Glycoside Hydrolase Family 39

• Author: Brian Rempel
• Responsible Curator: Stephen Withers

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Glycoside Hydrolase Family 30
Clan	GH-A
Mechanism	retaining
Active site residues	not known
CAZy DB link
http://www.cazy.org/fam/GH39.html

Substrate Specificities

This family contains two known enzyme activities: β-xylosidase and α-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 [1]. Enzyme from this family are currently found in bacteria and eukaryotes, although one gene sequence encoding a putative Family GH39 enzyme from archaea has been reported. The known β-xylosidase enzymes for which an enzyme activity has been experimentally established all come from bacteria, while the α-iduronidase enzymes all come from eukaryotes. Additionally, while there is a reasonable degree of sequence similarity within the β-xylosidases in GH39 and within the α-iduronidases in GH39, there is a much lower degree of homology between the β-xylosidases and α-iduronidases [2]. 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 enzymes that follow the classic 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 [3] and human α-iduronidase [4], and by covalent trapping of the enzymatic nucleophile (described below) for these two enzymes [2, 4]. These enzymes do not appear to require any sort of activator or cofactor for activity.

Catalytic Residues

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 [2]. A similar analysis performed on human α-iduronidase also successfully trapped the catalytic nucleophile and identified it as Glu-299 in the sequence IYNDEAD [4], which confirmed previous theoretical predictions [5]. The catalytic acid/base 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 [6].

Three-dimensional structures

The three-dimensional structure of the β-xylosidase from Thermoanaerobacterium saccharolyticum was first solved in 2004 [7]. Since then, the three dimensional structure for another GH39 β-xylosidase from Geobacillus stearothermophilus has also been solved [8, 9]. No experimentally determined three dimensional structure exists for the α-iduronidase enzymes, although a computer-generated homology model has been reported [10]. GH39 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).

References

1. 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. 1997;297(1):1-92. DOI:10.1016/s0008-6215(97)83449-0 |
2. 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. 1998;335 ( Pt 2)(Pt 2):449-55. DOI:10.1042/bj3350449 |
3. 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. 1996;236(2):706-13. DOI:10.1111/j.1432-1033.1996.00706.x |
4. 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. 2003;42(26):8054-65. DOI:10.1021/bi034293v |
6. 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. 2002;41(31):9736-46. DOI:10.1021/bi020078n |
7. 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. 2004;335(1):155-65. DOI:10.1016/j.jmb.2003.10.026 |
8. 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. 2005;353(4):838-46. DOI:10.1016/j.jmb.2005.09.003 |
9. 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. 2004;60(Pt 3):583-5. DOI:10.1107/S0907444904001088 |
10. Rempel BP, Clarke LA, and Withers SG. (2005). A homology model for human alpha-l-iduronidase: insights into human disease. Mol Genet Metab. 2005;85(1):28-37. DOI:10.1016/j.ymgme.2004.12.006 |