Glycoside Hydrolase Family 109 - Revision history

Spencer Williams at 02:22, 16 December 2023

2023-12-16T02:22:27Z

Harry Brumer: /* Substrate specificities */

2019-11-15T20:07:33Z

Substrate specificities

Spencer Williams at 03:20, 14 June 2011

2011-06-14T03:20:32Z

Harry Brumer at 13:24, 9 May 2011

2011-05-09T13:24:07Z

Harry Brumer: updated CAZy DB link

2011-05-09T13:23:43Z

updated CAZy DB link

Spencer Williams at 11:46, 5 July 2010

2010-07-05T11:46:42Z

Harry Brumer: /* Kinetics and Mechanism */

2010-04-27T14:46:45Z

Kinetics and Mechanism

Harry Brumer: /* Catalytic Residues */

2010-04-27T14:46:20Z

Catalytic Residues

Harry Brumer: /* Family Firsts */

2010-04-27T14:45:41Z

Family Firsts

Harry Brumer: /* Family Firsts */

2010-04-27T14:43:47Z

Family Firsts

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 |-
 |'''Mechanism'''
-|retaining
+|NAD-dependent hydrolysis
 |-
 |'''Active site residues'''

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 == Substrate specificities ==
-The only activity so far identified in this recently discovered family of [[glycoside hydrolases]] is that of &alpha;-''N''-acetylgalactosaminidase, although the lack of activity of several family members on GalNAc substrates suggests that other substrates might exist. The most characterized member of this family is the enzyme from ''Elizabethkingia meningosepticum''. Because it operates at neutral pH optimum, this enzyme was used succesfully for the removal of the A antigen on red blood cells thus opening the possibility of blood group conversion to universal group O <cite>1</cite>. The enzyme clearly prefers GalNAc over Gal as the aglycon as indicated by a 2,000-fold reduction in kcat for the hydrolysis of p-nitrophenyl &alpha;-galactoside compared with p-nitrophenyl &alpha;-''N''-acetyl-galactosaminide and by a more than tenfold increase in ''K''<sub>m</sub> <cite>1</cite>.
+The only activity so far identified in this recently discovered family of [[glycoside hydrolases]] is that of &alpha;-''N''-acetylgalactosaminidase, although the lack of activity of several family members on GalNAc substrates suggests that other substrates might exist. The most characterized member of this family is the enzyme from ''Elizabethkingia meningosepticum''. Because it operates at neutral pH optimum, this enzyme was used succesfully for the removal of the A antigen on red blood cells thus opening the possibility of blood group conversion to universal group O <cite>1</cite>. The enzyme clearly prefers GalNAc over Gal, as indicated by a 2,000-fold reduction in kcat for the hydrolysis of p-nitrophenyl &alpha;-galactoside compared with p-nitrophenyl &alpha;-''N''-acetyl-galactosaminide and by a more than tenfold increase in ''K''<sub>m</sub> <cite>1</cite>.
 == Kinetics and Mechanism ==

