Point mutations associated with insecticide resistance in theMarcel Amichot, Sophie Tare`s, Alexandra Brun-Barale, Laury Arthaud, Jean-Marc Bride and Jean-Baptiste Berge´ Unite´ Mixte de
Trang 1Point mutations associated with insecticide resistance in the
Marcel Amichot, Sophie Tare`s, 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,
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 the RDDTRstrain
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
Escheri-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 Two activities enhanced in the
RDDTR strain were measured with all recombinant
pro-teins, namely testosterone hydroxylation and DDT
meta-bolism 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, dichlorodiphenyl-dichloroethane and dichlorodiphenyl acetic acid The appar-ent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra Sequence align-ments 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 prominent role
in insecticide resistance
Keywords: cytochrome P450; mutation; insecticide; resist-ance; structure
Many cytochrome P450enzymes are known to be essential
for the protection of organisms against xenobiotics In
insects, the involvement of cytochrome P450enzymes in
plant toxin or insecticide resistance has already been
suggested or demonstrated [1–7], although high resistance
levels to insecticides still remain unexplained To date, only
three of the cytochrome P450enzymes linked to resistance
have been shown to be able to metabolize insecticides
Two were cloned from the house fly: CYP6A1 metabolizes
aldrin, heptachlor [8], terpenoids [9] and diazinon [10] and
CYP12A1 metabolizes aldrin, heptachlor, diazinon and
azinphosmethyl [11] The third is CYP6A2 from Drosophila
melanogaster.Baculovirus-directed production of wild-type
CYP6A2 showed metabolism of cyclodiene and organo-phosphorous insecticides, but 1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane (DDT) metabolism could not be detected [12] In addition, sequence polymorphism of CYP6A1 and CYP6D1 has been documented in the house fly, but there is no link between these instances of polymorphism and insecticide resistance [7,13,14] These results are in contrast with known instances of cytochrome P450polymorphisms in humans, which are well known to affect the metabolism of drugs [15,16] and even pesticides [17] In fact, only two examples of pesticide resistance linked
to mutations in a cytochrome P450have been described Single substitutions in CYP51 of Candida albicans (T315A) [18] and of Uncinula necator (F136Y) [19] confer resistance
to the fungicides fluconazole and to triadimenol, respect-ively Nevertheless, the situation is qualitatively very differ-ent from enhanced degradation of insecticides, as CYP51
is itself the target of the fungicides
Significant information is now available on the structure
of cytochrome P450 The majority of the structures des-cribed were those of cytochrome P450from bacteria (for the first descriptions see [20,21]) but two microsomal P450 structures have also been obtained [22,23] that are currently the only two structures publicly available for eukaryotes Although these structures were obtained from bacteria, rabbit or man, their overall similarity is striking Based
on these structures and on quantitative structure/activity relationships (QSAR) studies, several cytochrome P450or pharmacophore models from mammals were built either in
Correspondence to M Amichot, Unite´ Mixte de Recherche 1112,
Institut National de la Recherche Agronomique, 400 route des
Chappes, BP 167, 06903 Sophia Antipolis, France.
Fax: + 33 492386 401, Tel.: + 33 492386 409,
E-mail: amichot@antibes.inra.fr
Abbreviations: DDA, dichlorodiphenyl acetic acid; DDD,
dichlorodiphenyldichloroethane; DDT,
1,1,1-trichloro-2,2-bis-(4¢-chlorophenyl)ethane.
Database: The sequence of the CYP6A2vSVL allele has been
submitted to the GenBank database under the reference AY397730.
