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

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 the DXD 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 S_N2 reaction mechanism or in the stabilization of the oxocarbenium ion-like intermediate according to the D_N*A_Nss (S_Ni-like) reaction mechanism. We propose that either mechanism may occur in retaining glycosyltransferases with a GT-A fold, and, based on the gathered structural information, we identified an extended structural signature toward a common scaffold between the inverting and retaining glycosyltransferases.