Substrate specificity for the epoxidation of terpenoids and active site topology of house fly cytochrome P450 6A1

Article abstract of DOI:10.1021/tx9601162

Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450 6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids. In addition to the epoxidation of cyclodiene insecticides demonstrated previously [Andersen et al. (1994) Biochemistry 33, 2171-2177], this cytochrome P450 was shown to epoxidize a variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-cytochrome P450 reductase and phosphatidylcholine did not metabolize α-pinene, limonene, or the insect growth regulators hydroprene and methoprene. The four geometric isomers of methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also to the 6,7-epoxides and to the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxides of methyl farnesoate were metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited by a bulky substituted imidazole. The active site topology of P450 6A1 was studied by the reaction of the enzyme with phenyldiazene to form a phenyl-iron complex. Ferricyanide-induced in situ migration of the phenyl group showed formation of the N-phenylprotoporphyrinporphyrin IX adducts in a 17:25:33:24 ratio of the N(B):N(A):N(C):N(D) isomers. These experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed in insecticide-resistant strains of the house fly, is not severely limited by stereochemically constrained access to the active site.

Full text of DOI:10.1021/tx9601162

156
Chem. Res. Toxicol. 1997, 10, 156-164
Su bstr a te Sp ecificity for th e Ep oxid a tion of Ter p en oid s
a n d Active Site Top ology of Hou se F ly Cytoch r om e P 450
6A1
J ohn F. Andersen, J ennifer K. Walding, Philip H. Evans, William S. Bowers, and
Rene´ Feyereisen*
Department of Entomology, University of Arizona, Tucson, Arizona 85721
Received J uly 10, 1996X
Heterologous expression in Escherichia coli, purification, and reconstitution of house fly P450
6A1 and NADPH-cytochrome P450 reductase were used to study the metabolism of terpenoids.
In addition to the epoxidation of cyclodiene insecticides demonstrated previously [Andersen
et al. (1994) Biochemistry 33, 2171-2177], this cytochrome P450 was shown to epoxidize a
variety of terpenoids such as farnesyl, geranyl, and neryl methyl esters, juvenile hormones I
and III, and farnesal but not farnesol or farnesoic acid. P450 6A1 reconstituted with NADPH-
cytochrome P450 reductase and phosphatidylcholine did not metabolize R-pinene, limonene,
or the insect growth regulators hydroprene and methoprene. The four geometric isomers of
methyl farnesoate were metabolized predominantly to the 10,11-epoxides, but also to the 6,7-
epoxides and to the diepoxides. The 10,11-epoxide of methyl (2E,6E)-farnesoate was produced
in a 3:1 ratio of the (10S) and (10R) enantiomers. Monoepoxides of methyl farnesoate were
metabolized efficiently to the diepoxides. Methyl farnesoate epoxidation was strongly inhibited
by a bulky substituted imidazole. The active site topology of P450 6A1 was studied by the
reaction of the enzyme with phenyldiazene to form a phenyl-iron complex. Ferricyanide-
induced in situ migration of the phenyl group showed formation of the N-phenylprotopor-
phyrinporphyrin IX adducts in a 17:25:33:24 ratio of the NB:NA:NC:ND isomers. These
experiments suggest that metabolism of xenobiotics by this P450, constitutively overexpressed
in insecticide-resistant strains of the house fly, is not severely limited by stereochemically
constrained access to the active site.
juvenile hormone (Figure 1). Interest in these types of
compounds, long considered to be highly selective to
insects, has increased following the reports that insect
juvenile hormone III and farnesol activate the het-
erodimeric complex between the retinoid X receptor and
an orphan receptor (FXR) from mammals (6) and that
the juvenile hormone analog methoprene and its me-
tabolite methoprene acid specifically bind and activate
the retinoid X receptor (7).
In tr od u ction
CYP6A1 is a cytochrome P450 (P450)1 gene that is
constitutively overexpressed in some insecticide-resistant
strains of the house fly, Musca domestica (1, 2). When
produced in Escherichia coli and reconstituted with
NADPH-cytochrome P450 reductase, CYP6A1 (P450 6A1)
carries out the epoxidation of the cyclodiene insecticides
aldrin and heptachlor (3). We felt that a more extensive
study of the substrate specificity of P450 6A1 would
contribute to our understanding of insecticide resistance
and metabolic detoxification in insects and extend our
understanding of P450 substrate specificity in general.