Enzyme: Monooxygenases including cytochromes P450(EC 1.14.14.1)
(Received 10 December 2003, revised 3 February 2004,
accepted 6 February 2004)
Trang 2relation with xenobiotic metabolism or with metabolism of
endogenous compounds [24,25] Models for CYP51 were
also built in Saccharomyces cerevisiae [26] and C albicans
[27] Cytochrome P450structure modeling was found useful
to explore the functional consequences of sequence
poly-morphisms [28–30] Many of these theoretical models
were validated by site-directed mutagenesis The majority
of the mutagenesis studies focused in the vicinity or inside
the substrate recognition sites (SRS [31]) These areas
were proposed to interact with the substrates of
cyto-chrome P450and thus to be responsible for the specificity of
the reactions catalyzed In insects, little information is
available about the structure of cytochrome P450 A recent
report established some structure-activity relationships in
CYP6B1v1, an insect cytochrome P450involved in
furano-coumarin metabolism [32]
Some years ago, we selected a D melanogaster strain
for its resistance to DDT we called RDDTR Its
resistance level (ratio of LD50of the strains) is extremely
high (> 10 000) [33]) We have shown that the expression
of Cyp6a2 was increased [34,35] and that several
cyto-chrome P450associated enzyme activities were modified
(DDT, testosterone, lauric acid, ecdysone, ethoxycoumarin
and ethoxyresorufin metabolism) [33,36] Three point
mutations (R335S, L336V and V476L) have been found
in the variant of CYP6A2 from this dithiothreitol-resistant
strain and preliminary studies suggested an effect of these
mutations on DDT metabolism [6] We have expressed
several CYP6A2 variants in bacteria to study the effect of
these mutations on CYP6A2 function The structure
of CYP102A1 that is the closest known P450 structure to
CYP6A2 was used to infer positional information on the
mutations
Experimental procedures
CYP6A2 site-directed mutagenesis and bacterial
expression
Site-directed mutagenesis followed the protocol previously
described [37] The first step of the mutagenesis on the
CYP6A2 cDNA (GenBank U78088) was the insertion of an
NdeI restriction site at the first ATG codon (oligonucleotide
CA1 5¢-AGCTACGCCATATGTTTGTT-3¢, the
substi-tuted nucleotides are in bold) to subclone the cDNA in the
pCW plasmid vector [38] We then introduced the mutation
F2A (oligonucleotide CA3 5¢-CGCCATATGGCTGTTC
TAATA-3¢) to increase expression of CYP6A2 in E coli
[39] This sequence is hereafter called the wild-type enzyme
(CYP6A2wt) and was used for further mutagenesis We
obtained four new variants: CYP6A2vS, CYP6A2vV,
CYP6A2vSV and CYP6A2vL using the oligonucleotides
SLR (CAGGACAGCCTGCGCAACGAG), RVR (CAG
GACAGGGTGCGCAACGAG), SVR (CAGGACAG
CGTGCGCAACGAG) and SLC (AGGGTATCCCTC
TGCGATACG), respectively All the alleles were inserted
in pCW between the NdeI and XbaI restrictions sites The
CYP6A2vSVL enzyme was built by the replacement of the
CYP6A2vSV HindIII-HindIII fragment by its homologue
from the CYP6A2vL allele which contained the mutation
The CYP6A2wt and all the mutants obtained were verified
by sequencing These constructions were transfected by
electroporation (Easyject, Eurogentec) in the E coli strain DH5a
Cytochrome P450 extraction The procedure was the same as described in [39] At the end
of the production, the cultures were chilled on ice (15 min) then centrifuged (4000 g, 15 min, 4C) Bacteria were resuspended by 10mL of TSE buffer [100mMTris pH 7.6,
1 mM EDTA, 1 mM phenylmethanesulfonyl fluoride, 30% (v/v) glycerol] including 250 lg of lysozyme Cells were lysed at 4C for 1 h The spheroplasts were pelleted (4000 g, 15 min, 4C) and kept overnight at)80 C The pellet was then resuspended in 10mL of spheroplast buffer [100 mM potassium phosphate pH 7.6, 6 mM magnesium acetate, 20% (v/v) glycerol, 0.1 mM dithiothreitol] and lysed by sonication (six series of 20s at 50W, 4C) Unlysed spheroplasts were pelleted by centrifugation (4000 g, 15 min, 4C) The sonication and centrifugation steps were repeated once more The supernatant was finally centrifuged at 100 000 g for 1 h (4C) and the pelleted membranes were resuspended in 1.25 mL of TSE buffer The preparations were aliquoted in 125 lL fractions and kept at )70 C until used Protein concentration was measured as described in [40] This process was also applied
to bacteria transformed with the pCW vector
CYP6A2 concentrations
In order to assess the stability of the CYP6A2wt and mutant enzymes, we measured the apoenzyme and the holoenzyme amount for each one of them The CYP6A2 apoenzyme amount in each sample was determined by Western blotting using anti-CYP6A2 Igs [12] and the ECL system (Amer-sham Pharmacia Biotech) The photographic film was scanned and submitted to densitometry analysis using the IMAGEJ 1.29 software (http://rsb.info.nih.gov/ij/) The results were expressed as arbitrary units per litre The holoenzyme concentrations were measured metrically [41] Data from densitometry and spectrophoto-metry measurements for each construction were divided by the relevant data obtained with the wild-type enzyme These operations, for each construction, gave normalized values for the apo- and holoenzymes which were then used to calculate the holoenzyme/apoenzyme ratio This ratio indicates the proportion of functional cytochrome P450 produced and is thus an index of its stability
