title: Modulation of acceptor specificity of Ruminococcus albus cellobiose phosphorylase through site-directed mutagenesis.
abstract: Cellobiose phosphorylase (EC 2.4.1.20, CBP) catalyzes the reversible phosphorolysis of cellobiose to α-D-glucose 1-phosphate (Glc1P) and d-glucose. Cys485, Tyr648, and Glu653 of CBP from Ruminococcus albus, situated at the +1 subsite, were mutated to modulate acceptor specificity. C485A, Y648F, and Y648V were active enough for analysis. Their acceptor specificities were compared with the wild type based on the apparent kinetic parameters determined in the presence of 10 mM Glc1P. C485A showed higher preference for D-glucosamine than the wild type. Apparent kcat/Km values of Y648F for D-mannose and 2-deoxy-D-glucose were 8.2- and 4.0-fold higher than those of the wild type, respectively. Y648V had synthetic activity toward N-acetyl-D-glucosamine, while the other variants did not. The oligosaccharide production in the presence of the same concentrations of wild type and each mutant was compared. C485A produced 4-O-β-D-glucopyranosyl-D-glucosamine from 10 mM Glc1P and D-glucosamine at a rate similar to the wild type. Y648F and Y648V produced 4-O-β-D-glucopyranosyl-D-mannose and 4-O-β-D-glucopyranosyl-N-acetyl-D-glucosamine much more rapidly than the wild type when D-mannose and N-acetyl-D-glucosamine were used as acceptors, respectively. After a 4h reaction, the amounts of 4-O-β-D-glucopyranosyl-D-mannose and 4-O-β-D-glucopyranosyl-N-acetyl-D-glucosamine produced by Y648F and Y648V were 5.9- and 12-fold higher than the wild type, respectively.
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita-ku, Sapporo, Japan.
authors.forename: Ken
authors.initials: K
authors.lastname: Hamura
authors.forename: Wataru
authors.initials: W
authors.lastname: Saburi
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Matsui
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
mesh_terms: D055162:Biocatalysis; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000096:biosynthesis / Q000737:chemistry; D045857:Ruminococcus / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
chemical_list: D009844:Oligosaccharides; D005964:Glucosyltransferases; C034714:cellobiose phosphorylase
keywords: Acceptor specificity; CBGS; CBP; CBP from Ruminococcus albus; Cellobiose phosphorylase; ChBP; GH; Glc1P; Glycoside hydrolase family 94; LBP; NMR; Oligosaccharide synthesis; RaCBP; Site-directed mutagenesis; TLC; cellobiose phosphorylase; chitobiose phosphorylase; cyclic β-1,2-glucan synthase; glucose 1-phosphate; glycoside hydrolase; laminaribiose phosphorylase; nuclear magnetic resonance; thin layer chromatography