Insecticide-resistant strains which overexpress the
CYP6A1 gene are cross-resistant to synthetic structural
analogs of juvenile hormone (4), some of which are
commercially produced as insect growth regulators.
Furthermore, several studies have reported the oxidative
metabolism of juvenile hormone I [methyl (2E,6E,10Z)-
10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate] and
of insect growth regulators in insecticide-resistant strains
of the house fly, in addition to the well-described hydro-
lytic pathways involving juvenile hormone esterase and
epoxide hydrolase (5). It was therefore possible that P450
6A1 would metabolize terpenoid compounds related to
We have used a reconstituted system of P450 6A1,
NADPH-cytochrome P450 reductase and phospholipid to
characterize the regio- and stereoselectivity of the ep-
oxidation reactions of a number of terpenoids. We show
here that P450 6A1 metabolizes juvenile hormones I and
III to their diepoxides. The sequential epoxidations of
methyl farnesoate and related compounds by P450 6A1
have also allowed us to assess the features of the
substrate binding site of this enzyme by examining the
amount and type of products formed as a function of
chain length, geometric isomerism, and substitution
pattern. The regio- and stereochemistries of olefin ep-
oxidation observed in this study indicate that the vicinity
of the heme active site is quite spacious and can accom-
modate a variety of substrate geometries and orienta-
tions.
* Corresponding author: Department of Entomology, Forbes 410,
University of Arizona, Tucson, AZ 85721.
X Abstract published in Advance ACS Abstracts, J anuary 1, 1997.
Exp er im en ta l P r oced u r es
1
Abbreviations: P450, cytochrome P450; P450 6A1, CYP6A1;
BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; CHAPS, 3-[(3-chola-
midopropyl)dimethylammonio]-1-propanesulfonate; DTT, dithiothrei-
tol; EI-MS, electron impact mass spectrometry; MOPS, 4-morpho-
linepropanesulfonic acid; PCR, polymerase chain reaction; PMSF,
p-phenylmethanesulfonyl fluoride.
En zym e P r ep a r a tion . P450 6A1 was expressed in E. coli
as described previously (3) and purified from the membrane
fraction by a combination of hydrophobic interaction chroma-
tography on ω-aminooctyl agarose and chromatography on
S0893-228x(96)00116-6 CCC: $14.00 © 1997 American Chemical Society

P450 6A1 Metabolism of Terpenoids
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 157
purified material by chromatography on a small column of
DEAE-Sepharose as described above for P450 6A1.
Cytochrome P450 was quantified by measurement of its
dithionite-reduced vs reduced-CO bound difference spectrum by
the method of Omura and Sato (9). Spectra were taken at 4 °C
on a Perkin-Elmer Lambda 19 UV-visible spectrophotometer.
Previous studies (3) have shown that the reduced CO complex
of P450 6A1 is labile at higher temperatures. Reductase activity
was quantified by measurement of the reduction of cytochrome
c as described previously. Ligand binding was quantified by
measurement of type I or type II difference spectra (for the
ligands with an imidazole moiety) of oxidized cytochrome P450.
The spectra were measured in 100 mM 4-morpholinepropane-
sulfonic acid (MOPS) buffer (pH 7.4) at 25 °C, and the ligands
were added in ethanol. An equivalent amount of ethanol was
added to the reference cuvette, and the maximal amount of
ethanol did not exceed 1% of the total volume.
P r ep a r a tion of Su bstr a tessMeth yl (2E,6E)-F a r n esoa te,
Met h yl (2Z,6E)-F a r n esoa t e, Met h yl (2E,6Z)-F a r n esoa t e
a n d Meth yl (2Z,6Z)-F a r n esoa te. These compounds were
synthesized via the Wittig reaction (10). Trimethyl phospho-
noacetate (4.7 g) was added dropwise to 12 mL of dry dimeth-
ylformamide containing 1.5 g of NaH (60%) and stirred at room
temperature for 30 min. trans-Geranylacetone (5 g) in 12 mL
of dry dimethylformamide was addded dropwise and the result-
ant mixture was stirred at room temperature for 18 h. Water
(200 mL) was added, and the aqueous mixture was extracted
with hexane (2 × 100 mL). The organic extract was washed
SO4.
with water and brine and dried over anhydrous Na2
F igu r e 1. Terpenoid substrates tested as substrates with P450
6A1: (A) hydroprene; (B) methoprene; (C) juvenile hormone I;
(D) juvenile hormone III; (E) methyl (2E,6E)-farnesoate; (F)
methyl geranate; (G) (2E,6E)-farnesal; (H) (2E,6E)-farnesol; (I)
(2E,6E)-farnesoic acid.