Enzyme activities Each incubation initially contained house fly cytochrome b5 (1 nmol) [42], house fly cytochrome P450-reductase (1 nmol) [8], CHAPS (0.15%), dilauroylphosphatidyl cho-line (1 mgÆmL)1) and 100 pmoles of cytochrome P450 In the control experiments, we used 200 lg of membrane proteins from bacteria transformed with the pCW vector Membrane protein (200 lg) is the mean amount necessary
to get 100 pmol of CYP6A2vSVL, the least productive enzyme The enzyme mix was preincubated on ice for
15 min The reaction was then started by the addition of an NADPH regenerating system (80mMglucose-6-phosphate;
200 m NADP; 1 U glucose-6-phosphate dehydrogenase),
Trang 3the substrate, i.e either 0.5 lCi of [14C]4-4¢-DDT-Ring-UL
(82 mCiÆmmol)1, dissolved in ethanol; Amersham
Bio-sciences) or 0.25 lCi of [14C]testosterone (57 mCiÆmmol)1;
Sigma-Aldrich) and phosphate buffer (100 mM, pH 7.4) up
to 200 lL After 30min incubation at 30C, the reactions
were stopped by addition of 500 lL of methanol followed
by precipitation of the proteins and incubation at 4C for
15 min The mix was then centrifuged at 13 000 g for
15 min at 4C For DDT metabolism, 150 lL of the
supernatant were analyzed by HPLC [Column Altima C18,
5 lm Alltech (250· 4.6 mm) reverse phase] The mobile
phase consisted of a linear gradient from 50to 85% (v/v)
methanol in water, 0.2% (v/v) acetic acid (1.2 mL min)1)
DDT and its metabolites were detected with an in-line
Flow-one beta radioactivity detector (Radiomatic, Tampa,
FL, USA) We were not able to determine the Kmand Vmax
values because of the very high hydrophobicity of DDT
that did not allow an accurate determination of its effective
concentration The testosterone metabolites were resolved
as described earlier [36] using thin layer chromatography
[silica gel 60F254, Merck, first migration in
dichlorometh-ane/acetone (4 : 1; v/v), second migration in chloroform/
ethyl acetate/ethanol (40: 10: 7; v/v/v)] Cold markers
migrated alongside the testosterone metabolites After
autoradiography of the thin layer chromatography plates,
the metabolites were quantified by scraping the radioactive
areas and counting with a Wallac 1410counter
Substrate-induced binding spectra
Spectral titrations were conducted using a double-beam
spectrophotometer (Kontron Uvikon 860) with two
CYP6A2 enzymes: the wild-type and CYP6A2vSVL – the
one found in the RDDTRstrain Microlitre amounts (never
more than 1% of the final volume) of a dimethylsulfoxide
solution of DDT or of testosterone were added to the
experimental cuvette and an equal volume of
dimethylsulf-oxide to the reference cuvette so the final concentrations for
each ligand ranged from 10to 1000mM Each cuvette
contained 100 pmol of cytochrome P450 prepared as
des-cribed above (Cytochrome P450extraction) After the
addition of the substrate, the difference spectrum was
scanned from 375 to 500 nm We checked that
dimethyl-sulfoxide had no effect on the spectra The type of
substrate-induced binding spectra was determined by the positions
of the peak and the valley on the spectrum [43]
Sequence alignment The alignments between CYP6A2, CYP2C5, CYP2C9 and CYP102A1 were obtained with theCLUSTALXsoftware To obtain information about the spatial positions of the mutations, their similar positions were determined on the structure of CYP102A1, this protein is the most similar to CYP6A2 among those with a known structure The soft-ware used for this purpose wasDEEPVIEW/SWISS-PDBVIEWER
V3.7 (available at http://www.expasy.org/spdbv)
Results
Cytochrome P450 production inE coli The CYP6A2 apoenzyme was produced by the bacterial cells in lower amounts for all the enzymes than for CYP6A2wt but the differences had not statistical significant (Table 1) Most of the peptide was present in the membrane fraction but 20% of the total was soluble (data not shown) In addition, to address concerns over the produc-tion efficiency, the Western blots showed that no apo-enzyme degradation occurred during the preparation process (Fig 1) Furthermore, the apoenzymes have the same apparent molecular mass as the apoenzyme from Drosophilamicrosomes As the antibodies are polyclonal [12], we assume that they recognize equally the CYP6A2 enzymes tested here Spectral analysis of the preparations showed that there was a significant decrease in the specific contents of holoenzyme for the CYP6A2vSV and the CYP6A2vSVL enzymes (Table 1) The holoenzyme/apo-enzyme ratios of the variants, considered as a figure of the stability of the holoenzyme, are given in Table 1 Among the variants, those with a single substitution were the most stable (ratios ranging from 0.74 to 0.92) On the other hand, the CYP6A2vSV and the CYP6A2vSVL enzymes have a lower proportion of functional cytochrome P450(ratio values of 0.44 and 0.37, respectively) The membrane preparations from bacteria transformed with pCW did not reveal any cytochrome P450after a spectral analysis Metabolism studies
The low stability of the CYP6A2 mutants prevented us from purifying active CYP6A2 proteins to homogeneity and we used membrane preparations for metabolism studies
Table 1 Cytochrome P450 production in bacteria The apoenzyme and holoenzyme specific production of each mutant (mean ± SD, number of experiments in parentheses) was calculated For each mutant, the production of apoenzyme and holoenzyme was normalized relative to the wild-type enzyme The ratio of holoenzyme to apoenzyme normalized productions is an indication of the stability of the mutant.
Cytochrome
P450mutant
Apoenzyme production (arbitrary units)
Holoenzyme production (nmolÆL)1)
Normalized apoenzyme (production)
Normalized holoenzyme (production)
Holoenzyme/apoenzyme (normalized values)
* Statistically different from the reference (CYP6A2wt) (Dunnett test, P £ 0.01).
