title: Discovery of nigerose phosphorylase from Clostridium phytofermentans.
abstract: A novel phosphorylase from Clostridium phytofermentans belonging to the glycoside hydrolase family (GH) 65 (Cphy1874) was characterized. The recombinant Cphy1874 protein produced in Escherichia coli showed phosphorolytic activity on nigerose in the presence of inorganic phosphate, resulting in the release of D-glucose and β-D-glucose 1-phosphate (β-G1P) with the inversion of the anomeric configuration. Kinetic parameters of the phosphorolytic activity on nigerose were k(cat) = 67 s(-1) and K(m) = 1.7 mM. This enzyme did not phosphorolyze substrates for the typical GH65 enzymes such as trehalose, maltose, and trehalose 6-phosphate except for a weak phosphorolytic activity on kojibiose. It showed the highest reverse phosphorolytic activity in the reverse reaction using D-glucose as the acceptor and β-G1P as the donor, and the product was mostly nigerose at the early stage of the reaction. The enzyme also showed reverse phosphorolytic activity, in a decreasing order, on D-xylose, 1,5-anhydro-D-glucitol, D-galactose, and methyl-α-D-glucoside. All major products were α-1,3-glucosyl disaccharides, although the reaction with D-xylose and methyl-α-D-glucoside produced significant amounts of α-1,2-glucosides as by-products. We propose 3-α-D-glucosyl-D-glucose:phosphate β-D-glucosyltransferase as the systematic name and nigerose phosphorylase as the short name for this Cphy1874 protein.
authors.affiliation: National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Nihira
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Chiku
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
mesh_terms: D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology* / Q000235:genetics*; D004187:Disaccharides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D006005:Phosphorylases / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D004187:Disaccharides; D011994:Recombinant Proteins; C014637:laminaribiose; D006005:Phosphorylases; D006026:Glycoside Hydrolases