4ktp

Publication Abstract from PubMed

2-O-alpha-Glucosylglycerol phosphorylase (GGP) from Bacillus selenitireducens catalyzes both the reversible phosphorolysis of 2-O-alpha-glucosylglycerol (GG) and the hydrolysis of beta-D-glucose 1-phosphate (betaGlc1P). 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 with the glucose analogue 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, betaGlc1P 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 betaGlc1P hydrolysis. Y572F also showed significantly reduced activities for both of these reactions. In contrast, W381F showed significantly reduced reverse phosphorolytic activity but retained betaGlc1P 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 betaGlc1P 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.

Structural basis for reversible phosphorolysis and hydrolysis reactions of 2-O-alpha-Glucosylglycerol phosphorylase.,Touhara KK, Nihira T, Kitaoka M, Nakai H, Fushinobu S J Biol Chem. 2014 May 14. pii: jbc.M114.573212. PMID:24828502^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.