Trang 4instead First, we used testosterone to probe the activity
of the CYP6A2 enzymes All the variants were able to
hydroxylate testosterone to give a metabolite with no
significant differences in the specific activity for mutant
enzymes relative to CYP6A2wt (Table 2) We tentatively
identified this metabolite as 2b-hydroxy testosterone No
metabolism of testosterone was measured in control
experiments (bacteria with no plasmid or empty pCW)
The CYP6A2wt enzyme and four mutants metabolized
DDT to dicofol, DDD and DDA The CYP6A2vL variant
did not produce DDD in detectable amounts (Table 3)
The CYP6A2vV and CYP6A2vSV enzymes had the same
specific activity on DDT as the CYP6A2wt enzyme
Statistical analysis showed that only two enzymes were
significantly more efficient than CYP6A2wt in the
metabo-lism of DDT: CYP6A2vS and CYP6A2vSVL The former had a 4.79-fold higher specific activity than CYP6A2wt but only for dicofol production In contrast, the CYP6A2vSVL mutant had 8.59-, 5.81- and 21.00-fold higher specific activities than did CYP6A2wt for the production of dico-fol, dichlorodiphenyldichloroethane (DDD) and dichloro-diphenyl acetic acid (DDA), respectively Thus, only the CYP6A2vSVL enzyme, present in the insecticide resistant strain, was able to metabolize DDT efficiently As observed previously with testosterone, no metabolism of DDT was observed in control experiments
For each enzyme, dicofol is the major metabolite produced Nevertheless, a more careful analysis of the results demonstrated that the relative production of each metabolite varied among the enzymes Focusing on the enzymes with significant differences in the metabolism of DDT, i.e CYP6A2wt, CYP6A2vS and CYP6A2vSVL, the ratio dicofol : DDA (specific activities) is 70.00, 251.50 and 28.62, respectively, and the ratio DDD/DDA (specific activities) is 43.00, 62.25 and 11.90, respectively These variations of the ratios of the metabolites suggest modifi-cations in the catalytic mechanism responsible for the metabolism of DDT
Substrate binding The substrate induced binding spectra associated to DDT and to testosterone are type I spectra (data not shown) and
Fig 1 Production of the CYP6A2 variants in bacteria The lanes were
loaded with 5 mg of bacterial protein prepared as described in
Cyto-chrome P450extraction The arrow points to the CYP6A2 specific
signal, the star indicates unspecific signal observed in all lanes loaded
with bacterial protein The CYP6A2 variants have the same apparent
molecular mass as CYP6A2 from D melanogaster microsomes The
apoenzyme production varied among the variants No degradation
was observed for any of the apoenzymes.
Table 2 Specific production of 2b-hydroxy-testosterone by each of the CYP6A2 variants The mean ± SD and number of experiments (in parentheses) are presented for each variant No significant variation was observed relative to the specific activity of CYP6A2wt (Dunnett test, P > 0 0 5).
Cytochrome P450 mutant
Hydroxy-testosterone production (pmol per pmol P450per 30min) CYP6A2wt 5.39 ± 0.50 (3)
CYP6A2vS 5.40± 0.15 (3) CYP6A2vV 5.14 ± 0.12 (3) CYP6A2vL 5.44 ± 0.40 (3) CYP6A2vSV 3.86 ± 0.21 (3) CYP6A2vSVL 3.79 ± 0.95 (3)
Table 3 Specific production of the DDT metabolites by each of the CYP6A2 variants The mean ± SD and the number of experiments (in parentheses) are presented for each metabolite and variant Four specific activities are significantly different from the control, namely dicofol production by CYP6A2vS and DDA, and DDD and dicofol production by CYP6A2vSVL ND, not detected; NC, not calculated.
Membrane
preparation
DDT metabolite
pmol per pmol P450
per 30min
Ratio to CYP6A2wt
pmol per pmol P450 per 30min
Ratio to CYP6A2wt
pmol per pmol P450 per 30min
Ratio to CYP6A2wt CYP6A2wt 0.03 ± 0.02 (8) 1.00 1.29 ± 0.19 (8) 1.00 2.10 ± 1.19 (8) 1.00 CYP6A2vS 0.04 ± 0.03 (9) 1.33 2.49 ± 1.28 (9) 1.93 10.06 ± 5.80 (9)* 4.79 CYP6A2vV 0.05 ± 0.04 (8) 1.66 0.97 ± 0.46 (8) 0.75 3.27 ± 2.39 (8) 1.56
CYP6A2vSV 0.04 ± 0.03 (9) 1.33 2.19 ± 1.54 (9) 1.69 4.61 ± 2.46 (9) 2.19 CYP6A2vSVL 0.63 ± 0.42 (6)** 21.00 7.50 ± 5.17 (6)** 5.81 18.03 ± 11.66 (6)** 8.59
* Significantly different from CYP6A2wt P £ 0.05); ** significantly different from CYP6A2wt (P £ 0.001; Dunnett test).