Evaporation of the solvent yielded 3.1 g of a mixture of methyl
(2E,6E)-farnesoate (42.5%) and methyl (2Z,6E)-farnesoate (22.5%)
as indicated by gas chromatographic comparison with authentic
standards. After chromatography on 5% water-deactivated
Florisil (5% diethyl ether in hexane), the geometric isomers were
separated by centrifugal planar chromatography on a Chroma-
totron apparatus using a 2-mm silica gel layer eluted with 2%
diethyl ether in hexane at a flow rate of 6 mL/min. The
identities of the products were determined by GC/MS. Methyl
(2E,6Z)-farnesoate and methyl (2Z,6Z )-farnesoate were pre-
pared in an identical manner using cis-geranylacetone in place
of trans-geranylacetone. The yield in this case was 4.3 g of a
mixture containing 52 (2Z,6Z) and 26% (2Z,6E). Each of the
separated isomers was 97-99% pure as measured by GC/MS,
on a Hewlett-Packard 5890 gas chromatograph containing a 15
m × 0.3 mm i.d. HP-1 fused silica column, coupled with a
Hewlett-Packard 5970 mass-selective detector. The standard
temperature program was a 1 min hold at 100 °C followed by
an increase of 10 °C/ min to 250 °C. Under our GC conditions,
the retention times (tR) of the methyl farnesoates were 10.13,
9.85, 9.64, and 9.29 min, respectively, for the (2E,6E), (2E,6Z),
(2Z,6E), and (2Z,6Z) isomers.The olefinic methyl esters were
stored as neat liquids under nitrogen at -20 °C.
Meth yl Ger a n a te a n d Meth yl Ner a te. Active MnO2 (10g)
was added to 1.0 g of citral (65% geranial, 35% neral) in 125
mL of hexane and stirred at 0 °C for 30 min. The mixture was
filtered to remove MnO2, and the hexane evaporated. Acetic
acid (0.3 g), 0.8 g of NaCN, and 10 g of MnO2 in methanol were
added to the residue, and the mixture was stirred at room
temperature for 4 h. The mixture was filtered to remove MnO2,
and the methanol was evaporated. The residue was dissolved
in ethyl ether and washed with water and brine to give 0.57 g
of a 65:35 mixture of geranyl methyl ester and neryl methyl
ester. The mixture was purified by flash chromatography on
silica (10% ether in hexane), and the products were identified
by GC/MS. Retention times for methyl geranate and methyl
nerate were 4.43 and 3.85 min, respectively, under the condi-
tions described above.
DEAE-Sepharose. E. coli (pI-13C) cells were harvested, lysed
by sonication, and fractionated by centrifugation as described
previously. P450 6A1 was solubilized from membranes by
stirring with 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate (CHAPS) and the suspension was centrifuged
at 100000g. The supernatant was loaded onto an ω-aminooctyl
agarose column equilibrated with 100 mM sodium phosphate
buffer (pH 7.4) containing 0.2 mM dithiothreitol (DTT), 0.4%
sodium cholate, and 20% glycerol (buffer A). The column was
washed with buffer A, and P450 6A1 was eluted with buffer B
[10 mM sodium phosphate buffer (pH 7.4), 0.2 mM DTT, 0.4%
sodium cholate, 0.2% Emulgen 911, 20% glycerol]. The reddish
fractions were pooled and loaded onto a 20-mL column of DEAE-
Sepharose equilibrated with buffer C [10 mM Tris-HCl buffer
(pH 7.6), 0.2 mM DTT, 0.1% Emulgen 911], eluted with a
gradient of buffer C containing 0-0.3 M NaCl, and then dialyzed
against buffer C. Detergents were removed prior to use by
applying the purified P450 to a small DEAE-Sepharose column,
washing with buffer D [10 mM Tris-HCl buffer (pH 7.6), 0.2
mM DTT, 20% glycerol] until the absorbance of the eluent at
280 nm was equal to that of the wash buffer, and eluting with
buffer D containing 0.3 M NaCl. The specific content of P450
in these preparations was 12 nmol/mg of protein.
