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Glycoside hydrolases of GH63 are exo-acting α-glucosidases. Eukaryotic members of this family are processing α-glucosidase I enzymes (mannosyl-oligosaccharide glucosidase, EC 3.2.1.106), which specifically hydrolyze the terminal α-1,2-glucosidic linkage in the N-linked oligosaccharide precursor, Glc3Man9GlcNAc2, to produce β-glucose and Glc2Man9GlcNAc2. Processing α-glucosidase I thus plays a critical role in the maturation of eukaryotic N-glycans. The enzymatic properties of Cwh41p, a processing α-glucosidase I from Saccharomyces cerevisiae, have been intensively studied [1] (also reviewed in [2]).
Genes encoding GH63 enzymes have also been found in archaea and bacteria, but their natural substrates are still unclear, as these organisms are not known to produce eukaryotic N-linked oligosacharides. A bacterial GH63 enzyme, Escherichia coli YgjK, demonstrated the highest activity toward the α-1,3-glucosidic linkage of nigerose (Glc-α-1,3-Glc) among the commercially available sugars tested, but the Km value for nigerose was substantially higher than that for other typical α-glucosidases [3]. The aglycon specificity of YgjK was screened using its glycosynthase mutants (D324N and E727A), which synthesized 2-O-α-glucopyranosylgalactose from β-glucopyranosyl fluoride donor and galactose acceptor [4].
In 2013, the substrates of GH63 enzymes from Thermus thermophilus HB27 and Rubrobacter radiotolerans RSPS-4 were identified as compatible solutes, α-D-mannopyranosyl-1,2-D-glycerate (mannosylglycerate) and α-D-glucopyranosyl-1,2-D-glycerate (glucosylglycerate) [5]. Subsequently, glucosylglycerate hydrolase was identified in Mycobacterium hassiacum and was found to be involved in the recovery process from nitrogen starvation by hydrolyzing glucosylglycerate [6].
An orthologous gene for mannosyl/glucosylglycerate hydrolase was also found in the genome of plant Selaginella moellendorffii, and the gene product hydrolyzed these compatible solutes [7].
Kinetics and Mechanism
Family GH63 enzymes are inverting enzymes, as first shown by NMR on a processing α-glucosidase I from S. cerevisiae [8].
Catalytic Residues
The catalytic residues were inferred by comparing the catalytic (α/α)6 barrel domain of the GH63 enzyme, E. coli YgjK, with those of GH15 and GH37 enzymes. In the case of GH37 and GH63, both of which belong to clan GH-G, the catalytic general acid is predicted as an Asp residue (Asp501 in E. coli YgjK), and the general base is considered to be a Glu residue (Glu727 in E. coli YgjK) [3]. Although both of the corresponding residues of GH15, which belongs to clan GH-L, are identified as Glu residues, the positions of the catalytic residues of GH15, GH37, and GH63 are highly conserved [3, 9].
Three-dimensional structures
The crystal structures of the bacterial GH63 proteins, E. coli YgjK [3] (multiple PDB entries) and Thermus thermophilus uncharacterised protein TTHA0978 (PDB 2z07), have been reported. The catalytic domain consists of an (α/α)6 barrel fold. The main chain of the (α/α)6 barrel domain shares high structural similarity with those of GH15, GH37, GH65, and GH94 [3, 9]. This similarity had been predicted on the basis of sequence comparison, before their crystal structures were available [10]. The first crystal structure of the eukaryotic processing α-glucosidase I (PDB 4j5t) has been reported in 2013 [11].