Trang 5there was no qualitative difference between spectra obtained
with CYP6A2wt and CYP6A2vSVL For DDT, we did not
observe significant differences between the apparent affinity
of DDT to the CYP6A2 variants (Table 4) We found the
same qualitative results for testosterone and we concluded
that CYP6A2wt and CYP6A2vSVL bind DDT or
testo-sterone with the same apparent affinity
Sequence alignments and 3D localization
of the mutated positions
The sequence alignments of CYP6A2 with CYP2C5,
CYP2C9 and CYP102A1 are presented in Fig 2 R335S
is at a conserved position as a positive charge is found in the
four sequences L336V is at a position where an aliphatic
amino acid preferentially occurs, whereas, V476L is at a
nonconserved position The R335S and L336V mutations are located in helix J, and the V476L site is at the limit of the b3–3 sheet These structural elements are putative for CYP6A2 and deduced from the sequence alignment These three positions of CYP6A2 are similar to K289, A290and D425 of CYP102A1 Strikingly, these amino acids form a cluster distant from the active site, around the opening
of the pore containing helix I when placed on a spatial model (Fig 3) This cluster is located diametrically to the pole carrying the amino acids involved in substrate binding As far as we know, there has been no report about structure activity relationships in this area of the cytochrome P450s
Discussion
The D melanogaster insecticide resistant strain selected
in the laboratory, namely RDDTR, possesses a peculiar CYP6A2 enzyme: CYP6A2vSVL carrying three mutations Two are contiguous (R335S and L336V) and the third one (V476L) was found distal to the C451 which binds the heme
as described in preliminary work [6] We expressed five CYP6A2 mutants and the wild-type protein in bacteria to verify whether these mutations confer to CYP6A2 the ability to metabolize DDT First, the enzymes’ stability was addressed indirectly by spectrophotometry As deduced from the ratio of holoenzyme/apoenzyme (Table 1), the stability of the protein was affected by each of the mutations
Table 4 Apparent affinity of DDT and testosterone for CYP6A2wt and
CYP6A2vSVL For each apparent affinity calculated, the mean ± SD
and the number of experiments (in parentheses) are given Statistical
analysis of the results demonstrated that there was no significant
dif-ference among the values for each compound (t-test, P > 0 0 5).
DDT (l M ) Testosterone (l M ) CYP6A2wt 146 ± 31 (5) 100 ± 36 (5)
CYP6A2vSVL 173 ± 26 (5) 106 ± 47 (5)
Fig 2 Sequence alignments between CYP6A2, CYP102A1, CYP2C5 and CYP2C9 Identical or conserved amino acids in the four sequences are shaded black, identical or conserved amino acids in three sequences are shaded grey The secondary structures of CYP102A1 (labelled CYP102) are indicated below the alignment The mutations found in CYP6A2vSVL are indicated above the alignments and boxed.
Trang 6or their combination The CYP6A2vSV and CYP6A2vSVL
enzymes were particularly unstable We checked that this
was not the result of enhanced protein degradation This
first set of data demonstrates that these mutations alone or
in combination altered the stability of CYP6A2 It is likely
that this loss of stability is a consequence of structural
modifications in CYP6A2 As the CYP6A2vSVL enzyme is
the only one found in the insecticide-resistant Drosophila
strain, this suggests that the three mutations may be
important as a whole despite the instability they confer to
CYP6A2
Testosterone was found to be a useful substrate for
testing cytochrome P450activities in Drosophila as in
various other organisms All of the heterologously expressed
CYP6A2 enzymes were able to hydroxylate testosterone at
one position identified as 2b with no significant variation
in the specific activity As a consequence, we considered
testosterone hydroxylation at the 2b position as a
nondis-criminating activity for the CYP6A2 enzymes This activity
was already observed with Drosophila microsomes as one of
the major activities increased in the RDDTRstrain [36] As
CYP6A2 was able to hydroxylate testosterone only at
position 2b, it is likely that the other activities are carried by
additional cytochrome P450enzymes
CYP6A2wt was not able to metabolize DDT efficiently
In a previous work, no metabolism was detected [12]; this
may be explained by differences in the expression strategy
and metabolite analysis technique In contrast to what was
observed with testosterone, DDT metabolism clearly
dis-criminated the mutants The CYP6A2vSVL enzyme was
the most effective in the degradation of DDT to produce
dicofol, DDD and DDA These compounds are no longer efficient insecticides and dicofol is the main metabolite produced from DDT by microsomes from the DDT-resistant strain [33] As a first conclusion, these results strongly support that CYP6A2vSVL is a key enzyme for DDT metabolism and thus for resistance in the RDDTR strain Furthermore, the CYP6A2vSVL enzyme is also remarkable because the ratio of the DDT metabolites
it produces is different from the ratio observed for CYP6A2wt As the proportion of the metabolites produced
by an enzyme is a feature tightly associated to the catalytic mechanism, our results suggest that the active site may be modified or that the substrate may have access to the active site in different orientations according to the enzymes The apparent affinities of DDT and of testosterone for the CYP6A2wt and CYP6A2vSVL enzymes were assessed
to get more insights into the effects of the mutations on CYP6A2 We found no difference for these two substrates
in their apparent affinities for the CYP6A2 enzymes This was expected for testosterone as we did not observe any significant differences in its metabolism By contrast, DDT bonded equally to both enzymes although it was metabo-lized differently This suggests that the mutations induced
a modification of the catalytic properties of CYP6A2 that
is not caused by an alteration of substrate binding These results, taken together, suggest that the three mutations have a subtle effect on the structure of this cytochrome P450 and prompted us to address the spatial positions of the mutations According to the sequences alignment, the amino acids R335 and L336 would be in the
J helix and V476 near or in the b sheet 3–3 These structural
Fig 3 Ribbon image of the structure of CYP102A1 showing the positions of the amino acids similar to R335S, V336L and L476V of CYP6A2 In addition to the positions similar to those mutated in CYP6A2vSVL (blue), this figure also presents the amino acids interacting with the substrate (black, according to [44]) The heme is presented in green to localize the active site The I and J helices are labeled; the a helices are red and the b-sheets yellow.
