An active-site mutation causes enhanced reactivity and altered regiospecificity of transglucosylation catalyzed by the Bacillus sp SAM1606 alpha-glucosidase

Bacillus sp. SAM1606 alpha-glucosidase catalyzes the transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theande rose (6-O-G-glucosylsucrose) as the most abundant transfer product. To End the active-site amino acid residues which can affect the reactivity and reg iospecificity of the glycosyl transfer, 16 mutants with amino acid substitu tions near the active site were allowed to react with 1.75 M sucrose at 60 degrees C, pH 6.0, and the course of transglucosylation as well as the prod uct specificity were analyzed. The sites of the amino acid substitutions we re selected by comparing the conserved amino acid sequences located near th e active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-gl ucosidases (O16G), which have very high amino acid sequence similarities ne ar the active site but have a distinct substrate specificity. The results s howed that, among the mutated SAM1606 enzymes examined, only the mutants wi th substitution of Gly273 with Pro showed an altered reactivity and specifi city of transglucosylation; these mutants exhibited a significantly enhance d initial velocity of glucosyl transfer, yielding isomelezitose (6-O-F-gluc osylsucrose) instead of theanderose as the major transfer product. These re sults indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that the amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation t hat the Bacillus cereus O16G, which has a proline at the corresponding posi tion, produced isomelezitose as the major transfer product during transgluc osylation with sucrose.