The Retaining Mechanism of Xylose Transfer Catalyzed by Xyloside α-1,3-Xylosyltransferase (XXYLT1): a Quantum Mechanics/Molecular Mechanics Study.

Abstract

Glycosyltransferases (GTs) are a ubiquitous group of enzymes that catalyze the synthesis of glycosidic bonds. In this work, we focused on the retained reaction catalyzed by xyloside α-1,3-xylosyltransferase (XXYLT1) from Mus musculus. Our calculations revealed that the xylose transfer reaction follows the SNi-like mechanism, which involves a short-lived oxocarbenium-phosphate ion-pair intermediate (IP). The previously obtained crystal structure of the UDP-Xyl ternary Michaelis reaction complex was found to be an inactive form. Accordingly, the β-phosphate oxygen O3B of the donor should first undergo a conformational change to reach an active state where the donor forms a strong hydrogen bond with the acceptor, facilitating the departure of the phosphate group. Our calculations also revealed that two predicated transition states for the sugar-phosphate bond cleavage and glycosidic bond formation are structurally similar to the short-lived intermediate, which contains a three-member ring formed by the β-phosphate oxygen, the hydroxyl oxygen in the acceptor, and the anomeric carbon. It can be considered as a typical characteristic of the SNi-like mechanism. In addition, a nearby polar residue, Q330, is responsible for stabilizing the short-lived intermediate by electrostatic interactions. Thus, the Q330A mutant can abolish the activity of XXYLT1. In addition, using UDP-glucose as the donor, our calculations revealed that glucose transfer would correspond to a higher energy barrier owing to the steric repulsion between the glucosyl moiety and the nearby residue L327, indicating the requirement of active site architecture for glucose transfer. These findings not only explain the experimental observations but also are meaningful for clarifying the mechanism of GTs.