Trang 7elements have not been yet studied for their role in the
structure or the activity of cytochromes P450 We
high-lighted the similar positions in the structure of CYP102A1
as previous sequence analysis placed CYP6A2 in the
same clan as CYP102A1 (http://drnelson.utmem.edu/
CytochromeP450.html) As evidenced from Fig 3, these
positions are far from the active site and from the amino
acids interacting with the substrate but clustered around the
distal end of the I helix As testosterone metabolism is only
slightly modulated in two variants among five, we can
exclude any effect of these mutations on the electron
transfer process To elucidate the mechanism by which
mutations on the J helix and in the vicinity of the b sheet 3–3
can affect the catalytic properties of CYP6A2, the building
of a structural model appears necessary
The only other case in which the structure/activity
relationships was questioned in relation to protein sequence
in an insect cytochrome P450is CYP6B1v1 This
cyto-chrome P450from Papilio polyxenes is involved in
furano-coumarin metabolism It has been demonstrated that three
amino acids are involved in protein structure stability (F116,
H117 and F484) and two in substrate specificity (F116 and
F484) This was achieved after sequence alignment analyses
and site-directed mutagenesis [32] These amino acids
belong to the SRS1 and SRS6 and these locations (junction
between helices B¢ and C, junction between b sheets 4–1 and
4–2) are very different from the locations of the mutated
positions in CYP6A2 (helix J and vicinity of the b sheet 3–3)
These results are also original because this is the first
description of the role of mutations in the metabolism of an
insecticide by a cytochrome P450enzyme Indeed, a recent
paper described CYP6G1 as responsible in D melanogaster
for the resistance against imidacloprid and DDT [1] but no
direct metabolism of these insecticides by CYP6G1 has been
documented to date Furthermore, as the resistance ratios
measured in these field-collected strains (< 30) are lower
than that observed with the lab-selected RDDTR strain
(> 10 000), it is likely that the resistance mechanisms are
different between imidacloprid resistant strains and
RDDTR, although both relies on cytochrome P450s
In conclusion, this work is the first to describe mutations
in an insect cytochrome P450that directly affect insecticide
metabolism These results demonstrate that CYP6A2vSVL
should have a major role in the metabolism of DDT
observed with microsomes of resistant Drosophila and thus
support the hypothesis that mutations can be a resistance
mechanism leading to high resistance levels Further work is
needed to clarify the relationships between mutations and
overexpression affecting cytochrome P450s and their
relat-ive importance in insecticide resistance From the
localiza-tion of the mutalocaliza-tions on a spatial model of CYP102A1,
we also point out a new region of cytochrome P450that
appears to be important for structure-activity relationships
The building of a homology model for CYP6A2 should be
helpful to further understand the effects of the three
mutations on the structure and activity of this enzyme
Acknowledgements
We are very grateful to Dr Waters and Dr Ganguly for providing us
with the full-length CYP6A2 cDNA, to Dr T Friedberg for providing
us with the pCW vector and to Dr Rahmani for the facilities to analyze
the DDT metabolites We also thank Dr R Feyereisen for the house fly cytochrome P450reductase and cytochrome b 5 cDNAs and for their fruitful discussions.
The sequence of the CYP6A2vSVL allele is available at GenBank under the reference AY397730.
References
1 Daborn, P.J., Yen, J.L., Bogwitz, M.R., Le Goff, G., Feil, E., Jeffers, S., Tijet, N., Perry, T., Heckel, D., Batterham, P., Feyer-eisen, R., Wilson, T.G & ffrench-Constant, R.H (2002) A single p450allele associated with insecticide resistance in Drosophila Science 297, 2253–2256.
2 Brandt, A., Scharf, M., Pedra, J., Holmes, G., Dean, A., Kreit-man, M & Pittendrigh, B (2002) Differential expression and induction of two Drosophila cytochrome P450genes near the Rst (2) DDT locus Insect Mol Biol 11, 337–341.
3 Scott, J.G & Wen, Z (2001) Cytochromes P450 of insects: the tip
of the iceberg Pest Manag Sci 57, 958–967.
