Glycoside Hydrolase Family 123 - Revision history

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

2021-12-18T21:18:01Z

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

Harry Brumer: /* Family Firsts */ Corrected superscript tag

2016-09-06T16:30:55Z

Family Firsts: Corrected superscript tag

Spencer Williams at 23:16, 31 August 2016

2016-08-31T23:16:18Z

Spencer Williams at 23:16, 31 August 2016

2016-08-31T23:16:09Z

Spencer Williams: /* Substrate specificities */

2016-08-31T23:06:15Z

Substrate specificities

Spencer Williams at 23:03, 31 August 2016

2016-08-31T23:03:33Z

Spencer Williams at 22:56, 31 August 2016

2016-08-31T22:56:55Z

Spencer Williams at 22:55, 31 August 2016

2016-08-31T22:55:53Z

Spencer Williams at 22:54, 31 August 2016

2016-08-31T22:54:25Z

Spencer Williams at 22:53, 31 August 2016

2016-08-31T22:53:02Z

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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]]: ^^^Tomomi Sumida^^^, ^^^Spencer Williams^^^
+* [[Author]]: [[User:Tomomi Sumida|Tomomi Sumida]], [[User:Spencer Williams|Spencer Williams]]
-* [[Responsible Curator]]:  ^^^Tomomi Sumida^^^
+* [[Responsible Curator]]:  [[User:Tomomi Sumida|Tomomi Sumida]]
 ----

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 == Family Firsts ==
-;First stereochemistry determination: ''Bacteroides vulgatus'' GH123 ''N''-acetyl-β-galactosaminidase by ,sup>1</sup>H NMR <cite>Roth2016</cite>.
+;First stereochemistry determination: ''Bacteroides vulgatus'' GH123 ''N''-acetyl-β-galactosaminidase by <sup>1</sup>H NMR <cite>Roth2016</cite>.
 ;First catalytic nucleophile identification: It has been proposed that the carbonyl oxygen of the C-2 acetamido group of the substrate behaves as a nucleophile <cite>SumidaJBC2011</cite>.
 ;First general acid/base residue identification: Site-directed mutagenesis supports Glu608 acting as general acid/base for NgaP <cite>SumidaJBC2011</cite>.

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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]]: ^^^Tomomi Sumida^^^ ^^^Spencer Williams^^^
+* [[Author]]: ^^^Tomomi Sumida^^^, ^^^Spencer Williams^^^
 * [[Responsible Curator]]:  ^^^Tomomi Sumida^^^
 ----

@@ Line 1: / Line 1: @@
 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]: ^^^Tomomi Sumida^^^
+* [[Author]]: ^^^Tomomi Sumida^^^ ^^^Spencer Williams^^^
 * [[Responsible Curator]]:  ^^^Tomomi Sumida^^^
 ----

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 == Substrate specificities ==
-[[Glycoside hydrolase]] family 123 contains ''N''-acetyl-β-galactosaminidases (EC [{{EClink}}3.2.1.53 3.2.1.53]), which degrade glycosphingolipids. These enzymes hydrolyze the non-reducing terminal β-GalNAc linkage, but not β-GlcNAc linkages. ''N''-Acetyl-β-galactosaminidases (EC [{{EClink}}3.2.1.53 3.2.1.53]) are distinguished from β-hexosaminidases (EC [{{EClink}}3.2.1.52 3.2.1.52]) or ''N''-acetyl-β-glucosaminidases (EC [{{EClink}}3.2.1.52 3.2.1.52]) because ''N''-acetyl-β-galactosaminidases are selective for a β-GalNAc linkage while ''N''-acetyl-β-glucosaminidases are selective for a β-GlcNAc linkage; β-hexosaminidases hydrolyze both β-GlcNAc and β-GalNAc linkages at a non-reducing terminus. NgaP ''N''-acetyl-β-galactosaminidase from ''Paenibacillus'' sp., is the founding member of this family <cite>SumidaJBC2011</cite>.
+[[Glycoside hydrolase]] family 123 contains ''N''-acetyl-β-galactosaminidases (EC [{{EClink}}3.2.1.53 3.2.1.53]), which degrade glycosphingolipids. These enzymes hydrolyze the non-reducing terminal β-GalNAc linkage, but not β-GlcNAc linkages. ''N''-Acetyl-β-galactosaminidases (EC [{{EClink}}3.2.1.53 3.2.1.53]) are distinguished from β-hexosaminidases (EC [{{EClink}}3.2.1.52 3.2.1.52]) or ''N''-acetyl-β-glucosaminidases (EC [{{EClink}}3.2.1.52 3.2.1.52]) because ''N''-acetyl-β-galactosaminidases are selective for a β-GalNAc linkage while ''N''-acetyl-β-glucosaminidases are selective for a β-GlcNAc linkage; β-hexosaminidases hydrolyze both β-GlcNAc and β-GalNAc linkages at a non-reducing terminus. NgaP ''N''-acetyl-β-galactosaminidase from ''Paenibacillus'' sp., is the founding member of this family <cite>SumidaJBC2011</cite>.  Recombinant NgaP hydrolyzes ''p''NP-β-GalNAc but not ''p''NP-β-GlcNAc, ''p''NP-β-Gal, ''p''NP-α-GalNAc or other ''p''NP-glycosides, indicating that NgaP is a highly specific ''N''-acetyl-β-galactosaminidase. CpNga123 from ''Clostridium perfringens'' (CpNga123) is also a ''N''-acetyl-β-galactosaminidase with activity on the GA2 glycan <cite>Noach2016</cite>. Recombinant ''Bv''GH123 from ''Bacteroides vulgatus'' displayed a 23-fold preference for the hydrolysis of ''p''NP-β-GalNAc versus ''p''NP-β-GlcNAc, in terms of ''k''<sub>cat</sub>/''K''<sub>M</sub> <cite>Roth2016</cite>.
 == Kinetics and Mechanism ==

