Abstract
The substrate range of the haem monooxygenase cytochrome P450 cam (CYP101) has been broadened by site-directed mutagenesis. The hydroxylation selectivity of five mutants at the 96 position towards a range of substrates has been used to investigate P450 cam -substrate molecular recognition. The substrates contained aromatic and activated and unactivated aliphatic CH bonds, as well as reactive functional groups. Diphenylmethane, diphenylether, diphenylamine, and 1,1-di-phenylethylene were all hydroxylated regiospecifically at the para position, with no attack at the amine or the olefinic double bond. With benzylcyclohexane the activated benzylic and tertiary CH bonds were not attacked, and the reactions catalysed by the Y96G and Y96A mutants were highly diastereoselective, with 4- trans-benzylcyclohexanol constituting 90% of the products. 1-Phenyl-1-cyclohexylethylene was oxidised predominantly at the 4-position of the cyclohexane ring without attack at the olefinic double bond, and approximately equal amounts of cis- and trans-4-phenylethenylcyclohexanol were formed. These results show that P450 cam can be engineered to oxidise CH bonds without attacking more reactive functional groups.
Published Version
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