2-O-α-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on β-D-glucose 1-phosphate.
The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s(-1). No carbohydrate acted as acceptor for the reverse phosphorolysis using β-D-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a k cat of 2.8 s(-1); moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H2 (18)O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-D-glucosylglycerol (GG) at the rate of 180 s(-1). Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a k cat of 95 s(-1). We propose 2-O-α-D-glucopyranosylglycerol: phosphate β-D-glucosyltransferase as the systematic name and 2-O-α-D-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.
Graduate School of Science and Technology, Niigata University, Niigata, Japan.
Takanori
T
Nihira
Graduate School of Science and Technology, Niigata University, Niigata, Japan.
Yuka
Y
Saito
Graduate School of Science and Technology, Niigata University, Niigata, Japan.
Ken'ichi
K
Ohtsubo
Graduate School of Science and Technology, Niigata University, Niigata, Japan.
Hiroyuki
H
Nakai
National Food Research Institute, National Agriculture and Food Research Organization, Tsukuba, Ibaraki, Japan.
Motomitsu
M
Kitaoka
D001407:Bacillus / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D015870:Gene Expression; D005958:Glucosephosphates / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D005958:Glucosephosphates; D011994:Recombinant Proteins; C031590:glucose-1-phosphate; D006026:Glycoside Hydrolases