4 Wilson, T.G (2001) Resistance of Drosophila to toxins Annu Rev Entomol 46, 545–571.
5 Feyereisen, R (1999) Insect P450enzymes Annu Rev Entomol.
44, 507–533.
6 Berge, J.B., Feyereisen, R & Amichot, M (1998) Cytochrome P450monooxygenases and insecticide resistance in insects Philos Trans R Soc Lond B Biol Sci 353, 1701–1705.
7 Scott, J.G., Liu, N & Wen, Z (1998) Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 121, 147–155.
8 Andersen, J.F., Utermohlen, J.G & Feyereisen, R (1994) Expression of house fly CYP6A1 and NADPH-cytochrome P450 reductase in Escherichia coli and reconstitution of an insecticide-metabolizing P450system Biochemistry 33, 2171–2177.
9 Andersen, J.F., Walding, J.K., Evans, P.H., Bowers, W.S & Feyereisen, R (1997) Substrate specificity for the epoxidation of terpenoids and active site topology of house fly cytochrome P450 6A1 Chem Res Toxicol 10, 156–164.
10 Sabourault, C., Guzov, V.M., Koener, J.F., Claudianos, C., Plapp, F.W Jr & Feyereisen, R (2001) Overproduction of a P450 that metabolizes diazinon is linked to a loss- of-function in the chromosome 2 ali-esterase (MdalphaE7) gene in resistant house flies Insect Mol Biol 10, 609–618.
11 Guzov, V.M., Unnithan, G.C., Chernogolov, A.A & Feyereisen,
R (1998) CYP12A1, a mitochondrial cytochrome P450from the house fly Arch Biochem Biophys 359, 231–240.
12 Dunkov, B.C., Guzov, V.M., Mocelin, G., Shotkoski, F., Brun, A., Amichot, M., Ffrench-Constant, R.H & Feyereisen, R (1997) The Drosophila cytochrome P450gene Cyp6a2: structure, locali-zation, heterologous expression, and induction by phenobarbital DNA Cell Biol 16, 1345–1356.
13 Cohen, M.B., Koener, J.F & Feyereisen, R (1994) Structure and chromosomal localization of CYP6A1, a cytochrome P450-encoding gene from the house fly Gene 146, 267–272.
14 Tomita, T., Liu, N., Smith, F.F., Sridhar, P & Scott, J.G (1995) Molecular mechanisms involved in increased expression of a cytochrome P450responsible for pyrethroid resistance in the housefly, Musca domestica Insect Mol Biol 4, 135–140.
15 Ingelman-Sundberg, M (2001) Genetic variability in susceptibility and response to toxicants Toxicol Lett 120, 259–268.
16 Guengerich, F.P., Parikh, A., Turesky, R.J & Josephy, P.D (1999) Inter-individual differences in the metabolism of environ-mental toxicants: cytochrome P4501A2 as a prototype Mutat Res 428, 115–124.
17 Eaton, D.L (2000) Biotransformation enzyme polymorphism and pesticide susceptibility Neurotoxicology 21, 101–111.
Trang 818 Lamb, D.C., Kelly, D.E., Schunck, W.H., Shyadehi, A.Z.,
Akh-tar, M., Lowe, D.J., Baldwin, B.C & Kelly, S.L (1997) The
mutation T315A in Candida albicans sterol 14 alpha-demethylase
causes reduced enzyme activity and fluconazole resistance through
reduced affinity J Biol Chem 272, 5682–5688.
19 Delye, C., Bousset, L & Corio-Costet, M.F (1998) PCR cloning
and detection of point mutations in the eburicol
14alpha-demethylase (CYP51) gene from Erysiphe graminis f Sp Hordei,
a Recalcitrant Fungus Curr Genet 34, 399–403.
20 Ravichandran, K.G., Boddupalli, S.S., Hasermann, C.A.,
Peter-son, J.A & Deisenhofer, J (1993) Crystal structure of
hemopro-tein domain of P450BM-3, a prototype for microsomal P450s.
Science 261, 731–736.
21 Hasemann, C.A., Ravichandran, K.G., Peterson, J.A &
Dei-senhofer, J (1994) Crystal structure and refinement of cytochrome
P450terp at 2.3 A˚ resolution J Mol Biol 236, 1169–1185.
22 Williams, P.A., Cosme, J., Sridhar, V., Johnson, E.F & McRee,
D.E (2000) Mammalian microsomal cytochrome P450
mono-oxygenase: structural adaptations for membrane binding and
functional diversity Mol Cell 5, 121–131.
23 Williams, P.A., Cosme, J., Ward, A., Angove, H.C., Matak
Vinkovic, D & Jhoti, H (2003) Crystal structure of human
cytochrome P4502C9 with bound warfarin Nature 424, 464–468.
24 de Groot, M.J & Ekins, S (2002) Pharmacophore modeling of
cytochromes P450 Adv Drug Deliv Rev 54, 367–383.
