Quick-soaking of crystals reveals unprecedented insights into the catalytic mechanism of glycosyltransferases

Abstract

Glycosyltransferases (GTs) catalyze the transfer of a sugar moiety from nucleotide-sugar or lipid-phospho-sugar donors to a wide range of acceptor substrates, generating a remarkable amount of structural diversity in biological systems. Glycosyl transfer reactions can proceed with either inversion or retention of the anomeric configuration with respect to the sugar donor substrate. In this chapter, we discuss the application of a quick soaking method of substrates and products into protein crystals to visualize native ternary complexes of retaining glycosyltransferases. The crystal structures provide different snapshots of the catalytic cycle, including the Michaelis complex. During this sequence of events, we visualize how the enzyme guides the substrates into the reaction center where the glycosyl transfer reaction takes place, and unveil the mechanism of product release, involving multiple conformational changes not only in the substrates and products but also in the enzyme. The methodology described here provides unprecedented insights into the catalytic mechanism of glycosyltransferases at the molecular level, and can be applied to the study a myriad of enzymatic mediated reactions.

Keywords: Carbohydrate active enzymes; Enzyme catalysis; Glycosyltransferase; Soaking; X-ray crystallography.