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Difference between revisions of "Glycoside Hydrolase Family 89"

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== Substrate specificities ==
 
== Substrate specificities ==
  
Family 89 [[glycoside hydrolase]]s are α-N-acetylglucosaminidases <cite>1 2</cite>. CpGH89, produced by the animal pathogen ''Clostridium perfringens'', is active on the unusual α-D-GlcNAc-(1,4)-D-Galactose motif <cite>7</cite> displayed on the gastric mucosal class III mucins <cite>8</cite>.  The human lysosomal enzyme, NAGLU, is involved in the degradation of heparan sulfate <cite>3 4 </cite>.  Mutations in this enzyme can cause a devastating disease called Sanfilippo syndrome type B which is also called mucopolysaccharidosis IIIB <cite>1 2 3 4 </cite>.  
+
Family 89 [[glycoside hydrolase]]s are α-N-acetylglucosaminidases <cite>1 2</cite>. CpGH89, produced by the animal pathogen ''Clostridium perfringens'', is active on the unusual α-D-GlcNAc-(1,4)-D-Galactose motif <cite>7</cite> displayed on the gastric mucosal class III mucins <cite>8</cite>.  The human lysosomal enzyme, NAGLU, is involved in the degradation of heparan sulfate <cite>3 4 </cite>.  Mutations in this enzyme can cause a devastating disease called Sanfilippo syndrome type B which is also called mucopolysaccharidosis IIIB (MPS IIIB) <cite>1 2 3 4 </cite>.  
  
 
== Kinetics and Mechanism ==
 
== Kinetics and Mechanism ==
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The three dimensional structure was first described for CpGH89,  see PDB entries [{{PDBlink}}2vc9 2vc9], [{{PDBlink}}2vca 2vca], [{{PDBlink}}2vcb 2vcb],[{{PDBlink}}2vcc 2vcc] <cite>1</cite> followed by the structure of a CpGH89 double catalytic residue mutant in complex with the class III mucosal disaccharide α-D-GlcNAc-(1,4)-D-Galactose [{{PDBlink}}4a4a 4a4a]<cite>6 8</cite>.  The structure of the human enzyme NAGLU, [{{PDBlink}}4xwh 4xwh] was released in 2019 <cite>5</cite>.   
 
The three dimensional structure was first described for CpGH89,  see PDB entries [{{PDBlink}}2vc9 2vc9], [{{PDBlink}}2vca 2vca], [{{PDBlink}}2vcb 2vcb],[{{PDBlink}}2vcc 2vcc] <cite>1</cite> followed by the structure of a CpGH89 double catalytic residue mutant in complex with the class III mucosal disaccharide α-D-GlcNAc-(1,4)-D-Galactose [{{PDBlink}}4a4a 4a4a]<cite>6 8</cite>.  The structure of the human enzyme NAGLU, [{{PDBlink}}4xwh 4xwh] was released in 2019 <cite>5</cite>.   
  
CpGH89 is a multi-modular protein and quite large (2095 amino acids). Only residues 26-916 were crystallized <cite>1</cite>.  The N-terminal domain (residues 26-155) forms a β-sandwich fold and shares sequence identity to the family 32 carbohydrate-binding modules (CBMs).  This module is tightly packed against the rest of the protein through a number of hydrophobic and hydrogen bonding interactions. The catalytic region is comprised of a small mixed α/β domain (residues 170-280), a decorated (α/β)<sub>8</sub> core (residues 280-620), and an all α-helical domain (residues 621-916).  The active site is ''sock shaped''  which is able to accommodate the bend in the class III mucosal α-D-GlcNAc-(1,4)-D-Galactose motif caused by the alpha glycosidic linkage <cite>6</cite>.  
+
CpGH89 is a multi-modular protein and quite large (2095 amino acids). Only residues 26-916 were crystallized <cite>1</cite>.  The N-terminal domain (residues 26-155) forms a β-sandwich fold and shares sequence identity to the family 32 carbohydrate-binding modules (CBMs).  This module is tightly packed against the rest of the protein through a number of hydrophobic and hydrogen bonding interactions. The catalytic region is comprised of a small mixed α/β domain (residues 170-280), a decorated (α/β)<sub>8</sub> core (residues 280-620), and an all α-helical domain (residues 621-916).  The active site is sock shaped to accommodate the bend in the class III mucosal α-D-GlcNAc-(1,4)-D-Galactose motif caused by the alpha glycosidic linkage <cite>6</cite>. The mutations causing MPS IIIB were mapped onto the bacterial homologue, demonstrating that the mutations are mainly structural in nature and not
  
