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Glycoside Hydrolase Family 30
| Glycoside Hydrolase Family 30 | |
| Clan | GH-x |
| Mechanism | retaining |
| Active site residues | not known |
| CAZy DB link | |
| http://www.cazy.org/fam/GH30.html | |
Substrate specificities
This family contains three known enzyme activities: β-glucosylceramidase, β-1,6-glucanase, and β-xylosidase. This family enzymes currently contains enzymes from only bacteria and eukaryotes. The best-studied enzyme is human β-glucocerebrosidase whose deficiency causes Gauchers disease [1]. 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 first-formed sugar product, covalent trapping of the enzymatic 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 for optimal activity, [2] although the role of these activators is still not entirely clear. Neither the β-1,6-glucanases [3] nor the β-xylosidases [4] 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 [5]. The catalytic nucleophile had been previously mistakenly identified as Asp443 using a tritiated bromoconduritol epoxide [6, 7], although subsequent kinetic analyses of site-directed mutants of Asp443 were not consistent with its role as the catalytic nucleophile [8]. The catalytic acid/base has not been experimentally verified through analysis of variant proteins created by mutation of that site, although it is consistent with structural studies (below).
Three-dimensional structures
The three-dimensional structure of human β-glucocerebrosidase was first solved in 2003 [9], and since then a number of structures of this enzyme have been reported (reviewed in [10]). 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) [11].