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

• Published in: Communications biology 2022-01, Vol.5 (1), p.100-100, Article 100
• Main Authors: Zhang, Peng, Zhang, Lijuan, Jiang, Xukai, Diao, Xiao-tong, Li, Shuang, Li, Dan-dan, Zhang, Zheng, Fang, Junqiang, Tang, Ya-jie, Wu, Da-lei, Wu, Changsheng, Li, Yue-zhong
• Format: Article
• Language: English
• Subjects: 631/45/221; 631/45/607/1172; 631/92/603; 82/58; 82/80; 82/83; Alanine; Amino Acid Motifs; Amino Acid Sequence; Antitumor activity; Antitumor agents; Appendages; Biology; Biomedical and Life Sciences; Cytotoxicity; Epothilones - chemistry; Epothilones - metabolism; Gene Expression Regulation, Enzymologic; Glucosyltransferases - metabolism; Glycosides; Glycosyltransferase; Hep G2 Cells; Hepatocytes; Humans; Life Sciences; Molecular Docking Simulation; Mutagenesis; Mutation; Oligosaccharides; Permeability; Protein Engineering; Saturation mutagenesis
• ISSN: 2399-3642; 2399-3642
• DOI: 10.1038/s42003-022-03047-y

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. 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.