Efficient O‑Glycosylation of Triterpenes Enabled by Protein Engineering of Plant Glycosyltransferase UGT74AC1

• Published in: ACS catalysis 2020-03, Vol.10 (6), p.3629-3639
• Main Authors: Li, Jiao, Yang, Jiangang, Mu, Shicheng, Shang, Na, Liu, Cui, Zhu, Yueming, Cai, Yi, Liu, Pi, Lin, Jianping, Liu, Weidong, Sun, Yuanxia, Ma, Yanhe
• Format: Article
• Language: English
• ISSN: 2155-5435; 2155-5435
• DOI: 10.1021/acscatal.9b05232

Triterpene O-glycosylation has attracted significant interest from the pharmaceutical industry as a valuable means for drug design and development. Plant glycosyltransferases, which catalyze this glycosylation reaction, play a key step in preparing structure diverse and valuable triterpene glycosides. However, this class of enzymes usually suffers from low catalytic efficiency. To address this problem, triterpene glycosyltransferase UGT74AC1 from Siraitia grosvenorii was chosen and its crystal structure was solved and employed as the molecular basis to implement directed evolution and sequence/structure-based engineering. Several resultant uridine diphosphate (UDP) glycosyltransferases (UGTs) variants exhibit a 102- to 104-fold improvement in catalytic efficiency for triterpene glycosylation. Especially, one variant exhibited up to 4.17 × 104-fold increase in catalytic efficiency toward mogrol and 1.53 × 104-fold increase to UDP-glucose, respectively. Moreover, the mutants also displayed extended substrate promiscuity compared with wild-type enzyme and conserved regioselectivity. Based on the results of molecular docking and molecular dynamics simulations, it was proposed that the improved enzymatic activity and substrate promiscuity were likely owing to the stable hydrophobic interactions and favorite conformations between the enzyme and substrates. This work has also laid a foundation for the engineering of other plant UGTs for their practical application to the synthesis of valuable triterpene saponins.