Point mutations associated with insecticide resistance in the
Drosophila
cytochrome P450
Cyp6a2
enable DDT metabolism
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 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 Escheri-
chia 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 pro-
teins, namely testosterone hydroxylation and DDT meta-
bolism. Testosterone was hydroxylated at the 2b position

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
P450 polymorphisms 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 P450 have 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 P450 from 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 P450 or
pharmacophore models from mammals were built either in

CYP6B1v1, an insect cytochrome P450 involved in furano-
coumarin metabolism [32].
Some years ago, we selected a D. melanogaster strain
for its resistance to DDT we called RDDT
R
.Its
resistance level (ratio of LD
50
of 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 P450 associated 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

EDTA, 1 m
M
phenylmethanesulfonyl fluoride,
30% (v/v) glycerol] including 250 lg of lysozyme. Cells
were lysed at 4 °C for 1 h. The spheroplasts were pelleted
(4000 g, 15 min, 4 °C) and kept overnight at )80 °C. The
pellet was then resuspended in 10 mL of spheroplast buffer
[100 m
M
potassium phosphate pH 7.6, 6 m
M
magnesium
acetate, 20% (v/v) glycerol, 0.1 m
M
dithiothreitol] and
lysed by sonication (six series of 20 s at 50 W, 4 °C).
Unlysed spheroplasts were pelleted by centrifugation
(4000 g,15min,4°C). The sonication and centrifugation
steps were repeated once more. The supernatant was finally
centrifuged at 100 000 g for 1 h (4 °C) 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

M
glucose-6-phosphate;
200 m
M
NADP; 1 U glucose-6-phosphate dehydrogenase),
Ó FEBS 2004 DDT metabolism by a mutant CYP6A2 (Eur. J. Biochem. 271) 1251
the substrate, i.e. either 0.5 lCi of [
14
C]4-4¢-DDT-Ring-UL
(82 mCiÆmmol
)1
, dissolved in ethanol; Amersham Bio-
sciences) or 0.25 lCi of [
14
C]testosterone (57 mCiÆmmol
)1
;
Sigma-Aldrich) and phosphate buffer (100 m
M
,pH7.4)up
to 200 lL. After 30 min incubation at 30 °C, the reactions
were stopped by addition of 500 lL of methanol followed
by precipitation of the proteins and incubation at 4 °Cfor
15 min. The mix was then centrifuged at 13 000 g for
15 min at 4 °C. For DDT metabolism, 150 lLofthe
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 50 to 85% (v/v)
methanol in water, 0.2% (v/v) acetic acid (1.2 mL min
)1

oxide to the reference cuvette so the final concentrations for
each ligand ranged from 10 to 1000 m
M
.Eachcuvette
contained 100 pmol of cytochrome P450 prepared as des-
cribed above (Cytochrome P450 extraction). 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 the
CLUSTALX
software. 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 was
DEEPVIEW
/
SWISS
-
PDBVIEWER
V3.7 (available at />Results
Cytochrome P450 production in
E. coli
The CYP6A2 apoenzyme was produced by the bacterial

type enzyme. The ratio of holoenzyme to apoenzyme normalized productions is an indication of the stability of the mutant.
Cytochrome
P450 mutant
Apoenzyme
production
(arbitrary units)
Holoenzyme
production
(nmolÆL
)1
)
Normalized
apoenzyme
(production)
Normalized holoenzyme
(production)
Holoenzyme/apoenzyme
(normalized values)
CYP6A2wt 15.2 ± 3.6 (3) 960 ± 800 (16) 1.00 1.00 1.00
CYP6A2vS 8.3 ± 5.5 (3) 390 ± 255 (5) 0.55 0.40 0.74
CYP6A2vV 12.9 ± 4.6 (3) 650 ± 250 (6) 0.85 0.67 0.79
CYP6A2vL 12.8 ± 3.8 (3) 750 ± 105 (3) 0.84 0.78 0.92
CYP6A2vSV 8.7 ± 4.2 (3) 245 ± 100 (11)* 0.57 0.25 0.44
CYP6A2vSVL 8.1 ± 6.7 (3) 190 ± 130 (8)* 0.53 0.20 0.37
* Statistically different from the reference (CYP6A2wt) (Dunnett test, P £ 0.01).
1252 M. Amichot et al. (Eur. J. Biochem. 271) Ó FEBS 2004
instead. 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

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 P450 extraction. 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
molecularmassasCYP6A2fromD. 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 >0.05).
Cytochrome P450
mutant
Hydroxy-testosterone production
(pmol per pmol P450 per 30 min)
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)

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,
A290 and 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 RDDT
R
, possesses a peculiar
CYP6A2 enzyme: CYP6A2vSVL carrying three mutations.
Two are contiguous (R335S and L336V) and the third one

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 P450 activities 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 RDDT
R
strain [36]. As
CYP6A2 was able to hydroxylate testosterone only at
position 2b, it is likely that the other activities are carried by
additional cytochrome P450 enzymes.
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-

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.
Ó FEBS 2004 DDT metabolism by a mutant CYP6A2 (Eur. J. Biochem. 271) 1255
elements 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 ( />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 P450 is CYP6B1v1. This cyto-
chrome P450 from 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

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 P450 reductase and cytochrome b
5
cDNAsandfortheir
fruitful discussions.
The sequence of the CYP6A2vSVL allele is available at GenBank
under the reference AY397730.
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