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Glycoside Hydrolase Family 39
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|Glycoside Hydrolase Family 39|
|Active site residues||known|
|CAZy DB link|
This glycoside hydrolase family predominantly consists of two known enzyme activities: β-xylosidase and α-L-iduronidase. Both enzyme activities cleave equatorial glycosidic bonds: the 'α' designation of α-iduronidase is a consequence of the stereochemical designations used for carbohydrates in which the α/β designation is related to the D/L designation defined by the stereochemistry at C5 in hexopyranoses . In addition to β-xylosidase activity, the equatorial blond cleaving β-glucosidase, β-galactosidase, and xylanase activities have been identified in one family GH39 enzyme . Furthermore, recent studies have characterized a GH39 from Pseudomonas aeruginosa active on the exopolysaccharide Psl (composed of D-mannose, D-glucose, and L-rhamnose) [3, 4], and a group of fungal GH39 enzymes which possess α-L-(β-1,2)-arabinobiosidase activity and can release D-galactose-(α-1,2)-L-arabinose from arabinoxylans . Enzymes from this family are currently found in bacteria and eukaryotes, although eleven gene sequences encoding putative Family GH39 enzymes from archaea have been reported in the CAZy database. The known β-xylosidase enzymes for which an enzyme activity has been experimentally established all come from microbes, while the α-iduronidase enzymes all come from metazoan eukaryotes. Additionally, while there is a reasonable degree of sequence similarity within the bacterial β-xylosidases in GH39 and within the α-iduronidases in GH39 , there is a much lower degree of homology between enzymes with differing activities [3, 5, 6, 7]. The best-studied enzymes are human α-iduronidase, whose deficiency causes Mucopolysaccharidosis I (also known as Hurler-Scheie syndrome), and the β-xylosidase from Thermoanaerobacterium saccharolyticum.
Kinetics and Mechanism
Family GH39 enzymes are retaining glycoside hydrolases that follow the classical Koshland double-displacement mechanism. This has been demonstrated experimentally through NMR analysis of the first-formed sugar product produced by glycoside hydrolysis by the β-xylosidase from Thermoanaerobacterium saccharolyticum  and human α-iduronidase , and by covalent trapping of the catalytic nucleophile (described below) for these two enzymes [6, 9]. These enzymes do not appear to require any activator or cofactor for activity.
The catalytic nucleophile was first identified in the β-xylosidase from Thermoanaerobacterium saccharolyticum as Glu-277 in the sequence IILNSHFPNLPFHITEY by trapping of the 2-deoxy-2-fluoro-xylosyl-enzyme intermediate and subsequent peptide mapping by LC/MS-MS . A similar analysis performed on human α-iduronidase also successfully trapped the catalytic nucleophile and identified it as Glu-299 in the sequence IYNDEAD , which confirmed previous theoretical predictions . The general acid/base residue has been experimentally identified in the β-xylosidase from Thermoanaerobacterium saccharolyticum as Glu-160 through trapping using the affinity label N-bromoacetyl-β-D-xylopyranosylamine and analysis of variant proteins created by mutation of that site .
The three-dimensional structure of the β-xylosidase from Thermoanaerobacterium saccharolyticum was first solved in 2004 . Since then, the three dimensional structures for GH39 enzymes from Geobacillus stearothermophilus [13, 14], Homo sapiens [15, 16], Pseudomonas aeruginosa , Neocallimastix frontalis , and Bacteroides cellulosilyticus  have also been solved. GH39 enzymes are members of the clan GH-A 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).
- First stereochemistry determination
- Thermoanaerobacterium saccharolyticum β-xylosidase by NMR 
- First catalytic nucleophile identification
- Thermoanaerobacterium saccharolyticum β-xylosidase by 2-fluoroxylose labelling 
- First general acid/base residue identification
- Thermoanaerobacterium saccharolyticum β-xylosidase through labelling with N-bromoacetyl-β-D-xylopyranosylamine and kinetic analysis of mutants generated at the identified position 
- First 3-D structure of a GH39 enzyme
- Thermoanaerobacterium saccharolyticum β-xylosidase 
- McNaught AD (1997) International Union of Pure and Applied Chemistry and International Union of Biochemistry and Molecular Biology. Joint Commission on Biochemical Nomenclature. Nomenclature of carbohydrates. Carbohydr Res. 297, 1-92.
