Qingbo Deng , Zhen-Ming Lu, Zhaoting Yuan, Yinghui Feng , Lujia Zhang b , Jinsong Shi , Zhenghong Xu , M.A.G. Kofas , Hui Li
P450 dihydroxylase plays a crucial role in steroid drug synthesis by efficiently catalyzing two-step selective hydroxylation reactions. However, natural P450 dihydroxylases are scarce, with poor catalytic performance and intermediate accumulation limiting production. Here, we report a P450 dihydroxylase BM3-H that synthesizes 7α,15α-diOH-DHEA with 76 % selectivity. To enhance 7α,15α-diOH-DHEA synthesis, we engineered a novel exit channel for the intermediate by modifying key residues in the solvent channel. The triple mutant D182K/E143D/V178A exhibited significant improvements in product concentration (10.08-fold), enzymatic activity (2.16-fold), catalytic efficiency (kcat/Km, 42.32-fold), electron transfer rate (kET, 27.14-fold), and coupling efficiency (CE, 3.93-fold). Molecular dynamics simulations revealed that D182K/E143D/V178A created a novel exit channel for 7α-OH-DHEA, with channel length, polarity, and steric hindrance influencing enzyme performance. Our approach enhances the overall catalytic performance of P450BM3-H by excavating new intermediate product exit channels, providing theoretical guidance for the design of other enzyme molecules.