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 == Three-dimensional structures ==
-The three-dimensional structure of ''Cp''Nga123 from ''Clostridium perfringens'' <cite>Noach2016</cite> and ''Bv''GH123 from ''Bacteroides vulgatus'' <cite>Roth2016</cite> have been solved . The crystal structures of ''Cp''Nga123 (apo form and complex forms with β-GalNAc (product), GalNAc-F2, GA2 trisaccharide and Gb4 disaccharide) were determined. ''Cp''Nga123 has a catalytic (β/α)<sub>8</sub>-barrel domain and an N-terminal β-sandwich domain. It was also revealed that a structural change of the active site occurred upon binding the substrate, and to order the active site residues for the proposed substrate-assisted catalytic mechanism. Furthermore, the difference of the hydrolysis activity of the enzyme toward GA2 and Gb4 glycosphingolipids was explained by the structural difference of the complex structures.
+The three-dimensional structure of ''Cp''Nga123 from ''Clostridium perfringens'' <cite>Noach2016</cite> and ''Bv''GH123 from ''Bacteroides vulgatus'' <cite>Roth2016</cite> have been solved . The crystal structures of ''Cp''Nga123 (apo form and complex forms with β-GalNAc (product), GalNAc-F2, GA2 trisaccharide and Gb4 disaccharide) were determined. ''Cp''Nga123 has a catalytic (β/α)<sub>8</sub>-barrel domain and an N-terminal β-sandwich domain, with some similarity to so-called BACON domains. It was also revealed that a structural change of the active site occurred upon binding the substrate, and to order the active site residues for the proposed substrate-assisted catalytic mechanism. Furthermore, the difference of the hydrolysis activity of the enzyme toward GA2 and Gb4 glycosphingolipids was explained by the structural difference of the complex structures. An X-ray structure of ''Bv''GH123 in complex with Gal-thiazoline revealed movement of several active-site residues compared with the 'apo' structure. Residues Asp361 and Glu362 (equivalent to Asp607 and Glu608 in NgaP), were located in positions consistent with their proposed roles as transition state stabilizer and [[general acid/base]], respectively <cite>Roth2016</cite>.
 == Family Firsts ==

