Glycoside Hydrolase Family 30

• Author: Brian Rempel
• Responsible Curator: Stephen Withers

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Glycoside Hydrolase Family 30
Clan	GH-A
Mechanism	retaining
Active site residues	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 phospholipids 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 of human β-glucoerebrosidase has been predicted to be Glu-274 [9]. 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 [10], and since then a number of structures of this enzyme have been reported (reviewed in [11]). 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) [12].

Family Firsts

First catalytic nucleophile identification

Human β-glucocerebrosidase by 2-fluoroglucose labelling [1]

First 3-D structure of a GH30 enzyme

Human β-glucocerebrosidase [2]

References

1. Grabowski GA (2008). Phenotype, diagnosis, and treatment of Gaucher's disease. Lancet. 2008;372(9645):1263-71. DOI:10.1016/S0140-6736(08)61522-6 |
2. Grabowski GA, Gatt S, and Horowitz M. (1990). Acid beta-glucosidase: enzymology and molecular biology of Gaucher disease. Crit Rev Biochem Mol Biol. 1990;25(6):385-414. DOI:10.3109/10409239009090616 |
3. Oyama S, Yamagata Y, Abe K, and Nakajima T. (2002). Cloning and expression of an endo-1,6-beta-D-glucanase gene (neg1) from Neurospora crassa. Biosci Biotechnol Biochem. 2002;66(6):1378-81. DOI:10.1271/bbb.66.1378 |
4. Brunner F, Wirtz W, Rose JK, Darvill AG, Govers F, Scheel D, and Nürnberger T. (2002). A beta-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans. Phytochemistry. 2002;59(7):689-96. DOI:10.1016/s0031-9422(02)00045-6 |
5. Miao S, McCarter JD, Grace ME, Grabowski GA, Aebersold R, and Withers SG. (1994). Identification of Glu340 as the active-site nucleophile in human glucocerebrosidase by use of electrospray tandem mass spectrometry. J Biol Chem. 1994;269(15):10975-8. | Google Books | Open Library
6. Dinur T, Osiecki KM, Legler G, Gatt S, Desnick RJ, and Grabowski GA. (1986). Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site. Proc Natl Acad Sci U S A. 1986;83(6):1660-4. DOI:10.1073/pnas.83.6.1660 |
7. Legler G (1990). Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv Carbohydr Chem Biochem. 1990;48:319-84. DOI:10.1016/s0065-2318(08)60034-7 |
8. Grace ME, Newman KM, Scheinker V, Berg-Fussman A, and Grabowski GA. (1994). Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression. J Biol Chem. 1994;269(3):2283-91. | Google Books | Open Library
9. Dvir H, Harel M, McCarthy AA, Toker L, Silman I, Futerman AH, and Sussman JL. (2003). X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO Rep. 2003;4(7):704-9. DOI:10.1038/sj.embor.embor873 |
10. Kacher Y, Brumshtein B, Boldin-Adamsky S, Toker L, Shainskaya A, Silman I, Sussman JL, and Futerman AH. (2008). Acid beta-glucosidase: insights from structural analysis and relevance to Gaucher disease therapy. Biol Chem. 2008;389(11):1361-9. DOI:10.1515/BC.2008.163 |
11. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. (1995). Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995;92(15):7090-4. DOI:10.1073/pnas.92.15.7090 |