Probing the mechanism of a fungal glycosyltransferase essential for cell wall biosynthesis. UDP-Chitobiose is not a substrate for chitin synthase

Abstract

Chitin synthase is responsible for the biosynthesis of chitin, an essential component of the fungal cell wall. There is a long-standing question as to whether “processive” transferases such as chitin synthase operate in the same manner as non-processive transferases. The question arises from analysis of the polysaccharide structure – in chitin, for instance, each sugar residue is rotated ≈180° relative to the preceding sugar in the chain. This requires that the enzyme account for the alternating “up/down” configuration during biosynthesis. An enzyme with a single active site, analogous to the non-processive transferases – would have to accommodate a distorted glycosidic linkage at every other synthetic step. An alternative proposal is that the enzyme might assemble the disaccharide donor, addressing the “up/down” conformational problem prior to polymer synthesis. We present compelling evidence that this latter hypothesis is incorrect.