House fly NADPH-cytochrome P450 reductase (8) was also
expressed in E. coli and purified by a combination of chroma-
tography on DEAE-Sepharose followed by chromatography on
phenyl-Sepharose. Membranes were collected from E. coli (pI-
95), and proteins were solubilized with CHAPS as described
previously (3). The solubilized material was loaded onto a
DEAE-Sepharose column equilibrated with buffer E [25 mM
Tris-HCl buffer (pH 7.4) containing 0.5 mM DTT, 0.1% Emulgen
911, 10% glycerol]. The column was washed with buffer A and
eluted with a gradient of 0-0.4 M NaCl in buffer A. The yellow
fractions were pooled, diluted 2-fold with buffer F [20 mM Tris-
HCl buffer (pH 7.6), 0.5 mM DTT, 1.0 mM EDTA, 10% glycerol],
and loaded onto a column of phenyl-Sepharose equilibrated with
buffer F. The column was washed with buffer F and eluted with
a gradient from 0% detergent to 0.8% cholate and 0.15%
Emulgen 911 in buffer F. Detergents were removed from the
Mon o- a n d Diep oxid es of Meth yl F a r n esoa te. The
epoxides were synthesized by m-chloroperoxybenzoic acid oxida-
tion (11, 12). A 1.2 molar excess of m-chloroperoxybenzoic acid
was added to 75 mg each of methyl (2E,6E)- and (2Z,6E)-
farnesoate in 4 mL of methylene chloride. The reaction was
stirred on ice for 30 min and stopped by adding 8 mL of 5%

158 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Andersen et al.
Ta ble 1. Meta bolism of Ter p en oid Su bstr a tes by P 450
6A1
aqueous sodium bicarbonate. The organic phase was collected,
washed with water, and dried over anhydrous sodium sulfate.
Silica gel thin-layer chromatography developed with benzene/
ethyl acetate (85:15) showed the presence of products corre-
sponding to unreacted methyl farnesoate, its 10,11-epoxide, its
6,7-epoxide, and its 6,7,10,11-diepoxide. The epoxides were
preparatively separated by silica gel TLC using the same solvent
system, and the products were eluted from silica gel with ethyl
acetate. The purities of these isolated substrates, measured by
GC/MS, were as follows: methyl (2Z)-6,7-epoxyfarnesoate 95%
(tR 10.79 min); methyl (2Z)-10,11,-epoxyfarnesoate 99% (tR 10.74
min); methyl (2E)-6,7-epoxyfarnesoate 85% (tR 11.28 min);
methyl (2E)-10,11-epoxyfarnesoate 96% (tR 11.17 min). The
epoxides were stored in ethanol at -20 °C.
Deter m in a tion of th e Absolu te Con figu r a tion of 10,11-
Ep oxid es of Meth yl (2E,6E)-F a r n esoa te. Optically pure
juvenile hormone III [methyl (2E,6E )(10R )-10,11-epoxyfarne-
soate] was prepared by incubating excised corpora allata from
5-day-old adult mated female Diploptera punctata in TC199
medium containing 50 µM methionine (13). Thirty pairs of
corpora allata were incubated at 30 °C for 24 h. The incubation
medium was then extracted with five volumes of isooctane. The
isooctane extract was dried over anhydrous Na2SO4 and con-
centrated under a stream of nitrogen. J uvenile hormone III was
identified by comparison of its mass spectrum and retention
time with an authentic sample of racemic hormone.
products (nmol of product)-1
(nmol of P450)-1 min-1 b
substratea
hydroprene
10,11-epoxide 6,7-epoxide
other
ndc
nd
methoprene
juvenile hormone I
juvenile hormone III
methyl (2E,6E)-
farnesoate
0.96 ( 0.08 1.33 ( 0.21d
4.18 ( 0.19 1.83 ( 0.25d
2.84 ( 0.20 0.54 ( 0.03 0.71 ( 0.18e
methyl geranatef
(2E,6E)-farnesal
(2E,6E)-farnesol
(2E,6E)-farnesoic
acid
3.10 ( 0.15 nd
1.65 ( 0.30 0.89 ( 0.02 nd
nd
nd
nd
nd
nd
nd
R-pinene
limonene
nd
nd
a
b
All substrates at nominal concentration of 50 µM. (nmol of
product formed)-1 (nmol of P450)-1 (min-1 ( SE (mean of three
determinations). Time of incubation 20 min. c nd, no product
detected. Approximate limit of detection is 0.1% conversion to
d
product or
a
rate of 0.03. Unidentified product. e 6,7,10,11-
Diepoxide product. f 65:35 mixture of geranyl and neryl methyl
esters.