NAGLU has only 28.2% sequence identity with CpGH89 and lacks the [[CBM32]].  Otherwise the tertiary structures of CpGH89 and NAGLU are similar <cite>5</cite>. The predicted catalytic glutamates, E316 and E446, are present in NAGLU <cite>5</cite>.  NAGLU has six potential N-glycosylation sites and appears to be N-glycosylated in the crystal structure <cite>5</cite>. The recombinant enzyme was produced as a trimer in solution though the physiological relevance of the trimer is unknown <cite>5</cite>. The substrate binding pocket of NAGLU is more open when compared to the ''sock shaped'' active site of CpGH89 <cite>5 1</cite>.
+
NAGLU has only 28.2% sequence identity with CpGH89 and lacks the [[CBM32]].  Otherwise the tertiary structures of CpGH89 and NAGLU are similar <cite>5</cite>. The predicted catalytic glutamates, E316 and E446, are present in NAGLU <cite>5</cite>.  NAGLU has six potential N-glycosylation sites and appears to be N-glycosylated in the crystal structure <cite>5</cite>. The recombinant enzyme was produced as a trimer in solution though the physiological relevance of the trimer is unknown <cite>5</cite>. The substrate binding pocket of NAGLU is more open when compared to the sock shaped active site of CpGH89 <cite>5 1</cite>.
  
 
== Family Firsts ==
 
== Family Firsts ==

Revision as of 06:57, 25 May 2020

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Glycoside Hydrolase Family GH89
Clan none
Mechanism Retaining
Active site residues known
CAZy DB link
http://www.cazy.org/GH89.html

Substrate specificities

Family 89 glycoside hydrolases are α-N-acetylglucosaminidases [1, 2]. CpGH89, produced by the animal pathogen Clostridium perfringens, is active on the unusual α-D-GlcNAc-(1,4)-D-Galactose motif [3] displayed on the gastric mucosal class III mucins [4]. The human lysosomal enzyme, NAGLU, is involved in the degradation of heparan sulfate [5, 6]. Mutations in this enzyme can cause a devastating disease called Sanfilippo syndrome type B which is also called mucopolysaccharidosis IIIB (MPS IIIB) [1, 2, 5, 6].

Kinetics and Mechanism

Mechanistic data is available on CpGH89, a family 89 glycoside hydrolase produced by Clostridium perfringens. CpGH89 uses a classical Koshland retaining mechanism to hydrolyze the glycosidic bond. This results in hydrolysis with a net retention of stereochemistry at the anomeric carbon [1].

Catalytic Residues

Two catalytically important glutamate residues have been identified in CpGH89, Glu483 and Glu601 [1]. These residues are between 6.1-6.7Å apart, which is consistent with a retaining catalytic mechanism. Mutation of Glu601 to an alanine results in an apparent abolishment of activity suggesting this residue is active as the catalytic nucleophile. Glu601 resides below the A-face of the sugar ring and is 2.8-3.1Å from C1 and appears suitably placed for nucleophilic attack on the anomeric carbon. Mutation of Glu483 to alanine results in much less severe impairments in catalysis suggesting this residue is active as the general acid/base residue. Glu483 is ~3.6Å from C1 and appears to be positioned in such a way that it would be capable of forming a hydrogen bond with the glycosidic oxygen of the substrate.

Three-dimensional structures

The three dimensional structure was first described for CpGH89, see PDB entries 2vc9, 2vca, 2vcb,2vcc [1] followed by the structure of a CpGH89 double catalytic residue mutant in complex with the class III mucosal disaccharide α-D-GlcNAc-(1,4)-D-Galactose 4a4a[4, 7]. The structure of the human enzyme NAGLU, 4xwh was released in 2019 [8].

