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	known
CAZy DB link
http://www.cazy.org/fam/GH39.html

Substrate Specificities

This glycoside hydrolase 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 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 [3] and human α-iduronidase [4], and by covalent trapping of the catalytic nucleophile (described below) for these two enzymes [2, 4]. These enzymes do not appear to require any 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 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 [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).

Family Firsts

First stereochemistry determination

Thermoanaerobacterium saccharolyticum β-xylosidase by NMR [3]

First catalytic nucleophile identification

Thermoanaerobacterium saccharolyticum β-xylosidase by 2-fluoroxylose labelling [4]

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 [5]

First 3-D structure of a GH39 enzyme

Thermoanaerobacterium saccharolyticum β-xylosidase [6]

References

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