α1,4-N-Acetylhexosaminyltransferase EXTL2: The Missing Link for Understanding Glycosidic Bond Biosynthesis with Retention of Configuration

Abstract

Glycosyltransferases (GTs) are biocatalysts that synthesize a wide variety of glycans present in nature. The mechanism by which they form a new glycosidic bond, preserving the configuration at the anomeric carbon, has been strongly debated. In the last few years, new experimental and computational results have provided very valuable knowledge: the proposed front-side reaction seems to be the preferred path, whereas some systems (family GT6 members) can switch mechanism toward the double displacement. However, why would retaining GTs have evolved to catalyze sugar transfer by different mechanisms? The present QM(DFT)/MM work on α1,4-N-acetylhexosaminyltransferase (EXTL2) fills this gap in the understanding of retaining glycosyltransferases catalysis. We show that EXTL2, despite having a nucleophilic residue (Asp246) that is structurally analogous to the Glu nucleophile found in GT6, clearly follows a front-side mechanism. Our results show that Asp246 is used to facilitate UDP–GalNAc bond cleavage by stabil...