- Morrison JM, Elshahed MS, and Youssef N. (2016) A multifunctional GH39 glycoside hydrolase from the anaerobic gut fungus Orpinomyces sp. strain C1A. PeerJ. 4, e2289. DOI:10.7717/peerj.2289 |
- Baker P, Whitfield GB, Hill PJ, Little DJ, Pestrak MJ, Robinson H, Wozniak DJ, and Howell PL. (2015) Characterization of the Pseudomonas aeruginosa Glycoside Hydrolase PslG Reveals That Its Levels Are Critical for Psl Polysaccharide Biosynthesis and Biofilm Formation. J Biol Chem. 290, 28374-87. DOI:10.1074/jbc.M115.674929 |
- Byrd MS, Sadovskaya I, Vinogradov E, Lu H, Sprinkle AB, Richardson SH, Ma L, Ralston B, Parsek MR, Anderson EM, Lam JS, and Wozniak DJ. (2009) Genetic and biochemical analyses of the Pseudomonas aeruginosa Psl exopolysaccharide reveal overlapping roles for polysaccharide synthesis enzymes in Psl and LPS production. Mol Microbiol. 73, 622-38. DOI:10.1111/j.1365-2958.2009.06795.x |
- Jones DR, Uddin MS, Gruninger RJ, Pham TTM, Thomas D, Boraston AB, Briggs J, Pluvinage B, McAllister TA, Forster RJ, Tsang A, Selinger LB, and Abbott DW. (2017) Discovery and characterization of family 39 glycoside hydrolases from rumen anaerobic fungi with polyspecific activity on rare arabinosyl substrates. J Biol Chem. 292, 12606-12620. DOI:10.1074/jbc.M117.789008 |
- Vocadlo DJ, MacKenzie LF, He S, Zeikus GJ, and Withers SG. (1998) Identification of glu-277 as the catalytic nucleophile of Thermoanaerobacterium saccharolyticum beta-xylosidase using electrospray MS. Biochem J. 335 ( Pt 2), 449-55.
- Ali-Ahmad A, Garron ML, Zamboni V, Lenfant N, Nurizzo D, Henrissat B, Berrin JG, Bourne Y, and Vincent F. (2017) Structural insights into a family 39 glycoside hydrolase from the gut symbiont Bacteroides cellulosilyticus WH2. J Struct Biol. 197, 227-235. DOI:10.1016/j.jsb.2016.11.004 |
- Armand S, Vieille C, Gey C, Heyraud A, Zeikus JG, and Henrissat B. (1996) Stereochemical course and reaction products of the action of beta-xylosidase from Thermoanaerobacterium saccharolyticum strain B6A-RI. Eur J Biochem. 236, 706-13.
- Nieman CE, Wong AW, He S, Clarke L, Hopwood JJ, and Withers SG. (2003) Family 39 alpha-l-iduronidases and beta-D-xylosidases react through similar glycosyl-enzyme intermediates: identification of the human iduronidase nucleophile. Biochemistry. 42, 8054-65. DOI:10.1021/bi034293v |
- Durand P, Lehn P, Callebaut I, Fabrega S, Henrissat B, and Mornon JP. (1997) Active-site motifs of lysosomal acid hydrolases: invariant features of clan GH-A glycosyl hydrolases deduced from hydrophobic cluster analysis. Glycobiology. 7, 277-84.
- Vocadlo DJ, Wicki J, Rupitz K, and Withers SG. (2002) A case for reverse protonation: identification of Glu160 as an acid/base catalyst in Thermoanaerobacterium saccharolyticum beta-xylosidase and detailed kinetic analysis of a site-directed mutant. Biochemistry. 41, 9736-46.
- Yang JK, Yoon HJ, Ahn HJ, Lee BI, Pedelacq JD, Liong EC, Berendzen J, Laivenieks M, Vieille C, Zeikus GJ, Vocadlo DJ, Withers SG, and Suh SW. (2004) Crystal structure of beta-D-xylosidase from Thermoanaerobacterium saccharolyticum, a family 39 glycoside hydrolase. J Mol Biol. 335, 155-65.
- Czjzek M, Ben David A, Bravman T, Shoham G, Henrissat B, and Shoham Y. (2005) Enzyme-substrate complex structures of a GH39 beta-xylosidase from Geobacillus stearothermophilus. J Mol Biol. 353, 838-46. DOI:10.1016/j.jmb.2005.09.003 |
- Czjzek M, Bravman T, Henrissat B, and Shoham Y. (2004) Crystallization and preliminary X-ray analysis of family 39 beta-D-xylosidase from Geobacillus stearothermophilus T-6. Acta Crystallogr D Biol Crystallogr. 60, 583-5. DOI:10.1107/S0907444904001088 |
- Maita N, Tsukimura T, Taniguchi T, Saito S, Ohno K, Taniguchi H, and Sakuraba H. (2013) Human α-L-iduronidase uses its own N-glycan as a substrate-binding and catalytic module. Proc Natl Acad Sci U S A. 110, 14628-33. DOI:10.1073/pnas.1306939110 |
- Bie H, Yin J, He X, Kermode AR, Goddard-Borger ED, Withers SG, and James MN. (2013) Insights into mucopolysaccharidosis I from the structure and action of α-L-iduronidase. Nat Chem Biol. 9, 739-45. DOI:10.1038/nchembio.1357 |