CpGH89 is a multi-modular protein and quite large (2095 amino acids). Only residues 26-916 were crystallized [1]. The N-terminal domain (residues 26-155) forms a β-sandwich fold and shares sequence identity to the family 32 carbohydrate-binding modules (CBMs). This module is tightly packed against the rest of the protein through a number of hydrophobic and hydrogen bonding interactions. The catalytic region is comprised of a small mixed α/β domain (residues 170-280), a decorated (α/β)8 core (residues 280-620), and an all α-helical domain (residues 621-916). The active site is sock shaped to accommodate the bend in the class III mucosal α-D-GlcNAc-(1,4)-D-Galactose motif caused by the alpha glycosidic linkage [7]. The mutations causing MPS IIIB were mapped onto the bacterial homologue, demonstrating that the mutations are mainly structural in nature and not

NAGLU has only 28.2% sequence identity with CpGH89 and lacks the CBM32. Otherwise the tertiary structures of CpGH89 and NAGLU are similar [8]. The predicted catalytic glutamates, E316 and E446, are present in NAGLU [8]. NAGLU has six potential N-glycosylation sites and appears to be N-glycosylated in the crystal structure [8]. The recombinant enzyme was produced as a trimer in solution though the physiological relevance of the trimer is unknown [8]. The substrate binding pocket of NAGLU is more open when compared to the sock shaped active site of CpGH89 [1, 8].

Family Firsts

First sterochemistry determination
1H NMR spectroscopy reveals that CpGH89 acts with retention of stereochemistry [1].
First catalytic nucleophile identification
The catalytic nucleophile was revealed by site directed mutagenesis on CpGH89 Glu601 [1].
First general acid/base residue identification
The general acid/base was revealed by site directed mutagenesis on CpGH89 Glu483 [1].
First 3-D structure
See PDB entries 2vc9, 2vca, 2vcb and 2vcc [1].

References

  1. Ficko-Blean E, Stubbs KA, Nemirovsky O, Vocadlo DJ, and Boraston AB. (2008) Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci U S A. 105, 6560-5. DOI:10.1073/pnas.0711491105 | PubMed ID:18443291 | HubMed [1]
  2. Weber B, Hopwood JJ, and Yogalingam G. (2001) Expression and characterization of human recombinant and alpha-N-acetylglucosaminidase. Protein Expr Purif. 21, 251-9. DOI:10.1006/prep.2000.1361 | PubMed ID:11237686 | HubMed [2]
  3. Fujita M, Tsuchida A, Hirata A, Kobayashi N, Goto K, Osumi K, Hirose Y, Nakayama J, Yamanoi T, Ashida H, and Mizuno M. (2011) Glycoside hydrolase family 89 alpha-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAc alpha1,4Gal beta1R at the non-reducing terminus of O-glycans in gastric mucin. J Biol Chem. 286, 6479-89. DOI:10.1074/jbc.M110.206722 | PubMed ID:21177247 | HubMed [7]
  4. Ishihara K, Kurihara M, Goso Y, Urata T, Ota H, Katsuyama T, and Hotta K. (1996) Peripheral alpha-linked N-acetylglucosamine on the carbohydrate moiety of mucin derived from mammalian gastric gland mucous cells: epitope recognized by a newly characterized monoclonal antibody. Biochem J. 318 ( Pt 2), 409-16. DOI:10.1042/bj3180409 | PubMed ID:8809027 | HubMed [8]
  5. Yogalingam G and Hopwood JJ. (2001) Molecular genetics of mucopolysaccharidosis type IIIA and IIIB: Diagnostic, clinical, and biological implications. Hum Mutat. 18, 264-81. DOI:10.1002/humu.1189 | PubMed ID:11668611 | HubMed [3]
  6. Weber B, Guo XH, Kleijer WJ, van de Kamp JJ, Poorthuis BJ, and Hopwood JJ. (1999) Sanfilippo type B syndrome (mucopolysaccharidosis III B): allelic heterogeneity corresponds to the wide spectrum of clinical phenotypes. Eur J Hum Genet. 7, 34-44. DOI:10.1038/sj.ejhg.5200242 | PubMed ID:10094189 | HubMed [4]
  7. Ficko-Blean E and Boraston AB. (2012) Structural analysis of a bacterial exo-α-D-N-acetylglucosaminidase in complex with an unusual disaccharide found in class III mucin. Glycobiology. 22, 590-5. DOI:10.1093/glycob/cwr165 | PubMed ID:22090394 | HubMed [6]
  8. Birrane G, Dassier AL, Romashko A, Lundberg D, Holmes K, Cottle T, Norton AW, Zhang B, Concino MF, and Meiyappan M. (2019) Structural characterization of the α-N-acetylglucosaminidase, a key enzyme in the pathogenesis of Sanfilippo syndrome B. J Struct Biol. 205, 65-71. DOI:10.1016/j.jsb.2019.02.005 | PubMed ID:30802506 | HubMed [5]
All Medline abstracts: PubMed | HubMed