Clock-Turning Control of P450 TxtE Regioselectivity Enables Precise Site Functionalization of Aromatic Compounds

High Resolution Image Download MS PowerPoint Slide Aromatic nitration is a key transformation in the synthesis of pharmaceuticals, functional materials, and fine chemicals, yet selective functionalization of electronically deactivated sites remains a formidable challenge. The C6 position of l -tryptophan is particularly inert due to the high conjugation and low electron density of the indole ring, and no chemical or enzymatic system has achieved efficient single-step nitration at this site. Here we report a computer-aided regioselective design platform that precisely reprograms cytochrome P450 TxtE to enable C6-selective nitration. Guided by MD simulations, MM/PBSA energy decomposition, and QM/MM calculations, we identified key residues that control substrate orientation and engineered the H176F/A248F double mutant, which achieves >99% C6 selectivity with the highest total turnover number among all variants. High-resolution crystallographic analysis (1.53 Å) combined with QM/MM calculations revealed that, in the H176FA248F, steric repacking of the active pocket and substrate flipping lower the C6 nitration barrier from 54.3 kcal·mol –1 in the WT to 17.9 kcal·mol –1 . This work not only endows TxtE with unprecedented C6-selective nitration capability, but also reveals that the clock-like control of P450 TxtE regioselectivity enables precise site-selective functionalization of aromatic compounds, providing a new conceptual framework and a generalizable strategy for targeted functionalization of aromatic scaffolds.