Publications

• Structural basis for reversible phosphorolysis and hydrolysis reactions of 2-O-alpha-glucosylglycerol phosphorylase.

  Touhara K.K., Nihira T., Kitaoka M., Nakai H., Fushinobu S.

  2-O-α-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-α-glucosylglycerol (GG) and the hydrolysis of β-d-glucose 1-phosphate (βGlc1P). GGP belongs to the glycoside hydrolase (GH) family 65 and can efficiently and specifically p ... >> More

  2-O-α-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-α-glucosylglycerol (GG) and the hydrolysis of β-d-glucose 1-phosphate (βGlc1P). GGP belongs to the glycoside hydrolase (GH) family 65 and can efficiently and specifically produce GG. However, its structural basis has remained unclear. In this study, the crystal structures of GGP complexed with glucose and the glucose analog isofagomine and glycerol were determined. Subsite -1 of GGP is similar to those of other GH65 enzymes, maltose phosphorylase and kojibiose phosphorylase, whereas subsite +1 is largely different and is well designed for GG recognition. An automated docking analysis was performed to complement these crystal structures, βGlc1P being docked at an appropriate position. To investigate the importance of residues at subsite +1 in the bifunctionality of GGP, we constructed mutants at these residues. Y327F and K587A did not show detectable activities for either reverse phosphorolysis or βGlc1P hydrolysis. Y572F also showed significantly reduced activities for both of these reactions. In contrast, W381F showed significantly reduced reverse phosphorolytic activity but retained βGlc1P hydrolysis. The mode of substrate recognition and the reaction mechanisms of GGP were proposed based on these analyses. Specifically, an extensive hydrogen bond network formed by Tyr-327, Tyr-572, Lys-587, and water molecules contributes to fixing the acceptor molecule in both reverse phosphorolysis (glycerol) and βGlc1P hydrolysis (water) for a glycosyl transfer reaction. This study will contribute to the development of a large scale production system of GG by facilitating the rational engineering of GGP. << Less

  J. Biol. Chem. 289:18067-18075(2014) [PubMed] [EuropePMC]

• 2-O-alpha-D-glucosylglycerol phosphorylase from Bacillus selenitireducens MLS10 possessing hydrolytic activity on beta-D-glucose 1-phosphate.

  Nihira T., Saito Y., Ohtsubo K., Nakai H., Kitaoka M.

  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 ... >> More

  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. << Less

  PLoS ONE 9:E86548-E86548(2014) [PubMed] [EuropePMC]