Glycosyltransferase 1 from Bacillus cereus (BcGT1) catalyzes the transfer of a glucosyl moiety from uridine diphosphate glucose (UDP-glucose) to various acceptors; it was expressed and characterized. The specificity of acceptors was found to be broad: more than 20 compounds classified into O-, S-, and N-linkage glucosides can be prepared with BcGT1 catalysis. Based on this work, we conclude that the corresponding acceptors of these compounds must possess the following features: (1) the acceptors must contain at least one aromatic or fused-aromatic or heteroaromatic ring; (2) the reactive hydroxyl or sulfhydryl or amino group can attach either on the aromatic ring or on its aliphatic side chain; and (3) the acceptors can be a primary, secondary, or even a tertiary amine. Four representative acceptors-fluorescein methyl ester, 17-β-estradiol, 7-mercapto-4-methylcoumarin, and 6-benzylaminopurine-were chosen as a candidate acceptor for O-, S-, and N-glucosidation, respectively. These enzymatic products were purified and the structures were confirmed with mass and NMR spectra. As all isolated glucosides are β-anomers, BcGT1 is confirmed to be an inverting enzyme. This study not only demonstrates the substrate promiscuity of BcGT1 but also showed the great application prospect of this enzyme in bioconversion of valuable bioactive molecules.

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Glycosyltransferase-1 from Bacillus cereus (BcGT1) catalyzes a reaction that transfers a glucosyl moiety to flavonoids, such as quercetin, kaempferol, and myricetin. The enzymatic glucosidation shows a broad substrate specificity when the reaction is catalyzed by wild-type BcGT1. Preliminary assays demonstrated that the F240A mutant significantly improves the regioselectivity of enzymatic glucosidation toward quercetin. To unveil and further to control the catalytic function of BcGT1, mutation of F240 to other amino acids, such as C, E, G, R, Y, W, and K, was performed. Among these mutants, F240A, F240G, F240R, and F240K greatly altered the regioselectivity. The quercetin-3-O-glucoside, instead of quercetin-7-O-glucoside as for the wild-type enzyme, was obtained as the major product. Among these mutants, F240R showed nearly 100 % product specificity but only retained 25 % catalytic efficiency of wild-type enzyme. From an inspection of the protein structure, we found two other amino acids, F132 and F138, together with F240, are likely to form a hydrophobic binding region, which is sufficiently spacious to accommodate substrates with varied aromatic moieties. Through the replacement of a phenylalanine by a tyrosine residue in the substrate-binding region, the mutants may be able to fix the orientation of flavonoids, presumably through the formation of a hydrogen bond between substrates and mutants. Multiple mutants-F240R_F132Y, F240R_F138Y, and F240R_F132Y_F138Y-were thus constructed for further investigation. The multiple points of mutants not only maintained the high product specificity but also significantly improved the catalytic efficiency, relative to F240R. The same product specificity was obtained when kaempferol and myricetin were used as a substrate.