Supelco LC-18DB column (5 µm, 25 × 0.46 cm), with a 30-min
isocratic elution with 70% A/30% B, where solvent B was 10:1
methanol/acetic acid. This was followed by a 1-min gradient to
100% B and isocratic elution at 100% B for 19 min. The flow
rate was 1.5 mL/min, and absorbance of the eluant was
monitored at 416 nm with a Perkin-Elmer LC95 spectropho-
tometer. Fresh stock solutions of potassium ferricyanide and
phenyldiazene (prepared by adding 2.5 µL of phenyldiazenecar-
boxylate azo ester to 200 µL of fresh 1 N NaOH) were stored on
ice. The procedure was verified by preparing the adducts of
horse heart myoglobin under the same conditions.
Derivatives of the racemic 10,11-epoxide of methyl (2E,6E)-
farnesoate, of the reaction product with P450 6A1, or of the
product of cultured corpora allata were prepared as follows. The
epoxides were first converted to the methoxyhydrin by acid
methanolysis (14). The conversion to methoxyhydrin was
quantitative as indicated by GC/MS (EI-MS m/ z 266, 225, 193,
135, 73). The methoxyhydrins were reacted with (S )-(+)-R-
methoxy-R-(trifluoromethyl)phenylacetyl chloride in pyridine at
60 °C for 1 h. The diastereomeric esters were then analyzed
by capillary gas chromatography on a 15 m × 0.32 mm i.d. DB-5
fused silica capillary column. The temperature program was a
1-min hold at 170 °C followed by an increase of 5 °C/ min to
300 °C. The identity of the esters (tR 21.80 and 21.91 min) was
verified by GC/MS (70 eV EI-MS m/ z 248, 189, 135, 73).
Resu lts
En zym a tic Rea ction s. Enzymatic assays were performed
in siliconized glass tubes containing 10 µg of dilaurylphosphatid-
ylcholine, 1.2 mM NADP+, 100 mM glucose 6-phosphate, 0.5
unit of glucose-6-phosphate dehydrogenase, 0.03 nmol of NADPH-
cytochrome P450 reductase, and 0.017 nmol of P450 6A1 in 100
mM MOPS buffer. Nonionic detergent concentration remaining
from the purification procedure was below the spectrophoto-
metric detection limit. The reactions were started by the
addition of substrate in 1-2 µL of ethanol and brief vortexing.
The total volume of the reactions was 200 µL. After 20-30 min,
the reactions were stopped and the products extracted with
isooctane.
Meta bolism of Ter p en oid s by P 450 6A1/Red u c-
ta se Recon stitu ted System . The insect growth regula-
tors methoprene and hydroprene were not metabolized
by P450 6A1 in our reconstituted system (Table 1). Both
compounds are characterized by a pair of double bonds
conjugated to an alkyl ester. However, the insect juvenile
hormones that these terpenoids were designed to mimic
were metabolized to the diepoxides at their unconjugated
6,7-double bond.
J uvenile hormone III (tR 11.17 min) and its homolog
juvenile hormone I (tR 12.81 min), possessing ethyl
branches at positions 6 and 11, were each metabolized
to two products. The earlier eluting peak in each case
was the 6,7,10,11-diepoxide (Table 1) (tR 12.16 and 13.83
min, respectively) (the hormones are themselves 10,11-
epoxides). For juvenile hormone I, the mass spectrum
of the diepoxide showed characteristic ion fragments at
m/ z 125, 114, 109, 107, 95, and 82. The identity of the
later eluting peak (tR 14.22 and 12.79 min, respectively)
obtained with juvenile hormones I and III was not
rigorously determined. Relative to the diepoxide, it was
produced in smaller amounts with juvenile hormone III
than with juvenile hormone I (Table 1). This product did
not result from thermal rearrangement of the 6,7,10,11-
diepoxide in the gas chromatograph, because injection
of the synthetic diepoxide did not result in its formation.
The metabolite did not form a trimethylsilyl derivative
on reaction with N,O-bis(trimethylsilyl) trifluoroaceta-
mide (BSTFA), and its infrared spectrum showed no
indication of hydroxylation or carbonyl formation. With
Products of enzymatic reactions were analyzed by GC/MS on
a HP-1 fused silica column as described above, and product
quantitation was done by integrating the peak areas in the total
ion chromatogram. The relative responses to methyl farnesoate
and its mono- and diepoxides were determined using authentic
standards of these compounds.
