2-O-α-D-Glucosylglycerol Phosphorylase from Bacillus selenitireducens MLS10 Possessing Hydrolytic Activity on β-D-Glucose 1-Phosphate

Abstract

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 18O 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.

Structural Basis for Reversible Phosphorolysis and Hydrolysis Reactions of 2-O-α-Glucosylglycerol Phosphorylase

“…The expression plasmids for the mutants were verified by DNA sequencing. The mutant proteins were expressed, purified, and characterized as described previously (11). Extinction coefficients of ⑀ ϭ 122,620 and 118,610 M Ϫ1 cm Ϫ1 were used to determine the protein concentrations of the Tyr to Phe and Trp to Phe mutants, respectively.…”

“…The reverse phosphorolytic (synthetic) activity was measured by subtracting the increase in D-glucose (hydrolytic activity) from the increase in P i (hydrolytic plus synthetic activities) at 30°C in the reaction mixture containing 0 -100 mM glycerol, 10 mM ␤Glc1P, and 40 mM HEPES-NaOH (pH 7.5) (11). The hydrolytic activity was measured based on the increase in D-glucose at 30°C in the reaction mixture containing 0.1-10 mM ␤Glc1P and 40 mM HEPES-NaOH (pH 7.5) (11). At least 9 different substrate concentrations for measurement of each enzyme and activity were collected.…”

“…We previously identified Bsel_2816 from Bacillus selenitireducens MLS10 as a novel enzyme 2-O-␣-glucosylglycerol phosphorylase (GGP, EC 2.4.1.x) (11). B. selenitireducens is a haloalkaliphilic bacterium that grows optimally in a salinity of 1.5 M NaCl (12).…”

“…GGP belongs to GH65 and specifically produces GG from ␤-D-glucose 1-phosphate (␤Glc1P) and glycerol via reverse phosphorolysis. GGP can significantly hydrolyze ␤Glc1P with a k cat of 2.8 s Ϫ1 and reversibly phosphorolyze GG with k cat values of 95 s Ϫ1 (phosphorolysis) and 180 s Ϫ1 (synthetic reaction) (11). Therefore, the ␤Glc1P hydrolysis of GGP is a side reaction of the reversible phosphorylase reactions.…”

“…The ␤Glc1P hydrolysis is a glucosyl-transfer reaction to an acceptor water molecule that produces an anomer-inverted ␣-glucose, not a phosphatase-type reaction. The reaction of GH-type inverting phosphorylases, including GH65, GH94, and GH112, is thought to proceed via a single displacement mechanism with the assistance of a general acid residue (13), which is probably Glu-475 in the case of GGP (11 (20), and GGP. However, the crystal structures of only two enzymes in the GH65 family, Lactobacillus brevis maltose phosphorylase (LbMP) in complex with inorganic phosphate (P i ) (21) and Caldicellulosiruptor saccharolyticus kojibiose phosphorylase (CsKP) in complex with glucose and P i and in complex with kojibiose and sulfate (22), have been reported from a structural point of view.…”