Real-time monitoring of glycocarrier formation unravels cryptic details in glycosyl transfer

Abstract

Glycosyl transfer constitutes a key step in the functionalisation of bioactive molecules, including proteins and natural products. In vitro studies to dissect glycosyltransferase catalysis are often challenging due to poor enzyme solubility, enzyme instability, and the requirement of advanced analytical setups. Herein, we report the development and use of a polynucleotide phosphorylase (PNPase)-coupled assay for the real-time monitoring of mannosyl transfer to polyprenyl phosphate carriers. This, together with molecular modeling, dynamics and site-directed mutagenesis, has allowed us to gather in-depth insights into enzyme catalysis associated with health and disease.