Structural analysis of Thermus thermophilus HB27 mannosyl-3- phosphoglycerate synthase provides evidence for a second catalytic metal ion and new insight into the retaining mechanism of glycosyltransferases

Abstract

Mannosyl-3-phosphoglycerate synthase is a glycosyltransferase involved in the two-step synthetic pathway of mannosylglycerate, a compatible solute that accumulates in response to salt and/or heat stresses in many microorganisms thriving in hot environments. The three-dimensional structure of mannosyl-3-phosphoglycerate synthase from Thermus thermophilus HB27 in its binary complex form, with GDP-α-D-mannose and Mg2+, shows a second metal binding site, about 6 Å away from the mannose moiety. Kinetic and mutagenesis studies have shown that this metal site plays a role in catalysis. Additionally, Asp167 in theDXD motif is found within van der Waals contact distance of the C1′ atom in the mannopyranose ring, suggesting its action as a catalytic nucleophile, either in the formation of a glycosyl-enzyme intermediate according to the double-displacement SN2 reaction mechanism or in the stabilization of the oxocarbenium ion-like intermediate according to the DN*ANss (S Ni-like) reaction mechanism. We propose that either mechanism may occur in retaining glycosyltransferases with a GT-Afold, and, based on the gathered structural information, we identified an extended structural signature toward a common scaffold between the inverting and retaining glycosyltransferases. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.