Glycoside Hydrolase Family 84 - Revision history

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

2021-12-18T21:21:02Z

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

Karen Eddy at 20:21, 12 June 2012

2012-06-12T20:21:52Z

Spencer Williams: /* Family Firsts */

2011-03-10T09:12:25Z

Family Firsts

Spencer Williams: /* Kinetics and Mechanism */

2011-03-10T09:10:20Z

Kinetics and Mechanism

Spencer Williams at 09:08, 10 March 2011

2011-03-10T09:08:08Z

Harry Brumer at 08:05, 25 February 2011

2011-02-25T08:05:46Z

Harry Brumer: /* Three-dimensional structures */

2011-02-25T08:03:35Z

Three-dimensional structures

Harry Brumer: /* Catalytic Residues */

2011-02-25T08:02:16Z

Catalytic Residues

Harry Brumer: /* Catalytic Residues */

2011-02-25T08:02:08Z

Catalytic Residues

Harry Brumer: /* Kinetics and Mechanism */

2011-02-25T08:01:46Z

Kinetics and Mechanism

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 <!-- CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
 {{CuratorApproved}}
-* [[Author]]: ^^^Ian Greig^^^
+* [[Author]]: [[User:Ian Greig|Ian Greig]]
-* [[Responsible Curator]]:  ^^^David Vocadlo^^^
+* [[Responsible Curator]]:  [[User:David Vocadlo|David Vocadlo]]
 ----

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 #DJV2009 pmid=19715310
 #DJV2010 pmid=20067256
-#DJV2006 Cetinbaş N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. ''Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants.'' Biochemistry. 2006 Mar 21;45(11):3835-44. //''Note: Due to a problem with PubMed data, this reference is not automatically formatted.  Please see these links out:'' [http://dx.doi.org/10.1021/bi052370b DOI:10.1021/bi052370b] [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=pubmed&dopt=Abstract&list_uids=16533067  PMID:16533067]
+#DJV2006 pmid=16533067
 #GJD2006 pmid=16565725
 #GJD2009 pmid=19217614

@@ Line 52: / Line 52: @@
 == Family Firsts ==
-;First sterochemistry determination: <sup>1</sup>H-NMR studies of human ''O''-GlcNAcase established that it hydrolyzes the product with [[retention]] of anomeric stereochemistry to give the ''β''-configured hemiacetal product, which then anomerises in solution <cite>DJV2009</cite>.
+;First sterochemistry determination: <sup>1</sup>H-NMR studies of human ''O''-GlcNAcase established that it hydrolyzes the product with [[retaining|retention]] of anomeric stereochemistry to give the ''β''-configured hemiacetal product, which then anomerises in solution <cite>DJV2009</cite>.
 ;First [[catalytic nucleophile]] identification: This family of enzymes uses a mechanism of neighbouring group participation, in the the substrate ''N''-acetyl group acts as a nucleophile. This was first established through the use of free energy relationships studies <cite>DJV2005</cite>.
 ;First [[general acid/base]] residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identified reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic [[general acid/base]] <cite>DJV2006</cite>.

@@ Line 35: / Line 35: @@
 Members of GH84 are [[retaining]] enzymes that use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of the human enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''<sub>a</sub> below 11 (with the chemical step rate-determining for substrates with higher p''K''<sub>a</sub> values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (D<sub>N</sub>*A<sub>N</sub>) <cite>Guthrie89</cite> mechanism involving [[general acid]] catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''<sub>a</sub> greater than approximately 7; a concerted (A<sub>N</sub>D<sub>N</sub>) <cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''<sub>a</sub> values (consistent with prior studies showing hydrolysis of  1-''S''-glucosaminides <cite>DJV2005Thio</cite>).
-Numerous carbohydrate and carbohydrate like-scaffolds have been reported as potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has enabled the development of inhibitors that are not only highly potent but also highly selective in their inhibition of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the [[oxocarbenium ion]]-like transition states of glycosyl group transfer and, as such may loosely be termed '[[transition state]] analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> [[transition state analogues]]. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.
+Numerous carbohydrate and carbohydrate like-scaffolds have been reported as potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has enabled the development of inhibitors that are not only highly potent but also highly selective in their inhibition of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the [[oxocarbenium ion]]-like transition states of glycosyl group transfer and, as such may loosely be termed '[[transition state]] analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> [[transition state]] analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.
 == Catalytic Residues ==

