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

Area of Science:

• Biocatalysis and Enzyme Engineering
• Organic Synthesis
• Computational Chemistry

Background:

• Selective aromatic nitration is crucial for synthesizing valuable compounds.
• Electronically deactivated aromatic sites, like the C6 position of l-tryptophan, are difficult to functionalize.
• Existing methods lack efficiency for single-step nitration at inert positions.

Purpose of the Study:

• To develop a regioselective method for C6 nitration of l-tryptophan.
• To reprogram the cytochrome P450 enzyme TxtE for enhanced site-specific functionalization.
• To establish a computer-aided design strategy for enzyme engineering.

Main Methods:

• Computer-aided regioselective design platform.
• Molecular dynamics (MD) simulations.
• MM/PBSA energy decomposition and QM/MM calculations.
• Site-directed mutagenesis (H176F/A248F double mutant).
• High-resolution crystallographic analysis.

Main Results:

• Engineered H176F/A248F mutant achieved >99% C6 selectivity for l-tryptophan nitration.
• The mutant exhibited the highest total turnover number among engineered variants.
• Computational and crystallographic analyses revealed mechanisms for lowered nitration barrier at C6.
• Significant reduction in activation energy from 54.3 kcal·mol^-1 (WT) to 17.9 kcal·mol^-1 (mutant).

Conclusions:

• The engineered TxtE enzyme demonstrates unprecedented C6-selective nitration capability.
• Computer-aided design provides a generalizable strategy for targeted functionalization of aromatic scaffolds.
• This approach offers a new conceptual framework for precise site-selective modification of aromatic compounds.