Trick or treat: Cytochrome P450BSβ was transformed into a monooxygenase suitable for practical use by employing a simple substrate trick. The substrate specificity of P450BSβ was altered drastically by a decoy molecule, while its intrinsic advantage, the use of hydrogen peroxide, was retained. The catalytic activities and the enantioselectivity of the H2O2–P450BSβ system are highly dependent on the structure of the decoy molecule.
Cytochrome P450 SP␣ (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H 2 O 2 -dependent P450. P450 SP␣ hydroxylates fatty acids with high ␣-regioselectivity. Herein we report the crystal structure of P450 SP␣ with palmitic acid as a substrate at a resolution of 1.65 Å . The structure revealed that the C ␣ of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective ␣-hydroxylation of fatty acid observed in P450 SP␣ . Mutations at the active site and the F-G loop of P450 SP␣ did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450 BS (CYP152A1), which shows -regioselectivity. This implies that the high regioselectivity of P450 SP␣ is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450 BS .
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