ABSTRACT
Insufficient catalytic efficiency of flavonoid 6-hydroxylases in the fermentative production of scutellarin leads to the formation of at least about 18% of by-products. Here, the catalytic mechanisms of two flavonoid 6-hydroxylases, CYP82D4 and CYP706X, were investigated by molecular dynamics simulations and quantum chemical calculations. Our results show that CYP82D4 and CYP706X have almost identical energy barriers at the rate-determining step and thus similar reaction rates, while the relatively low substrate binding energy of CYP82D4 may facilitate product release, which is directly responsible for its higher catalytic efficiency. Based on the study of substrate entry and release processes, the catalytic efficiency of the L540A mutation of CYP82D4 increased by 1.37-fold, demonstrating the feasibility of theoretical calculations-guided engineering of flavonoid 6-hydroxylase. Overall, this study reveals the catalytic mechanism of flavonoid 6-hydroxylases, which may facilitate the modification and optimization of flavonoid 6-hydroxylases for efficient fermentative production of scutellarin.