@@ Line 25: / Line 25: @@
 == Substrate specificities ==
-The only activity so far identified in this recently discovered family of [[glycoside hydrolases]] is that of alpha-N-acetylgalactosaminidase, although the lack of activity of several family members on GalNAc substrates suggests that other substrates might exist. The most characterized member of this family is the enzyme from ''Elizabethkingia meningosepticum''. Because it operates at neutral pH optimum,  this enzyme was used succesfully for the removal of the A antigen on red blood cells thus opening the possibility of blood group conversion to universal group O <cite>1</cite>. The enzyme clearly prefers GalNAc over Gal as the aglycon as indicated by a 2,000-fold reduction in kcat for the hydrolysis of pNP-alpha-Gal compared with pNP-alpha-GalNAc and by a more than tenfold increase in Km <cite>1</cite>.
+The only activity so far identified in this recently discovered family of [[glycoside hydrolases]] is that of &alpha;-''N''-acetylgalactosaminidase, although the lack of activity of several family members on GalNAc substrates suggests that other substrates might exist. The most characterized member of this family is the enzyme from ''Elizabethkingia meningosepticum''. Because it operates at neutral pH optimum, this enzyme was used succesfully for the removal of the A antigen on red blood cells thus opening the possibility of blood group conversion to universal group O <cite>1</cite>. The enzyme clearly prefers GalNAc over Gal as the aglycon as indicated by a 2,000-fold reduction in kcat for the hydrolysis of p-nitrophenyl &alpha;-galactoside compared with p-nitrophenyl &alpha;-''N''-acetyl-galactosaminide and by a more than tenfold increase in ''K''<sub>m</sub> <cite>1</cite>.
 == Kinetics and Mechanism ==
-Family GH109 enzymes operate via the unusual [[NAD-dependent hydrolysis]]  mechanism involving an NAD<sup>+</sup> cofactor, that so far has been seen only in [[Glycoside Hydrolase Family 4]] ([[GH4]]), despite different overall folds between these families (see below). NMR monitoring of the reaction catalyzed by alpha-N-acetylgalactosaminidase indicated that the enzyme proceeds with retention of the anomeric configuration and concomitant exchange of the GalNAc H-2 atom for a solvent proton [1]. This, and the indispensable presence of NAD+, indicate that GH109 enzymes most likely operate by a similar [[retaining]] mechanism. In this mechanism, the NAD+ molecule oxidizes the substrate at C-3, thereby acidifying the proton at C-2 and producing NADH. Deprotonation of C-2 by an enzymatic base with concomitant elimination of the glycosidic oxygen generates a 1,2-unsaturated [[intermediate]]. The reaction is completed by addition of water to the Michael-like acceptor and reduction of the resulting ketone by the NADH molecule, which returns to the initial NAD<sup>+</sup> state, ready for another catalytic cycle. The mechanism of GH109 enzymes allows cleavage of thioglycosides and of glycosides of the opposite anomeric configuration (both at a comparatively slow rate), two features that are extremely rare among 'classical' glycosidases <cite>1</cite>.
