黄酮6位羟基化酶催化机制的理论研究与应用

Theoretical analysis and practical applications of the catalytic mechanism of flavonoid 6-hydroxylase

摘要:

灯盏乙素发酵生产过程中，黄酮6位羟基化酶催化效率不足，导致产生至少约18%的副产物。本研究以2种黄酮6位羟基化酶CYP82D4与CYP706X为研究目标，通过分子动力学模拟与量子化学计算，对两种黄酮6位羟基化酶的催化机制进行解析。结果表明，CYP82D4与CYP706X在反应决速步的能垒几乎相同，应当具有相似的反应速率，而CYP82D4相对较小的底物结合能可能有利于产物释放，是其具有更高催化效率的直接原因。最后，基于对底物进出过程的研究，CYP82D4的L540A突变将催化效率提高了1.37倍，证明了理论计算指导黄酮6位羟基化酶改造优化的可行性。本研究揭示了黄酮6位羟化酶的催化机制，为对其进行改造优化以提高灯盏乙素的发酵生产效率提供了参考。

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.