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

Abstract

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.

Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: Specificity and substrate encounter rate

“…Of the two P. canadensis proteins, CYP6B25, an in-group homologue to CYP6B4 (17), generated a major CO-difference peak at 450 nm and a minor peak at 420 nm and CYP6B26, an in-group homologue to CYP6B17 and seven amino acids shorter at its C terminus (16), generated a CO-difference peak only at 420 nm (P420 form), indicating that this enzyme is incorrectly folded. Molecular modeling and site-directed mutagenesis presented in Chen et al (20) have indicated that aromatic residues F116, H117, and F484 positions are required for correct folding of the P. polyxenes CYP6B1 enzyme, and that two of these modulate the range of furanocoumarins metabolized. This seems not to be the case for P. glaucus͞P.…”

“…In this latter protein, an aromatic network that involves residues Phe-116, His-117, Phe-484, and Phe-371 is critical for substrate binding affinity to the CYP6B1 active site (20,33). Three-dimensional models of the present day CYP6B4 and the ancestral Anc1 and Anc2 proteins that we have con- 3.…”

Allelic Variation in the Depressaria pastinacella CYP6AB3 Protein Enhances Metabolism of Plant Allelochemicals by Altering a Proximal Surface Residue and Potential Interactions with Cytochrome P450 Reductase

“…A molecular model of CYP6AB3v1 contains three-dimensional elements similar to those predicted in the catalytic sites of the furanocoumarin-metabolizing CYP6B proteins in P. polyxenes, P. glaucus, and H. zea (25)(26)(27)31) including residues in the B-helix and BЈ-C loop of SRS1, the I-helix of SRS4, and the ␤-turn in ␤-sheet 4 of SRS6 (30). Amino acids are also conserved in SRS1, SRS4, and SRS5 that contribute to form the catalytic site of CYP6B1 from the specialist P. polyxenes, CYP6B4 from the generalist P. (25,31,32).…”

“…Furanocoumarin-metabolizing P450s of polyphagous (generalist) species, such as H. zea and P. glaucus, catalyze metabolism of a wide range of furanocoumarins with lower activities than the P450s of specialist species, such as P. polyxenes, which feed exclusively on furanocoumarin-containing host plants (17,20,(25)(26)(27)(28)(29). Molecular modelings of these P450s have indicated that at least some of the P450s in generalist insects are structurally more flexible and capable of binding a more diverse array of compounds when compared with some of the P450s in specialist insects (26,27).…”

Characterization of Drosophila melanogaster cytochrome P450 genes

“…Insect P450s capable of metabolizing allelochemicals have been identified (30,31). For example, CYP6B1v1 and CYP6B3v1 from the black swallowtail butterfly, Papilio polyxenes, can metabolize the furanocoumarins it is exposed to in its diet (32,33). Numerous individual P450s have also been implicated in insecticide resistance in different insect populations (34)(35)(36)(37)(38).…”