Crystal Structure of H2O2-dependent Cytochrome P450SPα with Its Bound Fatty Acid Substrate

Abstract

Cytochrome P450 SP (CYP152B1) isolated from Sphingomo-nas paucimobilis is the first P450 to be classified as a H 2 O 2-dependent P450. P450 SP hydroxylates fatty acids with high-re-gioselectivity. Herein we report the crystal structure of P450 SP with palmitic acid as a substrate at a resolution of 1.65 A ˚. The structure revealed that the C of the bound palmitic acid in one of the alternative conformations is 4.5 A ˚ from the heme iron. This conformation explains the highly selective-hydroxyla-tion of fatty acid observed in P450 SP. Mutations at the active site and the F-G loop of P450 SP did not impair its regioselec-tivity. 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-regioselec-tivity. 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 .