Glycoside Hydrolase Family 30

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Authors: Brian Rempel and ^^^Franz St. John^^^
Responsible Curator: Stephen Withers

Glycoside Hydrolase Family 30
Clan	GH-A
Mechanism	retaining
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH30.html

Family Classification

The family classification of a number of GH30 members was revised in 2010 [1]. In several studies concerning glucuronoxylan xylanohydrolases previously classified into GH5, sequence analysis had suggested that these enzymes were more closely related to GH30 members [2, 3, 4, 5, 6]. In consideration of these observations, the revised classification of St. John et al. employed phylogenetics, primary amino acid sequence and tertiary structure analysis to show that the glucuronoxylan xylanohydrolases in question, as well as several other enzyme groups previously classified as GH5 members, were indeed better placed in GH30 [1].

Substrate Specificities

This family contains glycoside hydrolases with three known enzyme activities: β-glucosylceramidase, β-1,6-glucanase, and β-xylosidase. This family currently contains enzymes from only bacteria and eukaryotes. The best-studied enzyme is human β-glucocerebrosidase whose deficiency causes Gauchers disease [7]. This enzyme is responsible for hydrolyzing the β-glucoside from the glycolipid glucosylceramide.

Kinetics and Mechanism

Family GH30 enzymes are retaining enzymes. Although this has never been formally demonstrated experimentally through NMR analysis of the initially formed sugar product, covalent trapping of the catalytic nucleophile (described below) conclusively demonstrates that these enzymes follow the classic Koshland double-displacement mechanism. The β-glucosylceramidases require an activator protein and negatively charged phospholipids for optimal activity, [8] although the role of these activators is still not entirely clear. Neither the β-1,6-glucanases [9] nor the β-xylosidases [10] appear to require any activators.

Catalytic Residues

The catalytic nucleophile was first identified in human β-glucocerebrosidase as Glu340 in the sequence FASEA by trapping of the 2-deoxy-2-fluoro-glucosyl-enzyme intermediate and subsequent peptide mapping by LC/MS-MS [11]. The catalytic nucleophile had been previously been mistakenly identified as Asp443 using a tritiated bromoconduritol epoxide [12, 13], although subsequent kinetic analyses of site-directed mutants of Asp443 were not consistent with its role as the catalytic nucleophile [14]. The general acid/base residue of human β-glucoerebrosidase is predicted to be Glu-274 [15]. While this identification has not been experimentally verified through analysis of variant proteins created by mutation of that site, it is consistent with structural studies (below).

Three-Dimensional Structures

The three-dimensional structure of human β-glucocerebrosidase was first solved in 2003 [16], and since then several different structures of this enzyme have been reported (reviewed in [17]). GH30 enzymes are members of the GHA clan fold, consistent with the classic (α/β)₈ TIM barrel fold with the two key active site glutamic acids located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [18].

Family Firsts

First catalytic nucleophile identification: Human β-glucocerebrosidase by 2-fluoroglucose labelling [19]
First 3-D structure of a GH30 enzyme: Human β-glucocerebrosidase [20]

References

All Medline abstracts: PubMed