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 == Catalytic Residues ==
-Point mutation analysis provide support that Glu608 of NagP functions as [[general acid/base]] and Asp607 as a transition state stabilizer of the 2-acetamido group <cite>SumidaJBC2011</cite>.
+Point mutation analysis provide support that Glu608 of NgaP functions as [[general acid/base]] and Asp607 as a transition state stabilizer of the 2-acetamido group <cite>SumidaJBC2011</cite>.
 == Three-dimensional structures ==
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 == Family Firsts ==
 ;First stereochemistry determination: ''Bacteroides vulgatus'' GH123 ''N''-acetyl-β-galactosaminidase by ,sup>1</sup>H NMR <cite>Roth2016</cite>.
-;First catalytic nucleophile identification: Unknown. It has been proposed that the carbonyl oxygen of the C-2 acetamido group of the substrate behaves as a nucleophile <cite>SumidaJBC2011</cite>.
+;First catalytic nucleophile identification: It has been proposed that the carbonyl oxygen of the C-2 acetamido group of the substrate behaves as a nucleophile <cite>SumidaJBC2011</cite>.
-;First general acid/base residue identification: Site-directed mutagenesis indicated that Glu608 is an essential amino acid for the catalytic reaction in NgaP <cite>SumidaJBC2011</cite>.
+;First general acid/base residue identification: Site-directed mutagenesis supports Glu608 acting as general acid/base for NgaP <cite>SumidaJBC2011</cite>.
 ;First 3-D structure: ''Cp''Nga123 from ''Clostridium perfringens'' <cite>Noach2016</cite>.

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 |-
 |'''Mechanism'''
-|probably retaining
+|retaining
 |-
 |'''Active site residues'''
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 == Kinetics and Mechanism ==
-The stereochemical outcome of substrate hydrolysis catalyzed by GH123 enzymes has not been determined. However, NgaP was proposed to be a retaining enzyme and to use substrate-assisted catalysis <cite>SumidaJBC2011</cite>, based on its inhibition by Gal-thiazoline, a structural analog of the oxazolinium intermediate of a [[neighboring group participation]] mechanism <cite>SumidaJBC2011</cite>. In this proposed mechanism, the C2-acetamido group of the substrate is proposed to act as a nucleophile, with a mechanism proceeding through a oxazolinium ion intermediate. Other families of [[glycoside hydrolases]] that operate through neighboring group participation mechanisms include families [[GH18]], [[GH20]], [[GH56]], [[GH84]] and [[GH85]] are [[retaining]]. A comparison of secondary structure of NgaP with that of other enzymes that utilize substrate-assisted catalysis suggested that Glu608 and Asp607 of NgaP functions as a [[general acid/base]] and a stabilizer of the 2-acetamide group of the β-GalNAc at the transition state, respectively. Point mutation analysis confirmed that Glu608 and Asp607 are integral for the activity of NgaP.
+Family GH123 ''N''-acetyl-β-galactosaminidases are retaining enzymes, as first shown by <sup>1</sup>H NMR analysis of the hydrolysis of p-nitrophenyl ''N''-acetyl-β-galactosaminide by ''Bacteroides vulgatus'' ''Bv''GH123 <cite>Roth2016</cite>. NgaP and ''Bv''GH123 are strongly inhibited by Gal-thiazoline, a mimic of an oxazolinium ion; on this basis family GH123 enzymes are proposed to act through a [[neighboring group participation]] mechanism involving an oxazolinium ion intermediate <cite>SumidaJBC2011 Roth2016</cite>. In this proposed mechanism, the C2-acetamido group of the substrate is proposed to act as a nucleophile, with a mechanism proceeding through a oxazolinium ion intermediate. Other families of [[glycoside hydrolases]] that operate through neighboring group participation mechanisms include families [[GH18]], [[GH20]], [[GH56]], [[GH84]] and [[GH85]] are [[retaining]]. A comparison of secondary structure of NgaP with that of other enzymes that utilize substrate-assisted catalysis suggested that Glu608 and Asp607 of NgaP functions as a [[general acid/base]] and a stabilizer of the 2-acetamide group of the β-GalNAc at the transition state, respectively. Point mutation analysis confirmed that Glu608 and Asp607 are integral for the activity of NgaP.
 == Catalytic Residues ==
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 == Family Firsts ==
-;First stereochemistry determination: None reported.
+;First stereochemistry determination: ''Bacteroides vulgatus'' GH123 ''N''-acetyl-β-galactosaminidase by ,sup>1</sup>H NMR <cite>Roth2016</cite>.
 ;First catalytic nucleophile identification: Unknown. It has been proposed that the carbonyl oxygen of the C-2 acetamido group of the substrate behaves as a nucleophile <cite>SumidaJBC2011</cite>.
 ;First general acid/base residue identification: Site-directed mutagenesis indicated that Glu608 is an essential amino acid for the catalytic reaction in NgaP <cite>SumidaJBC2011</cite>.
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 #SumidaJBC2011 pmid=21297160
 #Noach2016 pmid=27038508
 </biblio>
 [[Category:Glycoside Hydrolase Families|GH123]]