Examining the role of phosphate in glycosyl transfer reactions of Cellulomonas uda cellobiose phosphorylase using d-glucal as donor substrate

Abstract

Cellobiose phosphorylase from Cellulomonas uda (CuCPase) is shown to utilize d-glucal as slow alternative donor substrate for stereospecific glycosyl transfer to inorganic phosphate, giving 2-deoxy-α-d-glucose 1-phosphate as the product. When performed in D2O, enzymatic phosphorolysis of d-glucal proceeds with incorporation of deuterium in equatorial position at C-2, implying a stereochemical course of reaction where substrate becomes protonated from below its six-membered ring through stereoselective re side attack at C-2. The proposed catalytic mechanism, which is supported by results of docking studies, involves direct protonation of d-glucal by the enzyme-bound phosphate, which then performs nucleophilic attack on the reactive C-1 of donor substrate. When offered d-glucose next to d-glucal and phosphate, CuCPase produces 2-deoxy-β-d-glucosyl-(1→4)-d-glucose and 2-deoxy-α-d-glucose 1-phosphate in a ratio governed by mass action of the two acceptor substrates present. Enzymatic synthesis of 2-deoxy-β-d-glucosyl-(1→4)-d-glucose is effectively promoted by catalytic concentrations of phosphate, suggesting that catalytic reaction proceeds through a quaternary complex of CuCPase, d-glucal, phosphate, and d-glucose. Conversion of d-glucal and phosphate presents a convenient single-step synthesis of 2-deoxy-α-d-glucose 1-phosphate that is difficult to prepare chemically.