25 Lewis, D.F (2002) Molecular modeling of human cytochrome
P450–substrate interactions Drug Metab Rev 34, 55–67.
26 Lewis, D.F., Wiseman, A & Tarbit, M.H (1999) Molecular
modelling of lanosterol 14 alpha-demethylase (CYP51) from
Saccharomyces cerevisiae via homology with CYP102, a unique
bacterial cytochrome P450isoform: quantitative structure-activity
relationships (QSARs) within two related series of antifungal azole
derivatives J Enzyme Inhib 14, 175–192.
27 Holtje, H.D & Fattorusso, C (1998) Construction of a model of
the Candida albicans lanosterol 14-alpha-demethylase active site
using the homology modelling technique Pharm Acta Helv 72,
271–277.
28 Mathieu, A.P., Auchus, R.J & LeHoux, J.G (2002) Comparison
of the hamster and human adrenal P450c17 (17
alpha-hydro-xylase/17,20-lyase) using site-directed mutagenesis and molecular
modeling J Steroid Biochem Mol Biol 80, 99–107.
29 Li, D.N., Seidel, A., Pritchard, M.P., Wolf, C.R & Friedberg, T.
(2000) Polymorphisms in P450 CYP1B1 affect the conversion of
estradiol to the potentially carcinogenic metabolite
4-hydro-xyestradiol Pharmacogenetics 10, 343–353.
30 Stoilov, I., Akarsu, A.N., Alozie, I., Child, A., Barsoum-Homsy,
M., Turacli, M.E., Or, M., Lewis, R.A., Ozdemir, N., Brice, G.,
Aktan, S.G., Chevrette, L., Coca-Prados, M & Sarfarazi, M.
(1998) Sequence analysis and homology modeling suggest that
primary congenital glaucoma on 2p21 results from mutations
disrupting either the hinge region or the conserved core structures
of cytochrome P4501B1 Am J Hum Genet 62, 573–584.
31 Gotoh, O (1992) Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences J Biol Chem 267, 83–90.
32 Chen, J.S., Berenbaum, M.R & Schuler, M.A (2002) Amino acids in SRS1 and SRS6 are critical for furanocoumarin meta-bolism by CYP6B1v1, a cytochrome P450monooxygenase Insect Mol Biol 11, 175–186.
33 Cuany, A., Pralavorio, M., Pauron, D., Berge´, J.B., Fournier, D., Blais, C., Lafont, R., Salau¨n, J.P., Weissbart, D., Larroque, C & Lange, R (1990) Characterization of microsomal oxydative activities in a wild-type and in a DDT resistant strain of Drosophila melanogaster Pesticide Biochem Physiol 37, 293–302.
34 Amichot, M., Brun, A., Cuany, A., Helvig, C., Salau¨n, J.P., Durst,
F & Berge´, J.B (1994) In Cytochrome P450 8th International Conference (Lechner, M.C., eds), pp 689–692 John Libbey Eurotext, Paris, France.
35 Brun, A., Cuany, A., Le Mouel, T., Berge, J & Amichot, M (1996) Inducibility of the Drosophila melanogaster cytochrome P450gene, CYP6A2, by phenobarbital in insecticide susceptible
or resistant strains Insect Biochem Mol Biol 26, 697–703.
36 Amichot, M., Brun, A., Cuany, A., De Souza, G., Le Mouel, T., Bride, J.M., Babault, M., Salaun, J.P., Rahmani, R & Berge, J.B (1998) Induction of cytochrome P450activities in Drosophila melanogaster strains susceptible or resistant to insecticides Comp Biochem Physiol CPharmacol Toxicol Endocrinol 121, 311–319.
37 Kunkel, T.A., Bebenek, K & McClary, J (1991) Efficient site-directed mutagenesis using uracil-containing DNA Methods Enzymol 204, 125–139.
38 Gillam, E.M., Baba, T., Kim, B.R., Ohmori, S & Guengerich, F.P (1993) Expression of modified human cytochrome P4503A4
in Escherichia coli and purification and reconstitution of the enzyme Arch Biochem Biophys 305, 123–131.
39 Waterman, M.R., Jenkins, C.M & Pikuleva, I (1995) Genetically engineered bacterial cells and applications Toxicol Lett 82–83, 807–813.
40 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254.
41 Omura, T & Sato, R (1964) Tha carbon monoxyde binding pigment of liver microsomes I Evidence for its hemoprotein nature J Biol Chem 239, 2379.
42 Guzov, V.M., Houston, H.L., Murataliev, M.B., Walker, F.A & Feyereisen, R (1996) Molecular cloning, overexpression in Escherichia coli, structural and functional characterization of house fly cytochrome b 5 J Biol Chem 271, 26637–26645.
43 Jefcoate, C.R (1978) Measurement of substrate and inhibitor binding to microsomal cytochrome P-450by optical-difference spectroscopy Methods Enzymol 52, 258–279.
44 Li, H & Poulos, T.L (1997) The structure of the cytochrome p450BM-3 haem domain complexed with the fatty acid substrate, palmitoleic acid Nat Struct Biol 4, 140–146.