Glycoside Hydrolase Family 30

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

Contents

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 [11]
First 3-D structure of a GH30 enzyme
Human β-glucocerebrosidase [16]

References

  1. St John FJ, González JM, and Pozharski E. Consolidation of glycosyl hydrolase family 30: a dual domain 4/7 hydrolase family consisting of two structurally distinct groups. FEBS Lett. 2010 Nov 5;584(21):4435-41. DOI:10.1016/j.febslet.2010.09.051 | PubMed ID:20932833 | HubMed [stjohn2010]
  2. Brumshtein B, Wormald MR, Silman I, Futerman AH, and Sussman JL. Structural comparison of differently glycosylated forms of acid-beta-glucosidase, the defective enzyme in Gaucher disease. Acta Crystallogr D Biol Crystallogr. 2006 Dec;62(Pt 12):1458-65. DOI:10.1107/S0907444906038303 | PubMed ID:17139081 | HubMed [brumshtein2006]
  3. Haegeman A, Vanholme B, and Gheysen G. Characterization of a putative endoxylanase in the migratory plant-parasitic nematode Radopholus similis. Mol Plant Pathol. 2009 May;10(3):389-401. DOI:10.1111/j.1364-3703.2009.00539.x | PubMed ID:19400841 | HubMed [haegeman2009]
  4. Keen NT, Boyd C, and Henrissat B. Cloning and characterization of a xylanase gene from corn strains of Erwinia chrysanthemi. Mol Plant Microbe Interact. 1996 Sep;9(7):651-7. PubMed ID:8810080 | HubMed [keen1996]
  5. Larson SB, Day J, Barba de la Rosa AP, Keen NT, and McPherson A. First crystallographic structure of a xylanase from glycoside hydrolase family 5: implications for catalysis. Biochemistry. 2003 Jul 22;42(28):8411-22. DOI:10.1021/bi034144c | PubMed ID:12859186 | HubMed [larson2003]
  6. Mitreva-Dautova M, Roze E, Overmars H, de Graaff L, Schots A, Helder J, Goverse A, Bakker J, and Smant G. A symbiont-independent endo-1,4-beta-xylanase from the plant-parasitic nematode Meloidogyne incognita. Mol Plant Microbe Interact. 2006 May;19(5):521-9. DOI:10.1094/MPMI-19-0521 | PubMed ID:16673939 | HubMed [mitreva-dautova2006]
  7. Grabowski GA. Phenotype, diagnosis, and treatment of Gaucher's disease. Lancet. 2008 Oct 4;372(9645):1263-71. DOI:10.1016/S0140-6736(08)61522-6 | PubMed ID:19094956 | HubMed [grabowski2008]
  8. Grabowski GA, Gatt S, and Horowitz M. Acid beta-glucosidase: enzymology and molecular biology of Gaucher disease. Crit Rev Biochem Mol Biol. 1990;25(6):385-414. DOI:10.3109/10409239009090616 | PubMed ID:2127241 | HubMed [grabowski1990]
  9. Oyama S, Yamagata Y, Abe K, and Nakajima T. Cloning and expression of an endo-1,6-beta-D-glucanase gene (neg1) from Neurospora crassa. Biosci Biotechnol Biochem. 2002 Jun;66(6):1378-81. PubMed ID:12162562 | HubMed [oyama2002]
  10. Brunner F, Wirtz W, Rose JK, Darvill AG, Govers F, Scheel D, and Nürnberger T. A beta-glucosidase/xylosidase from the phytopathogenic oomycete, Phytophthora infestans. Phytochemistry. 2002 Apr;59(7):689-96. PubMed ID:11909624 | HubMed [brunner2002]
  11. Miao S, McCarter JD, Grace ME, Grabowski GA, Aebersold R, and Withers SG. Identification of Glu340 as the active-site nucleophile in human glucocerebrosidase by use of electrospray tandem mass spectrometry. J Biol Chem. 1994 Apr 15;269(15):10975-8. PubMed ID:7908905 | HubMed [miao1994]
  12. Dinur T, Osiecki KM, Legler G, Gatt S, Desnick RJ, and Grabowski GA. Human acid beta-glucosidase: isolation and amino acid sequence of a peptide containing the catalytic site. Proc Natl Acad Sci U S A. 1986 Mar;83(6):1660-4. PubMed ID:3456607 | HubMed [dinur1986]
  13. Legler G. Glycoside hydrolases: mechanistic information from studies with reversible and irreversible inhibitors. Adv Carbohydr Chem Biochem. 1990;48:319-84. PubMed ID:2077872 | HubMed [legler1990]
  14. Grace ME, Newman KM, Scheinker V, Berg-Fussman A, and Grabowski GA. Analysis of human acid beta-glucosidase by site-directed mutagenesis and heterologous expression. J Biol Chem. 1994 Jan 21;269(3):2283-91. PubMed ID:8294487 | HubMed [grace1994]
  15. Durand P, Lehn P, Callebaut I, Fabrega S, Henrissat B, and Mornon JP. Active-site motifs of lysosomal acid hydrolases: invariant features of clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis. Glycobiology. 1997 Mar;7(2):277-84. PubMed ID:9134434 | HubMed [durand1997]
  16. Dvir H, Harel M, McCarthy AA, Toker L, Silman I, Futerman AH, and Sussman JL. X-ray structure of human acid-beta-glucosidase, the defective enzyme in Gaucher disease. EMBO Rep. 2003 Jul;4(7):704-9. DOI:10.1038/sj.embor.embor873 | PubMed ID:12792654 | HubMed [dvir2003]
  17. Kacher Y, Brumshtein B, Boldin-Adamsky S, Toker L, Shainskaya A, Silman I, Sussman JL, and Futerman AH. Acid beta-glucosidase: insights from structural analysis and relevance to Gaucher disease therapy. Biol Chem. 2008 Nov;389(11):1361-9. DOI:10.1515/BC.2008.163 | PubMed ID:18783340 | HubMed [kacher2008]
  18. Henrissat B, Callebaut I, Fabrega S, Lehn P, Mornon JP, and Davies G. Conserved catalytic machinery and the prediction of a common fold for several families of glycosyl hydrolases. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):7090-4. PubMed ID:7624375 | HubMed [henrissat1995]
All Medline abstracts: PubMed | HubMed
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