title: Characterization of a laminaribiose phosphorylase from Acholeplasma laidlawii PG-8A and production of 1,3-β-D-glucosyl disaccharides.
abstract: We identified a glycoside hydrolase family 94 homolog (ACL0729) from Acholeplasma laidlawii PG-8A as a laminaribiose (1,3-β-D-glucobiose) phosphorylase (EC 2.4.1.31). The recombinant ACL0729 produced in Escherichia coli catalyzed phosphorolysis of laminaribiose with inversion of the anomeric configuration in a typical sequential bi bi mechanism releasing α-D-glucose 1-phosphate and D-glucose. Laminaritriose (1,3-β-D-glucotriose) was not an efficient substrate for ACL0729. The phosphorolysis is reversible, enabling synthesis of 1,3-β-D-glucosyl disaccharides by reverse phosphorolysis with strict regioselectivity from α-D-glucose 1-phosphate as the donor and suitable monosaccharide acceptors (D-glucose, 2-deoxy-D-arabino-hexopyranose, D-xylose, D-glucuronic acid, 1,5-anhydro-D-glucitol, and D-mannose) with C-3 and C-4 equatorial hydroxyl groups. The D-glucose and 2-deoxy-D-arabino-hexopyranose caused significantly strong competitive substrate inhibition compared with other glucobiose phosphorylases reported, in which the acceptor competitively inhibited the binding of the donor substrate. By contrast, none of the examined disaccharides served as acceptor in the synthetic reaction.
authors.affiliation: Faculty of Agriculture, Niigata University, Niigata 950-2181, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Nihira
authors.forename: Yuka
authors.initials: Y
authors.lastname: Saito
authors.forename: Motomitsu
authors.initials: M
authors.lastname: Kitaoka
authors.forename: Mamoru
authors.initials: M
authors.lastname: Nishimoto
authors.forename: Ken'ichi
authors.initials: K
authors.lastname: Otsubo
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
mesh_terms: D000128:Acholeplasma laidlawii / Q000201:enzymology*; D003001:Cloning, Molecular; D004187:Disaccharides / Q000096:biosynthesis* / Q000737:chemistry; D004789:Enzyme Activation; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D004187:Disaccharides; D011994:Recombinant Proteins; D005964:Glucosyltransferases; C082478:laminaribiose phosphorylase