM1 ([{{PDBlink}}6FKX PDB ID 6FKX]).
-Enzymes in the CE7 family contain high levels of multimerization, with quaternary structures containing 4, 5, 6 and 8 subunits described<cite>Vincent2003</cite>. For each of the  six resolved structures from the CE7 family, individual subunits adopt a classic α/β-hydrolase fold that consists of a central eight-stranded β-sheet surrounded by α-helices on both sides (See Fig. 1A)<cite>Levisson2012</cite>. Together, the multimerized subunits adopt a donut-like shape that results in the formation of a tunnel leading to the centre of the protein (See Fig. 1B). Each of the active sites from the individual multimers are arranged so that they face into the centre of the tunnel<cite>Vincent2003</cite>.
+Enzymes in the CE7 family contain high levels of multimerization, with quaternary structures containing 4, 5, 6 and 8 subunits described <cite>Vincent2003</cite>. For each of the  six resolved structures from the CE7 family, individual subunits adopt a classic α/β-hydrolase fold that consists of a central eight-stranded β-sheet surrounded by α-helices on both sides (See Fig. 1A) <cite>Levisson2012</cite>. Together, the multimerized subunits adopt a donut-like shape that results in the formation of a tunnel leading to the centre of the protein (See Fig. 1B). Each of the active sites from the individual multimers are arranged so that they face into the centre of the tunnel <cite>Vincent2003</cite>.
 == Family Firsts ==
-;First Characterized:The first cephalosporin-C deacetylase (CAH) of this family to be characterized was from ''B. subtilis'' strain 168 in 1975<cite>Abbot1975</cite>. CAH is able to deacetylate cephalosporin-C, as well as cephalosporin-C derivatives (eg., cephalosporanic acid and cephalothin) at the C-3 position<cite>Abbot1975</cite>.  The first acetyl xylan esterase to be characterized was Axe1, from the anaerobic thermophilic bacterium ''T. saccharolyticum'' strain JW/SL-YS485 in 1995<cite>Shao1995</cite>.  When in the presence of acetylated xylan, Axe1 showed esterase activity that liberated acetate, leading to its name<cite>Shao1995</cite>. However, Axe1 has also demonstrated activity against other acetylated sugars<cite>Shao1995</cite>.
+;First Characterized:The first cephalosporin-C deacetylase (CAH) of this family to be characterized was from ''B. subtilis'' strain 168 in 1975 <cite>Abbot1975</cite>. CAH is able to deacetylate cephalosporin-C, as well as cephalosporin-C derivatives (eg., cephalosporanic acid and cephalothin) at the C-3 position <cite>Abbot1975</cite>.  The first acetyl xylan esterase to be characterized was Axe1, from the anaerobic thermophilic bacterium ''T. saccharolyticum'' strain JW/SL-YS485 in 1995 <cite>Shao1995</cite>.  When in the presence of acetylated xylan, Axe1 showed esterase activity that liberated acetate, leading to its name <cite>Shao1995</cite>. However, Axe1 has also demonstrated activity against other acetylated sugars <cite>Shao1995</cite>.
-;First Mechanistic Insight: The first mechanistic insight into the CE7 family came from the CAH enzyme from ''B. subtilis'' in 1994 when Takimoto and coworkers tested the effects of various chemicals (''eg.'', EDTA, PMSF, diisopropyl flurophosphate) on CAH activity and concluded that serine is likely important for catalysis<cite>Takimoto1994</cite>. Almost 10 years later in 2003, Vincent and colleagues resolved the crystal structure of CAH, confirming that serine was present in the active site and that an aspartate and histidine completed the catalytic triad<cite>Vincent2003</cite>. More recently, co-crystallization of the AXE (TM0077) from ''T. martitima'' with PMSF and paraoxan ([{{PDBlink}}3M82 PDB ID 3M82] and [{{PDBlink}}3M83 PDB ID 3M83], respectively),  two known serine protease inhibitors, led to trapping of the tetrahedral intermediate; thereby providing further support for the serine-histidine-aspartate mediated mechanism described for this family.
+;First Mechanistic Insight: The first mechanistic insight into the CE7 family came from the CAH enzyme from ''B. subtilis'' in 1994 when Takimoto and coworkers tested the effects of various chemicals (''eg.'', EDTA, PMSF, diisopropyl flurophosphate) on CAH activity and concluded that serine is likely important for catalysis <cite>Takimoto1994</cite>. Almost 10 years later in 2003, Vincent and colleagues resolved the crystal structure of CAH, confirming that serine was present in the active site and that an aspartate and histidine completed the catalytic triad <cite>Vincent2003</cite>. More recently, co-crystallization of the AXE (TM0077) from ''T. martitima'' with PMSF and paraoxan ([{{PDBlink}}3M82 PDB ID 3M82] and [{{PDBlink}}3M83 PDB ID 3M83], respectively),  two known serine protease inhibitors, led to trapping of the tetrahedral intermediate; thereby providing further support for the serine-histidine-aspartate mediated mechanism described for this family.
-;First 3-D Structure:The first resolved structure in this family was the cephalosporin-C deacetylase, CAH ([{{PDBlink}}1ODS PDB ID 1ODS]) from ''B. subtilis'' strain 168 in 2003<cite>Vincent2003</cite>. The quaternary structure consisted of a trimer of dimers to make up a donut-shaped homohexamer with 32kDa subunits that each adopt the α/β hydrolase fold (See Fig. 1)<cite>Vincent2003</cite>. The hydrophobic core containing the six active site centers is thought to help exclude large substrates and is shielded from solvents through a lid-like domain<cite>Vincent2003</cite>. In order for the hexamer to form, there are α-helix interactions as well as a β-strand-like interface associating between monomers (See Fig. 1A)<cite>Vincent2003,Sista_Kameshwar2018</cite>.
+;First 3-D Structure:The first resolved structure in this family was the cephalosporin-C deacetylase, CAH ([{{PDBlink}}1ODS PDB ID 1ODS]) from ''B. subtilis'' strain 168 in 2003 <cite>Vincent2003</cite>. The quaternary structure consisted of a trimer of dimers to make up a donut-shaped homohexamer with 32kDa subunits that each adopt the α/β hydrolase fold (See Fig. 1) <cite>Vincent2003</cite>. The hydrophobic core containing the six active site centers is thought to help exclude large substrates and is shielded from solvents through a lid-like domain <cite>Vincent2003</cite>. In order for the hexamer to form, there are α-helix interactions as well as a β-strand-like interface associating between monomers (See Fig. 1A) <cite>Vincent2003,Sista_Kameshwar2018</cite>.
 == References ==
 <biblio>