F or m a tion a n d An a lysis of N-P h en ylp r otop or p h yr in IX
Isom er s. The formation of an iron-phenyl complex of P450
6A1 and the subsequent formation and HPLC analysis of
N-phenylprotoporphyrin IX adducts were adapted from the
procedures outlined by Tuck et al. (15). Briefly, 5 nmol of P450
6A1 in 10 mM sodium phosphate buffer (pH 7.4) containing 1
mM DTT, 0.4% sodium cholate, 0.2% Emulgen 911, and 20%
glycerol was reacted with 0.8 mM phenyldiazene for 15 min at
15 °C. The solution was oxidized with 1 mM potassium
ferricyanide for 2 min at 15 °C. The mixture was added to 5
mL of 5% aqueous sulfuric acid and kept at 4 °C for 2-4 h. The
porphyrin adducts were extracted with dichloromethane, the
solvent was removed, and the residue was dissolved in HPLC
solvent A (6:4:1 methanol/water/acetic acid). HPLC separation
of the N-phenylprotoporphyrin IX isomers was done on
a

P450 6A1 Metabolism of Terpenoids
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 159
F igu r e 2. Conversion of methyl (2E,6E)-farnesoate to its mono- and diepoxides by P450 6A1. Molecular ions and fragmentation
pattern observed by mass spectrometry are indicated for each compound.
juvenile hormone III, the product gave a mass spectrum
with a molecular ion at m/ z 282, a base ion at m/ z 165,
and other prominent ions at m/ z 71, 85, and 114. The
fragments at m/ z 71 and 85 were attributed to cleavages
R and â to the 10,11-epoxy group, which indicate that
this moiety was intact. The fragment at m/ z 114 showed
that no modification of the 2,3-unsaturation occurred.
The infrared spectrum of the unknown compound was
nearly identical to that of the 6,7,10,11-diepoxide, sug-
gesting that the product may be a rearranged diepoxide.
The product seen with juvenile hormone I showed ions
at m/ z 85, 99, and 179 that were homologous to the
fragments at m/ z 71, 85, and 165, respectively, in the
juvenile hormone III product and were indicative of the
ethyl side chains.
The sesquiterpenoid aldehyde (2E,6E)-farnesal was
metabolized to the 6,7-and 10,11-monoepoxide products
(tR 10.69 and 10.63 min) in a 1:2 ratio (Table 1). Unlike
the aldehyde, the sesquiterpenoid alcohol (2E,6E)-farne-
sol was not metabolized by P450 6A1, nor was the
corresponding carboxylic acid, farnesoic acid (Table 1).
P r od u cts fr om Meth yl F a r n esoa te a n d Its Rela -
tives. Gas chromatographic analysis showed that meth-
yl (2E,6E)-farnesoate was converted to three detectable
products by P450 6A1 (Table 1, Figure 2). The mass
spectrum of each contained an ion at m/ z 114 (CH2C-
(CH3)dCHC(OH)OCH3+) that is a characteristic fragment
in EI-MS spectra of farnesyl methyl esters (16). The
major product showed prominent ions at m/ z 71 and 85
which are indicative of cleavage R and â to a 10,11-epoxy
group in the farnesyl skeleton and suggested that this
compound was the 10,11-epoxide of methyl farnesoate.
Chromatographic and spectral comparison with an au-
thentic sample of methyl (2E,6E )-10,11-epoxyfarnesoate
verified this assignment. The optically pure (10R) form
of this compound is insect juvenile hormone III.
The second product peak, less prominent than the
10,11-epoxide and with a slightly longer retention time,
showed a base ion at m/ z 69 in its EI-MS and showed
ions at m/ z 95 and 135. These spectral features are
indicative of a lack of modification of the terminal prenyl
unit of the farnesyl skeleton and would be consistent with
the 6,7-epoxide of methyl (2E,6E)-farnesoate (Table 1).
Comparison with an authentic standard verified this
assignment. The third peak showed an apparent molec-
ular ion at m/ z 282 with major fragment ions at m/ z
71, 85, 93, 108, and 111. This molecular mass and
fragmentation pattern are suggestive of the 6,7,10,11-
diepoxide of methyl (2E,6E)-farnesoate, and a sample of
synthetic diepoxide was used to verify this assignment
(Table 1). The (6S,7S,10R)-enantiomer of this compound
is the major product secreted by the corpora allata, and
may be the active juvenile hormone, in a number of
species of higher Diptera (17).
