Difference between revisions of "Glycoside Hydrolase Family 89"

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Author: ^^^Elizabeth Ficko-Blean^^^
Responsible Curator: ^^^Alisdair Boraston^^^

Glycoside Hydrolase Family GH89
Clan	none
Mechanism	Retaining
Active site residues	known
CAZy DB link
https://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.

Figure 1. A secondary structure and surface representation of the C. perfringens CpGH89 4a4a double catalytic mutant Michaelis complex []. The E483Q and E601Q mutations are shown in yellow stick interacting with the α-D-GlcNAc-(1,4)-D-Galactose substrate in pink stick within the sock-shaped active site.

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 (Figure 1) [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 (CBM32s). 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 (Figure 1) [7]. The mutations causing MPS IIIB were mapped onto the bacterial homologue, demonstrating that the mutations are mainly structural in nature [1].

NAGLU has only 28.2% sequence identity with CpGH89 and lacks the CBM32-like domain. 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]. In comparison, CpGH89 crystallized as a monomer in the asymmetric unit [1]. The substrate binding pocket of NAGLU is more open when compared to the sock-shaped active site of CpGH89 [1, 8]. The mutations causing MPS IIIB were also mapped onto NAGLU, confirming that the mutations are mainly structural in nature [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

All Medline abstracts: PubMed

@@ Line 37: / Line 37: @@
 == Three-dimensional structures ==
-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] (Figure 1) <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 ([[CBM32]]s).  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 <cite>1</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 ([[CBM32]]s).  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 (Figure 1) <cite>6</cite>. The mutations causing MPS IIIB were mapped onto the bacterial homologue, demonstrating that the mutations are mainly structural in nature <cite>1</cite>.
-NAGLU has only 28.2% sequence identity with CpGH89 and lacks the [[CBM32]]-like domain.  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>. In comparison, CpGH89 crystallized as a monomer in the asymmetric unit <cite>1</cite>. The substrate binding pocket of NAGLU is more open when compared to the sock shaped active site of CpGH89 <cite>5 1</cite>. The mutations causing MPS IIIB were also mapped onto NAGLU, confirming that the mutations are mainly structural in nature <cite>5</cite>.
+NAGLU has only 28.2% sequence identity with CpGH89 and lacks the [[CBM32]]-like domain.  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>. In comparison, CpGH89 crystallized as a monomer in the asymmetric unit <cite>1</cite>. The substrate binding pocket of NAGLU is more open when compared to the sock-shaped active site of CpGH89 <cite>5 1</cite>. The mutations causing MPS IIIB were also mapped onto NAGLU, confirming that the mutations are mainly structural in nature <cite>5</cite>.
 == Family Firsts ==