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 == Substrate specificities ==
-GH84 contains ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to  ''β''-''N''-acetylhyaluronidases <cite>Heckel98</cite>. Biochemical analysis of a GH84 enzyme annotated in this way, however, revealed that it had no such activity <cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process hyaluronan. Humans possess only one enzyme belonging to GH84 first defined by its unique biochemical properties, including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human [[GH20]] lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5) <cite>Heckel98</cite>. It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues <cite>Gao01 Dong94</cite>.  A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes <cite>DJV2009Trunc</cite>. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.
+[[Glycoside hydrolases]] of GH84 include ''β''-''N''-acetylglucosaminidases but members have often also been annotated as ''β''-''N''-acetylhyaluronidases. This second annotation arises from the initial cloning of the enzyme and its early sequence analysis, which suggested sequence similarity to  ''β''-''N''-acetylhyaluronidases <cite>Heckel98</cite>. Biochemical analysis of a GH84 enzyme annotated in as a hyaluronidase, however, revealed that it had no such activity <cite>Black2006</cite> and structural studies of other homologues have suggested the active site would be unable to process polymeric hyaluronan. Humans possess only one enzyme belonging to GH84, first defined by its unique biochemical properties including activity at neutral pH and selectivity for ''N''-acetylglucosamine residues. The human GH84 enzyme was labelled as HexC to distinguish it from the human [[GH20]] lysosomal enzymes HexA and HexB. HexC was first cloned from a meningoma library using autologous serum and defined as meningoma expressed antigen 5 (MGEA5) <cite>Heckel98</cite>. It was later cloned and biochemically characterized as ''O''-GlcNAcase, a nuclear and cytoplasmic enzyme targeting glycoprotein substrates modified by ''β''-linked GlcNAc residues linked to serine and threonine residues <cite>Gao01 Dong94</cite>.  A truncated, nuclear-localized isoform of human ''O''-GlcNAcase lacking a putative C-terminal histone acetyl transferase domain retains similar kinetic properties (aside from a much lower activity) and inhibition patterns as the full-length cytosolic isoform and is consistent with hexosaminidase activity residing in the ''N''-terminal domain, which shares similarity with other GH84 enzymes <cite>DJV2009Trunc</cite>. In contrast to the ''β''-hexosaminidases of [[GH20]] a relaxed specificity for substitutions of the ''N''-acyl group is observed with residues significantly more bulky than the ''N''-acyl group being tolerated <cite>DJV2005</cite>.
 == Kinetics and Mechanism ==
-Members of GH84 use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of this enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''<sub>a</sub> below 11 (with the chemical step rate-determining for substrates with higher p''K''<sub>a</sub> values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (D<sub>N</sub>*A<sub>N</sub>) <cite>Guthrie89</cite> mechanism involving general acid catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''<sub>a</sub> greater than approximately 7; a concerted (A<sub>N</sub>D<sub>N</sub>) <cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''<sub>a</sub> values (consistent with prior studies showing hydrolysis of  1-''S''-glucosaminides <cite>DJV2005Thio</cite>).
+Members of GH84 are [[retaining]] enzymes that use a catalytic mechanism of [[neighboring group participation]], this originally being established through the use of free-energy relationship-based studies of human ''O''-GlcNAcase <cite>DJV2005</cite>. More recent studies of the human enzyme have investigated variations in rates of reaction (''V/K'') with both nucleophile and leaving group structures <cite>DJV2009</cite>. For substrates possessing the naturally-occurring acetyl nucleophile a pre-chemical step is rate-determining on ''V/K'' for leaving groups with a p''K''<sub>a</sub> below 11 (with the chemical step rate-determining for substrates with higher p''K''<sub>a</sub> values). Studies of substrates possessing fluoroacetyl nucleophiles highlighted that a dissociative (D<sub>N</sub>*A<sub>N</sub>) <cite>Guthrie89</cite> mechanism involving [[general acid]] catalysis operates for the hydrolysis of substrates possessing leaving groups with a p''K''<sub>a</sub> greater than approximately 7; a concerted (A<sub>N</sub>D<sub>N</sub>) <cite>Guthrie89</cite> mechanism, not employing general acid catalysis was found for substrates possessing leaving groups with lower p''K''<sub>a</sub> values (consistent with prior studies showing hydrolysis of  1-''S''-glucosaminides <cite>DJV2005Thio</cite>).
