Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio

A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GIcNAc)(2) (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GIcNAc1-P] and G1cNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with a-D-glucose 1-phosphate as the donor substrate as well as GIcNAc-1-P to produce beta-D-glucosyl-(1 --> 4)-2-acetamide-2-deoxy-D-glucose with G1cNAc as the acceptor substrate. The enzyme allowed aryl-beta-glycosides of GIcNAc as the acceptor substrate with 10-20% activities of GIcNAc. Kinetic parameters of (G1cNAc)(2) in the phosphorolysis and GIcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0) = 5.5 s(-1), K-m = 2.0 mM; synthetic reaction, ko = 10 s(-1), K-m = 14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.