Molecular mechanism of the substrate promiscuity in GT1 glucosyltransferases: Insights from molecular dynamics simulations and experimental validation

Glycosyltransferases (GTs) catalyze the transfer of sugar moieties from activated donors to acceptor molecules, which are widely-used biocatalysts in the industrial biosynthesis of natural products. Members of the GT1 family exhibit remarkable substrate promiscuity, enabling glycosylation of diverse natural products; however, the underlying molecular mechanisms remain poorly understood. Here we integrated all-atom molecular dynamics simulations, bioinformatics and experimental mutations to elucidate the structural and dynamics mechanisms governing the substrate promiscuity of GT1 enzymes. Using the Bs YjiC from Bacillus subtilis as a representative, we interestingly found that its acceptor-binding pocket harbored two flexible binding subsites with distinct geometric features, which enabled structurally diverse substrates to be specifically recognized in different subsites. The site-directed mutagenesis at the subsites and in vitro glycosylation assays further demonstrated the critical role of subsite-mediated substrate recognition. Importantly, the existence of two subsites in the acceptor-binding pocket was found to be a general feature across nine different members of GT1 family. Overall, the characteristic features of the acceptor-binding pocket represent a conserved trait across the GT1 family and provide potential guidance for engineering GTs with tailored substrate specificities.