+Family GH109 enzymes operate via the unusual [[NAD-dependent hydrolysis]] mechanism involving an NAD<sup>+</sup> cofactor, that so far has been seen only in [[Glycoside Hydrolase Family 4]] ([[GH4]]), despite different overall folds between these families (see below). NMR monitoring of the reaction catalyzed by &alpha;-''N''-acetylgalactosaminidase indicated that the enzyme proceeds with retention of the anomeric configuration and concomitant exchange of the GalNAc H-2 atom for a solvent proton [1]. This, and the indispensable presence of NAD+, indicate that GH109 enzymes most likely operate by a similar [[retaining]] mechanism. In this mechanism, the NAD+ molecule oxidizes the substrate at C-3, thereby acidifying the proton at C-2 and producing NADH. Deprotonation of C-2 by an enzymatic base with concomitant elimination of the glycosidic oxygen generates a 1,2-unsaturated [[intermediate]]. The reaction is completed by addition of water to the Michael-like acceptor and reduction of the resulting ketone by the NADH molecule, which returns to the initial NAD<sup>+</sup> state, ready for another catalytic cycle. The mechanism of GH109 enzymes allows cleavage of thioglycosides and of glycosides of the opposite anomeric configuration (both at a comparatively slow rate), two features that are extremely rare among 'classical' glycosidases <cite>1</cite>.
 == Catalytic Residues ==
@@ Line 34: / Line 34: @@
 == Three-dimensional structures ==
-The three-dimensional structure of ''Elizabethkingia meningosepticum'' alpha-N-acetylgalactosaminidase has been reported in 2007 <cite>1</cite>. The closest structural relatives belong to the Gfo/Idh/MocA oxidoreductase family (Z-score of 29.4 and r.m.s. deviation of 3.0 A for 329 equivalent Ca-atoms for ''Zymomonas mobilis'' glucose-fructose oxidoreductase ([{{PDBlink}}1ofg PDB 1ofg]). More distant structural homologs are identified by means of the classical Rossmann fold. The structural similarity includes the active-site architecture, where the spatial arrangement of NAD+ and several other residues is conserved, suggesting a common ancestor that has evolved its NAD+-based molecular mechanism to adapt to diverse metabolic requirements <cite>1</cite>.
+The three-dimensional structure of ''Elizabethkingia meningosepticum'' &alpha;-''N''-acetylgalactosaminidase has been reported in 2007 <cite>1</cite>. The closest structural relatives belong to the Gfo/Idh/MocA oxidoreductase family (Z-score of 29.4 and r.m.s. deviation of 3.0 A for 329 equivalent Ca-atoms for ''Zymomonas mobilis'' glucose-fructose oxidoreductase ([{{PDBlink}}1ofg PDB 1ofg]). More distant structural homologs are identified by means of the classical Rossmann fold. The structural similarity includes the active-site architecture, where the spatial arrangement of NAD+ and several other residues is conserved, suggesting a common ancestor that has evolved its NAD+-based molecular mechanism to adapt to diverse metabolic requirements <cite>1</cite>.
 == Family Firsts ==
-;First sterochemistry determination: ''Elizabethkingia meningosepticum'' alpha-N-acetylgalactosaminidase by <sup>1</sup>H NMR <cite>1</cite>.
+;First sterochemistry determination: ''Elizabethkingia meningosepticum'' &alpha;-''N''-acetylgalactosaminidase by <sup>1</sup>H NMR <cite>1</cite>.
-;First mechanistic identification: ''Elizabethkingia meningosepticum'' alpha-N-acetylgalactosaminidase, by deuterium exchange of H-2, involvement of NAD<sup>+</sup> and structural similarity with GH4 enzymes <cite>1</cite>.
+;First mechanistic identification: ''Elizabethkingia meningosepticum'' &alpha;-''N''-acetylgalactosaminidase, by deuterium exchange of H-2, involvement of NAD<sup>+</sup> and structural similarity with GH4 enzymes <cite>1</cite>.
-;First 3-D structure: ''Elizabethkingia meningosepticum'' alpha-N-acetylgalactosaminidase <cite>1</cite> ([{{PDBlink}}2ixa PDB 2ixa]) and ([{{PDBlink}}2ixb PDB 2ixb]).
+;First 3-D structure: ''Elizabethkingia meningosepticum'' &alpha;-''N''-acetylgalactosaminidase <cite>1</cite> ([{{PDBlink}}2ixa PDB 2ixa]) and ([{{PDBlink}}2ixb PDB 2ixb]).
 == References ==

