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

Abstract

CYP6AB3v1, a cytochrome P450 monooxygenase in Depressaria pastinacella (parsnip webworm), is highly specialized for metabolizing imperatorin, a toxic furanocoumarin in the apiaceous host plants of this insect. Cloning and heterologous expression of CYP6AB3v2, an allelic variant identified in D. pastinacella, reveals that it metabolizes imperatorin at a rate (V max of 10.02 pmol/min/pmol of cytochrome P450 monooxygenase (P450)) significantly higher than CYP6AB3v1 (V max of 2.41 pmol/min/pmol) when supplemented with even low levels of cytochrome P450 reductase. Comparisons of the NADPH consumption rates for these variants indicate that CYP6AB3v2 utilizes this electron source at a faster rate than does CYP6AB3v1. Molecular modeling of the five amino acid differences between these variants and their potential interactions with P450 reductase suggests that replacement of Val 92 on the proximal face of CYP6AB3v1 with Ala 92 in CYP6AB3v2 affects interactions with P450 reductase so as to enhance its catalytic activity. Allelic variation at this locus potentially allows D. pastinacella to adapt to both intraspecific and interspecific variation in imperatorin concentrations in its host plants. Cytochrome P450 monooxygenases (P450s) 2 are hemecontaining enzymes that catalyze the NADPH-dependent reductive cleavage of molecular oxygen to produce functionalized organic products and a molecule of water, making them crucial for phase I metabolism in a wide range of organisms. Although considerable allelic variation in both the coding and the regulatory regions is known to occur in many vertebrate P450 genes (1-3), the extent of allelic variation in insect P450 loci is not clear. Much of what is known of allelic variation in insect P450sresults from studies of insecticide resistance. Here, most examples of P450-mediated resistance to insecticides involve regulatory changes, often transposon-mediated (4). Only a few examples exist where point mutations in coding regions affect reactivities toward insecticides. Perhaps the best evidence of such allelic variation is the existence of three amino acid variations in CYP6A2 (R335S, L336V, V476L) in the DDT-resistant RDDT R strain of Drosophila melanogaster; these substitutions occur near the CYP6A2 active site and increase catalytic efficiency of the CYP6A2 enzyme against DDT, 7-ethoxycoumarin, and 7-benzoyloxycoumarin (5). Allelic variation in the form of amino acid substitutions has also been documented in CYP6D1, CYP6D3, and CYP6X1 as well as CYP6A2 (6 -8), but the functional contributions of these to resistance have not yet been determined.In addition to contributing to insecticide resistance, P450s play an important role in mediating resistance to plant allelochemicals (defense compounds) in herbivorous insects (9). However, these allelochemicals present a toxicological challenge to insects that differs in several fundamental ways from that presented by insecticides. Although insecticides are generally single compounds designed to kill and applied in a broadcast fashion, plant d...

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“…The potential association between some amino acid changes such as F63L and Q66K for CYP6P9a and V103I and S384N for CYP6P9b and the resistance phenotype is similar to the case observed for the CYP6AB3 gene in the insect Depressaria pastinacella (parsnip webworm) in which five amino acid changes enhance metabolism of plant allelochemicals (25). Similarly, in humans, allelic variations in P450 (CYP2D6, CYP2C9, CYP2C19, and CYP2A6) are known to affect drug metabolism with some alleles conferring a low metabolism to certain drugs and others conferring a fast rate of metabolism (26).…”

Facing the Future of Plant-Insect Interaction Research: Le Retour à la “Raison d'Être”

“…This high activity is due in part to the ability of CYP6AB3 to selectively metabolize imperatorin, the furanocoumarin that is most abundant in its two host plant genera (Li et al, 2004;Mao et al, 2006Mao et al, , 2007, and myristicin, a methylenedioxyphenyl compound that, in other insect species, is an effective P450 inhibitor (Mao et al, 2007) and thus acts, as do many methylenedioxy-containing compounds, as a synergist, enhancing toxicity. CYP6AB3 epoxidizes the four-carbon isoprenoid chain on imperatorin and the three-carbon allyl group on myristicin but not any of the other tested furanocoumarins containing only small modifications of the core psoralen structure (Mao et al, 2006).…”

Metabolism of myristicin by Depressaria pastinacella CYP6AB3v2 and inhibition by its metabolite

“…In view of the existence of multiple pathways for MDP metabolism, MDP inhibition mechanisms hypothesized to exist in vertebrates may differ from those potentiating insecticide action in insects. In this study, we demonstrate that myristicin, present in high quantities in the hostplants of the parsnip webworm Depressaria pastinacella, is a substrate for the D. pastinacella CYP6AB3v2 (Mao et al, 2007). With low amounts of heterologously expressed CYP6AB3v2 protein incubated with myristicin for short times, myristicin is effectively transformed into an epoxide metabolite that has been characterized by GC-MS and NMR analyses.…”

“…All of the CYP6AB3v2 V32A protein used in this study was coexpressed in Sf9 cells with housefly P450 reductase at a multiplicity of infection (MOI) ratio of 1:0.25 (P450:P450 reductase) using insect baculovirus expression systems as described in Mao et al (2007). For the purposes of this paper, this protein, which contains a single amino acid replacement in the linker region preceding its proline hinge, is referred to as the CYP6AB3v2 protein.…”