期刊
ACS CATALYSIS
卷 10, 期 6, 页码 3629-3639出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscatal.9b05232
关键词
biocatalysis; catalytic efficiency; protein engineering; plant glycosyltransferase; triterpene saponins
资金
- National Key Research and Development Program of China [2019YFA0905100]
- Natural Science Foundation of China [31771909]
- Tianjin synthetic biotechnology innovation capacity improvement action
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 10(2)- to 10(4)-fold improvement in catalytic efficiency for triterpene glycosylation. Especially, one variant exhibited up to 4.17 x 10(4)-fold increase in catalytic efficiency toward mogrol and 1.53 x 10(4)-fold increase to UDP-glucose, respectively. Moreover, the mutants also displayed extended substrate promiscuity compared with wildtype 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.
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