tailieunhanh - Báo cáo khóa học: Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism

Three point mutations R335S, L336V and V476L, distin-guish the sequence of a cytochrome P450CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) resistance in theRDDT R strain ofDrosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) ofCyp6a2was expressed inEscheri-chia colias 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. . | Eur. J. Biochem. 271 1250-1257 2004 FEBS 2004 doi Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism Marcel Amichot Sophie Tares Alexandra Brun-Barale Laury Arthaud Jean-Marc Bride and Jean-Baptiste Berge Unite Mixte de Recherche 1112 Institut National de la Recherche Agronomique Sophia Antipolis France Three point mutations R335S L336V and V476L distinguish the sequence of a cytochrome P450 C YP6A2 variant assumed to be responsible for 1 1 1-trichloro-2 2-bis- 4 -chlorophenyl ethane DDT resistance in the RDDTr 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 RDDTr strain were measured with all recombinant proteins namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2b 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 P450can have a .