Characterization of Two α-1,3-Glucoside Phosphorylases from Clostridium phytofermentans

Abstract

We characterized two α-1,3-glucoside phosphorylases that belonged to glycoside hydrolase family 65 from Clostridium phytofermentans: Cphy_3313 and Cphy_3314. Cphy_3313 was a typical nigerose phosphorylase that phosphorolyzed nigerose into D-glucose and β-D-glucose 1-phosphate (βGlc1P). Cphy_3314 catalyzed the synthesis of a series of α-1,3-oligoglucans using nigerose as the acceptor and βGlc1P as the donor. Kinetic analyses of their phosphorolytic reactions with α-1,3-oligoglucans (DP = 3 and 4) revealed that Cphy_3314 utilized a typical sequential Bi Bi mechanism, while this enzyme did not exhibit any significant phosphorolytic activity for nigerose. These results suggest that Cphy_3314 is a novel inverting phosphorylase that catalyzes reversible phosphorolysis of α-1,3-oligoglucans with DP of 3 or higher. In this study, we propose 3-O-α-D-oligoglucan: phosphate β-D-glucosyltransferase as the systematic name and α-1,3-oligoglucan phosphorylase as the short name for Cphy_3314.