Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome P450 monooxygenase

Abstract

Furanocoumarins represent plant toxins that are used in the treatment of a variety of skin diseases and are metabolized by cytochrome p450 monooxygenases (p450s) existing in insects such as Papilio polyxenes (the black swallowtail). To elucidate the active site in the CYP6B1 protein that is the principal p450 existing in this species, we have constructed a homology model of it based on sequence and structure alignments with the bacterial CYP102 protein whose crystal structure has been defined and with the insect CYP6B4 protein that also metabolizes furanocoumarins. In the derived CYP6B1 model, Phe116 and His117 in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to the formation of a resonant network that stabilizes the p450's catalytic site and allows for interactions with its furanocoumarin substrates. The first two of these residues are absolutely conserved in all members of the insect CYP6B subfamily and the last two are variable in different members of the CYP6B subfamily. A combination of theoretical and experimental docking analyses of two substrates (xanthotoxin and bergapten) and two inhibitors (coumarin and pilocarpine) of this p450 provide significant information on the positioning of furanocoumarins within this catalytic pocket. Molecular replacement models based on the results of variations at two of these critical amino acids provide support for our furanocoumarin-docked model and begin to rationalize the altered substrate reactivities observed in experimental analyses.

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

“…With all previous studies identifying residues in insect P450 catalytic sites affect- ing substrate recognition, turnover, and product release (25,31,38,39), this study is the first to identify a residue affecting interactions with P450 reductase, the one electron transfer partner essential to endoplasmic reticulum-localized P450s. The natural evolution of residue 92 in CYP6AB3v1 from valine to the smaller alanine side chain enhances the NADPH consumption rate of the CYP6AB3v2 protein 2.4-fold when the initial slopes on these curves are compared.…”

“…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).…”

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

“…Phe298 and Thr303 are observed within the active site itself and thus likely to interact with substrates. Although none of the docking studies with aromatic compounds binding CYP2E1 implicated SRS-5, the analysis of a homology model for CYP6B1 did suggest SRS-5 was critical in the recognition of furanocoumarins (43). Taken together, these different docking solutions for CYP2E1 reflect the uncertainty inherent with homology models and therefore justify other techniques, such as our labeling efforts, to complement and further refine the modeling of interactions between molecules and proteins.…”

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

“…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.…”