Chimeragenesis of the fatty acid binding site of cytochrome P450BM3. Replacement of residues 73-84 with the homologous residues from the insect cytochrome P450 CYP4C7

A protein fragment of P450BM3 (residues 73−84) which participates in palmitoleate binding was subjected to scanning chimeragenesis. Amino acids 73−84, 73−78, 75−80, and 78−82 were replaced with the homologous fragments of the insect terpenoid hydroxylase CYP4C7. The four chimeric proteins, C(73−84), C(73−78), C(75−80), and C(78−82), were expressed, purified, and characterized. All the chimeric proteins contained all the cofactors and catalyzed monooxygenation of palmitate and of the sesquiterpene farnesol. Chimeragenesis altered substrate binding as shown by the changes in the amplitude of the palmitate-induced type I spectral shift. C(78−82) had monooxygenase activities close to those of P450BM3, while the rest of the chimeric proteins had monooxygenase activities that were inhibited relative to that of wild-type P450BM3. The extent of inhibition of the chimeric proteins varied depending on the substrate, and in the case of C(73−84), farnesol and palmitate oxidation was inhibited by 1 and 4 orders of mag...