-Numerous carbohydrate and carbohydrate like-scaffolds have been reported as yielding potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has led to the development of inhibitors that are not only highly potent but also highly selective in their binging of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the oxacarbenium ion-like transition states of glycosyl group transfer and, as such may loosely be termed 'transition state analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> transition state analogues. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.
+Numerous carbohydrate and carbohydrate like-scaffolds have been reported as potent inhibitors of GH84 enzymes. These include "NAG-thiazolines" <cite>DJV2005</cite>, PUGNAc (''O''-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-''N''-phenylcarbamate) <cite>Stubbs06</cite>, GlcNAcstatins <cite>Dorf2006 Vasella2006</cite>, 6-''epi''-valeinamines <cite>Stick2007</cite>, and 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite>. The greater tolerance of the ''N''-acyl binding pockets of GH84 enzymes for bulky sidechains compared to those of [[GH20]] enzymes has enabled the development of inhibitors that are not only highly potent but also highly selective in their inhibition of human nucleocytoplasmic ''O''-GlcNAcase (GH84) over human lysosomal ''β''-hexosaminidases <cite>DJV2005 Whitworth2007 Dorf2010</cite>. Many of these families of inhibitors possess structural characteristics reminiscent of the [[oxocarbenium ion]]-like transition states of glycosyl group transfer and, as such may loosely be termed '[[transition state]] analogues'. An analysis of NAG-thiazoline- and PUGNAc-derived inhibitors of human ''O''-GlcNAcase has shown that only the NAG-thiazolines position the inhibitors and their ''N''-acyl side-chains within the hydrophobic binding pocket in a manner consistent with the species found along the reaction coordinate <cite>Whitworth2007</cite>. As such NAG-thiazoline inhibitors may be termed 'Bartlett-type' (free-energy relationship-based) <cite>Bartlett1997</cite> [[transition state analogues]]. Significantly streptozotocin, a widely used diabetogenic compound whose toxicity towards pancreatic β-cells was hypothesized to arise from its ability to act as an inhibitor of human ''O''-GlcNAcase, neither binds covalently to GH84 enzymes <cite>DJV2005 DvA2008 GJD2009</cite>, nor is a particularly potent inhibitor of human ''O''-GlcNAcase <cite>DJV2005 Kudlow2001 Sato1999</cite>.
 == Catalytic Residues ==
 Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.
-The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results clearly identified Asp175 as the general acid catalyst.
+The mutant Asp175Ala displayed marked reductions in activity (''V'' and (''V/K'')) towards aryl ''N''-acetylglucosaminides possessing poor leaving groups with smaller reductions being observed for both ''O''-aryl and ''S''-aryl ''N''-acetylglucosaminides substrates possessing better leaving groups. Exogenous azide was found to partially rescue the activity of human ''O''-GlcNAcase towards 3,4-dinitrophenylglucosaminide. These results clearly identified Asp175 as the [[general acid]] catalyst.
 The mutant Asp174Ala showed significantly decreased activity towards ''O''-aryl ''N''-acetylglucosaminides possessing either good or poor leaving groups and it was argued that this is consistent with its role as a residue responsible for the orientation and polarization of the ''N''-acyl nucleophile.
@@ Line 52: / Line 52: @@
 == Family Firsts ==
-;First sterochemistry determination: <sup>1</sup>H-NMR studies of human ''O''-GlcNAcase established that the ''β''-configured hemiacetal product is formed by the enzyme prior to anomerisation in solution <cite>DJV2009</cite>.
+;First sterochemistry determination: <sup>1</sup>H-NMR studies of human ''O''-GlcNAcase established that it hydrolyzes the product with [[retention]] of anomeric stereochemistry to give the ''β''-configured hemiacetal product, which then anomerises in solution <cite>DJV2009</cite>.
-;First catalytic nucleophile identification: This family of enzymes uses a mechanism of neighbouring group participation, which was first establishes through the use of free energy relationships studies <cite>DJV2005</cite>.
+;First [[catalytic nucleophile]] identification: This family of enzymes uses a mechanism of neighbouring group participation, in the the substrate ''N''-acetyl group acts as a nucleophile. This was first established through the use of free energy relationships studies <cite>DJV2005</cite>.
-;First general acid/base residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identify reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic general acid/base <cite>DJV2006</cite>.
+;First [[general acid/base]] residue identification: Studies of human ''O''-GlcNAcase mutant Asp175Ala identified reactivity patterns (free energy relationships, pH-activity profiles) consistent with the action of Asp175 as the catalytic [[general acid/base]] <cite>DJV2006</cite>.
 ;First 3-D structure: The structures of ''Bacteroides thetaiotaomicron'' ''O''-GlcNAcase <cite>GJD2006</cite> and ''Clostridium perfringens'' NagJ <cite>DvA2006</cite>.

