Structure-guided engineering of CYP82D213 for enhanced triptonide biosynthesis.

Triptonide (TN) is a highly oxidized, bioactive diterpenoid triepoxide from Tripterygium wilfordii with promising pharmaceutical applications. While the early steps of its biosynthesis have been elucidated, the mechanism responsible for the critical terminal tri-epoxidation remains to be elucidated. Here, to address the limitation posed by the low intrinsic activity of CYP82D213, we employed a comprehensive protein engineering strategy. Structure-guided mutagenesis pinpointed the key residues responsible for substrate recognition (G128, W129) and dynamic conformation (L396). Motif-driven engineering yielded the synergistic double mutant H425Q/L459M, which enhanced activity 2.30-fold. Subsequent surface charge optimization, further improved performance. The combination of these beneficial mutations in the triple mutant H425Q/L459M/T365R resulted in a 2.63-fold increase in TN production compared to the wild-type and revealed a putative dynamic catalytic mechanism that may involve substrate repositioning. These results provide a rational design strategy for complex multi-step enzyme catalysis.