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

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.