@@ Line 59: / Line 59: @@
 == References ==
 <biblio>
 #Gao01 pmid=11148210
 #Guthrie89 Guthrie RD, Jencks WP. ''IUPAC Recommendations for the Representation of Reaction Mechanisms'' Acc. Chem. Res. 1989; 22(10): 343-349.
@@ Line 89: / Line 88: @@
 #Sato1999 pmid=9917327
 #Mac2010 pmid=20851343
 </biblio>
 [[Category:Glycoside Hydrolase Families|GH084]]

@@ Line 45: / Line 45: @@
 == Three-dimensional structures ==
-The reported crystallization of ''Clostridium perfringens'' NagJ (''Cp''OGA)<cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-glucosaminidase (''Bt''OGA)<cite>DvA2006</cite>. In common with the chitinases of family GH18 and the ''exo''-acting ''β''-hexosaminidases of GH20 the catalytic domain is a (''βα'')<sub>8</sub>-barrel structure. The structure of human ''O''-GlcNAcase  has not been solved but both bacterial homologues are good models of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess good sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain, which separates the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
+The reported crystallization of ''Clostridium perfringens'' NagJ (''Cp''OGA) <cite>ABB2005</cite> was followed by solved structures for that enzyme <cite>DvA2006</cite> and ''Bacteroides thetaiotaomicron'' ''β''-glucosaminidase (''Bt''OGA) <cite>DvA2006</cite>. In common with the chitinases of family [[GH18]] and the ''exo''-acting ''β''-hexosaminidases of [[GH20]] the catalytic domain is a (''βα'')<sub>8</sub>-barrel structure. The structure of human ''O''-GlcNAcase  has not been solved but both bacterial homologues are good models of both the active site and catalytic domain of human ''O''-GlcNAcase. More recently the structure of the GH84 ''β''-hexosaminidase from ''Oceanicola granulosus'' has been solved and shown to possess good sequence identity with the catalytic domain of the human enzyme <cite>DvA2010</cite>. Furthermore, the ''C''-terminal domain of this protein also displays notable sequence identity with the spacer domain, which separates the domains possessing ''O''-GlcNAcase and histone acetyltransferase activities) of the human enzyme.
 [[Image:GH84conf.jpg|thumb|450px|'''Defining the conformational itinerary of a GH84 enzyme.''' Azepane (i)  binds with a boat-like conformation to ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase (''Bt''OG). This binding mode is confirmed by the structure of a bound substrate (ii). Thiazoline (iii) binds to wild-type ''Bt''OG, 5-fluoro-oxazoline (iv) binds to the Asp243Asn mutant of ''Bt''OG, and oxazoline (v) binds to the Asp242Asn mutant of ''Bt''OG in the <sup>4</sup>C<sub>1</sub> conformation.]]
-A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define the conformational itinerary for this family of enzymes. Substrate distortion from the stable <sup>4</sup>C<sub>1</sub> conformation found in solution to a bound <sup>1,4</sup>B / <sup>1</sup>S<sub>3</sub> conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>.  This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OGA-bound thiazoline show that this intermediate is found in a <sup>4</sup>C<sub>1</sub>conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound in this conformation to ''Bt''OGA active site mutants<cite>GJD2010</cite>.
+A series of crystallographic studies on ''Bacteroides thetaiotaomicron'' ''β''-hexosaminidase have used a variety of small molecules to define the conformational itinerary for this family of enzymes. Substrate distortion from the stable <sup>4</sup>C<sub>1</sub> conformation found in solution to a bound <sup>1,4</sup>B / <sup>1</sup>S<sub>3</sub> conformation was supported by the crystal structure of the wild-type enzyme in complex with 6-acetamido-6-deoxy-castanospermine <cite>Mac2010</cite> and the 7-membered ring-containing azepane <cite>Ble2009</cite>.  This distortion was confirmed by the structure the wild-type enzyme in complex with the substrate 3,4-difluorophenyl 2-deoxy-2-difluoroacetamido-''β''-D-glucopyranoside <cite>GJD2010</cite>. Early studies of the wild-type ''Bt''OGA-bound thiazoline show that this intermediate is found in a <sup>4</sup>C<sub>1</sub>conformation <cite>DJV2005</cite>; subsequent studies have shown that oxazoline intermediates are bound in this conformation to ''Bt''OGA active site mutants <cite>GJD2010</cite>.
 == Family Firsts ==

← Older revision		Revision as of 08:02, 25 February 2011
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	== Catalytic Residues ==		== Catalytic Residues ==
−
	Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.		Studies of two mutants of human ''O''-GlcNAcase established that adjacent aspartate residues, Asp174 and Asp175, act as critical components of the catalytic machinery of this enzyme <cite>DJV2006</cite>.