Structure-guided engineering of P450BM3 as a cofactor-free peroxygenase for efficient biotransformation of cresol...

Cresols, widely used industrial intermediates and common constituents of wastewater, are of environmental concern due to their acute toxicity and frequent occurrence. In this study, we engineered cytochrome P450BM3 to operate as a cofactor-free peroxygenase directly driven by H₂O₂. Introduction of an aspartic acid residue at position 268 (T268D) endowed the enzyme with enhanced peroxidase activity, while additional substitutions at positions 78, 87, and 328 reshaped the active site to improve substrate binding and stabilization. The resulting quadruple mutant V78F/F87A/T268D/A328F exhibited rapid and nearly complete biotransformation of o-, m-, and p-cresol within 2 min under mild conditions (25 °C, pH 7.0), achieving catalytic efficiencies of 56000, 48333, and 57464 M^-1 s^-1, respectively. The enzyme maintained >98 % activity in simulated wastewater, highlighting its robustness in complex matrices. UPLC-MS analysis confirmed that cresol conversion mainly proceeds through hydroxylation and oxidation pathways rather than mineralization. Structural analysis revealed that the synergistic arrangement of residues at positions 78, 87, 268, and 328 enhanced H₂O₂ activation and facilitated π-π interactions with aromatic substrates. These results establish a robust and generalizable platform for peroxygenase-based biotransformation of phenolic pollutants, offering a sustainable and efficient strategy for wastewater treatment.