It is clear from these data that methyl farnesoate is
converted to its 6,7,10,11-diepoxide through a monoep-
oxide intermediate (Figure 2). The major intermediate,
based on its abundance in the reaction mixtures is methyl
(2E,6E) 10,11-epoxyfarnesoate (Table 1). When the
conversion of methyl (2E,6E)-farnesoate to mono- and
diepoxide was examined as a function of substrate
concentration, it was found that the amount of diepoxide
in the total product increased with increasing substrate
concentration (Figure 3).

160 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Andersen et al.
Ta ble 2. Con ver sion of Geom etr ic Isom er s of Meth yl
F a r n esoa te to Ep oxid e P r od u cts by P 450 6A1a
products (nmol of product)-1 (nmol of P450)-1 min-1
substrate
isomer
10,11-epoxide
6,7-epoxide
diepoxide
(2E,6E)
(2Z,6E)
(2Z,6Z)
(2E,6Z)
2.84 ( 0.20b
1.76 ( 0.30c
1.93 ( 0.20
2.83 ( 0.10
0.54 ( 0.03
c
0.45 ( 0.04
1.81 ( 0.07
0.71 ( 0.18
0.24 ( 0.04
0.43 ( 0.05
0.65 ( 0.08
a
The four geometric isomers of methyl farnesoate at a nominal
concentration of 50 µM were incubated with 85 nM P450 6A1 for
b
20 min, and the products were analyzed by GC/MS. Mean of
three determinations ( SE. c 10,11- and 6,7-epoxides were not
separated on GC but mass spectrum indicated mainly 10,11-
epoxide.
Ta ble 3. Con ver sion of Meth yl F a r n esoa te Ep oxid es to
Diep oxid es by P 450 6A1a
F igu r e 3. Production of mono- and diepoxide from methyl
(2E,6E)-farnesoate by P450 6A1 in relation to the concentration
of substrate: (9) 6,7-monoepoxide; (b) 10,11-monoepoxide; (0)
diepoxide. The P450 6A1 concentration was 85 nM, and the
reaction products were measured after 30 min. Each point is
the mean of two separate experiments.
substrate
diepoxidea
methyl (2E,6E )-6,7-epoxyfarnesoate
methyl (2E,6E )-10,11-epoxyfarnesoate
methyl (2Z,6E )-6,7-epoxyfarnesoate
methyl (2Z,6E )-10,11-epoxyfarnesoate
41.7 ( 8.2b
14.0 ( 2.9c
17.0 ( 0.3
11.8 ( 0.7
a
The four geometric isomers of methyl farnesoate at a nominal
concentration of 250 µM were incubated with 85 nM P450 6A1
b
for 20 min, and the products were analyzed by GC/MS. (nmol of
product formed)-1 (nmol of P450)-1 min-1
.
c Mean of three deter-
d
minations ( SE. In these experiments, conversion of methyl
(2E,6E )-10,11-epoxyfarnesoate to the unknown metabolite (see
Table 1) was less than 2%. The other substrates were not
metabolized to metabolites other than the diepoxide.
farnesoate is also converted mainly to 10,11-epoxide,
methyl (2E,6Z)-farnesoate is converted to a 1.5:1 ratio
of the 10,11- and 6,7-monoepoxides (Table 2).
Deter m in a tion of th e Absolu te Con figu r a tion of
th e 10,11-Ep oxid a tion Ca ta lyzed by P 450 6A1. A
comparison of the reaction product of P450 6A1 with
optically pure juvenile hormone III obtained by culture
of corpora allata from the cockroach Diploptera punctata
and with racemic synthetic methyl (2E,6E)-10,11-epoxy-
farnesoate allowed us to determine the absolute config-
uration of the 10,11-epoxide product from methyl (2E,6E)-
farnesoate. These epoxides were converted to methoxy-
hydrins containing a free hydroxyl group at C10. The
methoxyhydrins were then reacted with a chiral reagent,
and the resulting diastereomers were separated by
capillary gas chromatography. The derivative prepared
from the synthetic racemic methyl (2E,6E)-10,11-epoxy-
farnesoate showed two peaks with near-identical areas
separated by 0.1 min. A derivative produced from
authentic (10R) juvenile hormone III obtained from D.
punctata corpora allata gland cultures produced a single
peak coincident with the more slowly eluting peak of the
racemic material. The derivative of the reaction product
from P450 6A1 contained a ratio of ∼3:1 in favor of the
more rapidly eluting form. P450 6A1 therefore converts
methyl (2E,6E)-farnesoate predominantly to the antipode
(10S) of natural juvenile hormone III (10R) (18, 19).
