Docking-guided rational engineering of a macrolide glycosyltransferase glycodiversifies epothilone B

Zhang et al. identify three hot regions of macrolide glycosyltransferase BsGT-1, that can play a role in epothilone B and sugar donor specificity. Further mutagenesis of BsGT-1 showed that specificity could be engineered and when the mutations were introduced into homologous GTs, promiscuity was improved, setting the stage for further rational glycodiversification of macrolides. Glycosyltransferases typically display acceptor substrate flexibility but more stringent donor specificity. BsGT-1 is a highly effective glycosyltransferase to glycosylate macrolides, including epothilones, promising antitumor compounds. Here, we show that BsGT-1 has three major regions significantly influencing the glycodiversification of epothilone B based on structural molecular docking, hot spots alanine scanning, and site saturation mutagenesis. Mutations in the PSPG-like motif region and the C2 loop region are more likely to expand donor preference; mutations of the flexible N3 loop region located at the mouth of the substrate-binding cavity produce novel epothilone oligosaccharides. These hot spots also functioned in homologues of BsGT-1. The glycosides showed significantly enhanced water solubility and decreased cytotoxicity, although the glycosyl appendages of epothilone B also reduced drug permeability and attenuated antitumor efficacy. This study laid a foundation for the rational engineering of other GTs to synthesize valuable small molecules.

Automated summary

Zhang et al. identify three hot regions of macrolide glycosyltransferase BsGT-1 that influence the specificity of epothilone B and sugar donor. Further study showed that mutations in these regions could expand donor preference and produce novel epothilone oligosaccharides. This research lays a foundation for rational engineering of other glycosyltransferases to synthesize valuable small molecules.