Mechanistic Insights into the Selective C–S Bond Formation by P450 TleB

The P450 monooxygenase TleB (CYP107E48) catalyzes intramolecular C–S bond formation in a thiol-containing substrate, yielding two sulfur-containing indolactam derivatives (P1 and P2). However, the key sites influencing TleB’s product selectivity and the molecular mechanisms underlying the selective C–S bond formation are not fully understood. To address this, we created an artificial self-sufficient P450, TleB-CYP116B46, by fusing TleB with the reductase domain of CYP116B46. Structure-guided engineering of TleB-CYP116B46 generates variant L85G with 99% selectivity for P1 and variant I282L/Q387L/I234F with 95% selectivity for P2. Exploring TleB homologues and generating corresponding mutants elucidate the identified sites’ crucial role in product selectivity. Computational studies suggest a diradical mechanism for C–S bond formation for both P1 and P2 products. Intriguingly, we found that the substrate radical could undergo conformational changes in both the S–H and indole groups. The L85G variant facilitates the conformational switch of the indole radical group, thereby leading to the selective C–S bond formation for the P1 product. By contrast, the I282L/Q387L/I234F variant barricades the conformational switch of the indole radical group, affording the P2 product. Our simulations highlight that the protein environment can dictate the dynamics and positioning of the substrate radical, thereby leading to the selective C–S bond formation in P450s.