title: 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 D(2)O, 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.
authors.affiliation: Institute of Biotechnology and Biochemical Engineering, Graz University of Technology, Petersgasse 12/1, A-8010 Graz, Austria.
authors.forename: Patricia
authors.initials: P
authors.lastname: Wildberger
authors.forename: Lothar
authors.initials: L
authors.lastname: Brecker
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D055162:Biocatalysis; D002236:Carbohydrate Conformation; D040143:Cellulomonas / Q000737:chemistry / Q000201:enzymology*; D003847:Deoxyglucose / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D003903:Deuterium; D004926:Escherichia coli; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D005958:Glucosephosphates / Q000096:biosynthesis / Q000737:chemistry*; D005964:Glucosyltransferases / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D005958:Glucosephosphates; D010710:Phosphates; D011522:Protons; D011994:Recombinant Proteins; C001289:d-glucal; C081196:2-deoxyglucose-1-phosphate; D003847:Deoxyglucose; D003903:Deuterium; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase; D005947:Glucose