Two GH43 β-xylosidases, RS223-BX from a rice straw metagenomic library, and BoXA from Bacteroides ovatus, that share similar amino acid sequences (81% identical) and 19 of 20 active-site residues, were compared by using site-directed mutagenesis of Asp and His residues implicated in metal binding. Thus, RS223-BX is strongly activated by divalent-metal cations and the previously published X-ray structure of this enzyme shows that a Ca2+ cation is chelated by an active-site Asp carboxyl group and an active-site His. Mutation to Ala causes 90% loss of activity for the Asp mutant and 98% loss of activity for the His mutant, indicating their importance to catalysis. For the other enzyme (BoXA), mutation to Ala causes 20% loss of activity for the His mutant and 40% gain of activity for the Asp mutant, indicating the lack of importance for activity of the native residues and the lack of metal-dependency, given that the Asp residue occupies the active site to secure the metal cation in known metal ion dependent GH43 xylosidases. The high activity of the BoXA mutants compared to that of the analogous RS223-BX mutants further undermines the possibility that BoXA maintains a tightly bound metal cofactor resistant to EDTA extraction. The results strengthen our conclusion that the very similar proteins differ in one being metal ion dependent and one not.
Keywords: Biofuels; BoXA; GH43; Metal ion activation; Xylan; Xylosidase.
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