Modification of an Enzyme Biocatalyst for the Efficient and Selective Oxidative Demethylation of para-Substituted Benzene Derivatives

The bacterial CYP199A4 enzyme is able to oxidise a narrow range of aromatic acids, which includes 4‐methoxybenzoic acid, efficiently. A serine 244 to aspartate variant was identified with enhanced activity for a wide range of para‐methoxy‐substituted benzenes. Substrates in which the acidic benzoic acid moiety is replaced with a phenol and the amide, aldehyde and bromide analogues were all oxidised with high activity by the S244D mutant (product formation rate >600 nmol nmolCYP−1 min−1) with turnover numbers of up to 20 000. If the carboxylate moiety was modified to a nitro, ketone, boronic acid, hydroxymethyl or nitrile group, these substrates were also oxidised at a significantly higher activity by S244D than the wild‐type enzyme. 3,4‐Dimethoxybenzaldehyde was demethylated selectively and oxidatively to 3‐methoxy‐4‐hydroxybenzaldehyde by the S244D mutant 84‐fold more rapidly than with the wild‐type enzyme. CYP199A4 would have applications in the catalytic regioselective oxidative demethylation of suitably substituted benzene substrates under mild conditions and in the presence of more oxidatively sensitive functional groups, such as an aldehyde. Mutant ninja catalysts: The cytochrome P450 enzyme, CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2, is modified to expand its substrate range beyond benzoic acids. The S244D mutant is able to act on a range of para‐substituted benzenes with high activity and total turnover to result in the selective catalytic oxidative demethylation under mild conditions.