title: Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism.
abstract: Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane (DDT) resistance in the RDDT(R) strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT(R) strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2beta position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.
authors.affiliation: Unité Mixte de Recherche 1112, Institut National de la Recherche Agronomique, Sophia Antipolis, France. amichot@antibes.inra.fr
authors.forename: Marcel
authors.initials: M
authors.lastname: Amichot
authors.forename: Sophie
authors.initials: S
authors.lastname: Tarès
authors.forename: Alexandra
authors.initials: A
authors.lastname: Brun-Barale
authors.forename: Laury
authors.initials: L
authors.lastname: Arthaud
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Bride
authors.forename: Jean-Baptiste
authors.initials: JB
authors.lastname: Bergé
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072472:Cytochrome P450 Family 6; D003634:DDT / Q000378:metabolism*; D003632:Dichlorodiphenyldichloroethane / Q000378:metabolism; D004010:Dicofol / Q000378:metabolism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D006900:Hydroxylation; D006915:Hydroxytestosterones / Q000378:metabolism; D007305:Insecticide Resistance / Q000235:genetics*; D007306:Insecticides / Q000494:pharmacology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017354:Point Mutation; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship; D013739:Testosterone / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; D006915:Hydroxytestosterones; D007306:Insecticides; D011994:Recombinant Proteins; D013739:Testosterone; D003577:Cytochrome P-450 Enzyme System; D003634:DDT; C109499:Cyp6a2 protein, Drosophila; D000072472:Cytochrome P450 Family 6; D003632:Dichlorodiphenyldichloroethane; D004010:Dicofol