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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 [1]. In addition to β-xylosidase activity, the equatorial bond cleaving β-glucosidase, β-galactosidase, and xylanase activities have been identified in one family GH39 enzyme [2]. 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 [5]. 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 [6], 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 retainingglycoside 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 [8] and human α-iduronidase [9], 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.
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 [6]. A similar analysis performed on human α-iduronidase also successfully trapped the catalytic nucleophile and identified it as Glu-299 in the sequence IYNDEAD [9], which confirmed previous theoretical predictions [10]. 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 [11].
Three-dimensional structures
The three-dimensional structure of the β-xylosidase from Thermoanaerobacterium saccharolyticum was first solved in 2004 [12]. Since then, the three dimensional structures for GH39 enzymes from Geobacillus stearothermophilus [13, 14], Homo sapiens [15, 16], Pseudomonas aeruginosa [3], Neocallimastix frontalis [5], and Bacteroides cellulosilyticus [7] 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).
Family Firsts
First stereochemistry determination
Thermoanaerobacterium saccharolyticum β-xylosidase by NMR [8]
Thermoanaerobacterium saccharolyticum β-xylosidase through labelling with N-bromoacetyl-β-D-xylopyranosylamine and kinetic analysis of mutants generated at the identified position [10]
