Amino acids in SRS1 and SRS6 are critical for furanocoumarin metabolism by CYP6B1v1, a cytochrome P450 monooxygenase.

CYP6B1v1 is the principal cytochrome P450 monooxygenase (P450) that detoxifies dietary furanocoumarins in the guts of Papilio polyxenes, the black swallowtail caterpillar. Sequence alignments and structure comparisons of CYP6B1v1 with the mouse CYP2A5 and bacterial CYP102 proteins, which are also capable of metabolizing the linear furanocoumarin xanthotoxin (8-methoxypsoralen), suggested that Phe116, His117, Val368 and Phe484 might be active site residues. In a homology model developed for CYP6B1v1, the side chains of Phe116 and His117 located in the B'-C loop of SRS1 are predicted to be positioned above the haem plane, while the side chain of Phe484 located in SRS6 is predicted near the entrance of the catalytic pocket. Site-directed mutagenesis of residues Phe116, His117 and Phe484 indicated that these residues represent several of those that determine this protein's stability and substrate specificity. Whereas all aromatic mutants of Phe116 and Phe484 generated CO-difference spectra with maxima at 450 nm indicative of correctly configured monooxygenases, aromatic mutants of Phe116 exhibited reduced reactivities toward some furanocoumarins and aromatic mutants of Phe484 eliminated all reactivities toward furanocoumarins. All single and double aliphatic mutants of Phe116, His117 and Phe484 and aromatic mutants of His117 generated carbon monoxide (CO) difference spectra with maxima at 420 nm (P420) indicative of incorrectly configured monooxygenases. These studies define residues Phe116, His117 and Phe484 as determinants of this insect P450's catalytic site integrity and residues Phe116 and Phe484 as determinants of its substrate specificity. Conservation of Phe116 and His117 in an array of lepidopteran CYP6B proteins implies that these amino acids serve a similar function in other monooxygenases of the insect CYP6B subfamily.