@@ Line 26: / Line 26: @@
 == Substrate specificities ==
 The only activity so far identified in this recently discovered family of [[glycoside hydrolases]] is that of alpha-N-acetylgalactosaminidase, although the lack of activity of several family members on GalNAc substrates suggests that other substrates might exist. The most characterized member of this family is the enzyme from ''Elizabethkingia meningosepticum''. Because it operates at neutral pH optimum,  this enzyme was used succesfully for the removal of the A antigen on red blood cells thus opening the possibility of blood group conversion to universal group O <cite>1</cite>. The enzyme clearly prefers GalNAc over Gal as the aglycon as indicated by a 2,000-fold reduction in kcat for the hydrolysis of pNP-alpha-Gal compared with pNP-alpha-GalNAc and by a more than tenfold increase in Km <cite>1</cite>.
 == Kinetics and Mechanism ==

@@ Line 20: / Line 20: @@
 |{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
 |-
-| colspan="2" |http://www.cazy.org/fam/GH109.html
+| colspan="2" |{{CAZyDBlink}}GH109.html
 |}
 </div>

@@ Line 29: / Line 29: @@
 == Kinetics and Mechanism ==
-Family GH109 enzymes operate via the unusual [[NAD-dependent hydrolysis]]  mechanism involving an NAD<sup>+</sup> cofactor, that so far has been seen only in [[Glycoside Hydrolase Family 4]] (GH4), despite different overall folds between these families (see below). NMR monitoring of the reaction catalyzed by alpha-N-acetylgalactosaminidase indicated that the enzyme proceeds with retention of the anomeric configuration and concomitant exchange of the GalNAc H-2 atom for a solvent proton [1]. This, and the indispensable presence of NAD+, indicate that GH109 enzymes most likely operate by a similar [[retaining]] mechanism. In this mechanism, the NAD+ molecule oxidizes the substrate at C-3, thereby acidifying the proton at C-2 and producing NADH. Deprotonation of C-2 by an enzymatic base with concomitant elimination of the glycosidic oxygen generates a 1,2-unsaturated intermediate. The reaction is completed by addition of water to the Michael-like acceptor and reduction of the resulting ketone by the NADH molecule, which returns to the initial NAD<sup>+</sup> state, ready for another catalytic cycle. The mechanism of GH109 enzymes allows cleavage of thioglycosides and of glycosides of the opposite anomeric configuration (both at a comparatively slow rate), two features that are extremely rare among 'classical' glycosidases <cite>1</cite>.
+Family GH109 enzymes operate via the unusual [[NAD-dependent hydrolysis]]  mechanism involving an NAD<sup>+</sup> cofactor, that so far has been seen only in [[Glycoside Hydrolase Family 4]] ([[GH4]]), despite different overall folds between these families (see below). NMR monitoring of the reaction catalyzed by alpha-N-acetylgalactosaminidase indicated that the enzyme proceeds with retention of the anomeric configuration and concomitant exchange of the GalNAc H-2 atom for a solvent proton [1]. This, and the indispensable presence of NAD+, indicate that GH109 enzymes most likely operate by a similar [[retaining]] mechanism. In this mechanism, the NAD+ molecule oxidizes the substrate at C-3, thereby acidifying the proton at C-2 and producing NADH. Deprotonation of C-2 by an enzymatic base with concomitant elimination of the glycosidic oxygen generates a 1,2-unsaturated intermediate. The reaction is completed by addition of water to the Michael-like acceptor and reduction of the resulting ketone by the NADH molecule, which returns to the initial NAD<sup>+</sup> state, ready for another catalytic cycle. The mechanism of GH109 enzymes allows cleavage of thioglycosides and of glycosides of the opposite anomeric configuration (both at a comparatively slow rate), two features that are extremely rare among 'classical' glycosidases <cite>1</cite>.
 == Catalytic Residues ==

@@ Line 32: / Line 32: @@
 == Catalytic Residues ==
-A stated above the enzymes of this family do not use a classical acid/base catalysis, but instead use a rare catalytic mechanism involving [[NAD-dependent hydrolysis]] with an NAD+ cofactor, highly similar to that seen in family [[Glycoside Hydrolase Family 4]]. The catalytic machinery therefore comprises NAD<sup>+</sup> and Tyr-179, which abstracts H-2 to form the unsaturated intermediate. Subtle differences with family GH4 exist, however, such as the absence in GH109 of an identifiable acid to assist glycosidic bond cleavage. It is believed that this enables the hydrolysis of the 'wrong' anomer.
+A stated above the enzymes of this family do not use a classical acid/base catalysis, but instead use a rare catalytic mechanism involving [[NAD-dependent hydrolysis]] with an NAD+ cofactor, highly similar to that seen in family [[Glycoside Hydrolase Family 4]]. The catalytic machinery therefore comprises NAD<sup>+</sup> and Tyr-179, which abstracts H-2 to form the unsaturated intermediate. Subtle differences with family [[GH4]] exist, however, such as the absence in GH109 of an identifiable acid to assist glycosidic bond cleavage. It is believed that this enables the hydrolysis of the 'wrong' anomer.
 == Three-dimensional structures ==