Con ver sion of Mon oep oxid es of Meth yl F a r n e-
soa te to Diep oxid es. The monoepoxides of methyl
(2E,6E)- and (2Z,6E)-farnesoate were compared as sub-
strates of P450 6A1 for conversion to their diepoxides in
the reconstituted system. Conversion of the monoep-
oxides to diepoxides occurred regardless of the position
of the epoxide moiety or the geometry of the substrate
at the C2 double bond (Table 3). With the (2E,6E)
isomer, epoxidation of the 10,11-double bond (6,7-epoxide
as a substrate) occurred at a substantially higher rate
than at the 6,7-double bond (10,11-epoxide as a sub-
F igu r e 4. Structures of the four geometric isomers of methyl
farnesoate.
The C10 monoterpenoid homologs of methyl farnesoate
were also converted to monoepoxides by P450 6A1. A
65:35 mixture of the methyl esters of neric (2Z) and
geranic (2E) acids was converted by P450 6A1 to a
mixture of products having abundant ions in the EI mass
spectrum at m/ z 59, 81, 85, and 112 that were consistent
with the 6,7-epoxides of these two geometric isomers
(Table 1). Comparison with a synthetic mixture of the
two epoxides verified the identity of the products (tR 5.04
and 5.66 min, respectively).
Rea ction of P 450 6A1 w ith th e Geom etr ic Isom er s
of Meth yl F a r n esoa te. The metabolism of the four
geometric isomers of methyl farnesoate by the reconsti-
tuted system of P450 6A1 and NADPH-cytochrome P450
reductase were examined to determine the amount of
epoxide formation for each (Figure 4). Incubation with
P450 6A1 resulted in the formation of detectable levels
of monoepoxide and diepoxide with each of the isomers
(Table 2). The overall reaction rates were similar, but
the distribution of products between the 6,7- and 10,11-
epoxides varied somewhat. The 6,7- and 10,11-epoxides
of methyl (2Z,6E)-farnesoate did not separate well on the
columns used in this study, but the mass spectrum of
the monoepoxide indicated that the product was pre-
dominantly the 10,11-epoxide. While methyl (2Z,6Z)-

P450 6A1 Metabolism of Terpenoids
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 161
F igu r e 5. Concentration dependence of the epoxidation of
methyl (6E)-epoxyfarnesoates to their respective diepoxides.
F igu r e 7. Absorption spectrum of P450 6A1 after reaction with
phenyldiazene. P450 6A1 (0.5 nmol) was titrated with phenyl-
diazene at a final concentration of 0.13, 0.27, 0.54, and 0.80 mM.
The decrease in the heme Soret band at 416 nm corresponds to
the formation of the phenyl-iron complex which absorbs at 481
nm.
Ta ble 5. HP LC Elu tion Tim es a n d Ra tios of
N-P h en ylp r otop or p h yr in Regioisom er s F or m ed a fter
Rea ction of Myoglobin or P 450 6A1 w ith P h en yld ia zen e
myoglobina
P450 6A1b
elution
elution
isomer time (min) % of total isomer time (min) % of total
NB
NA
NC
ND
19.5 ( 0.2 13.4 ( 2.2
21.5 ( 0.3 11.7 ( 3.2
27.1 ( 0.4 38.3 ( 2.5
30.5 ( 1.0 36.5 ( 2.9
NB
NA
NC
ND
19.9 ( 0.4 17.1 ( 1.9
22.1 ( 0.5 25.1 ( 2.8
28.1 ( 0.9 33.3 ( 4.1
30.9 ( 1.0 24.4 ( 4.5
F igu r e 6. Structures of substituted imidazole inhibitors of
P450 6A1.
a
b
Ta ble 4. In ter a ction of P 450 6A1 w ith Im id a zole
In h ibitor s
Mean ( SD of three determinations. Mean ( SD of four
determinations.
compound
IC50 (µM)a
Ks (µM)b
TH27. Both of these inhibitors produced type II differ-
ence spectra when incubated with P450 6A1, with a peak
at 432.0 nm and a trough at 411.5 nm. The spectral
dissociation constants were ∼25-fold lower than the IC50
values for inhibition of the catalytic reaction under the
conditions described in Table 4.
TH27
TH76
2.3 ( 0.2
0.08 ( 0.01
2.3 ( 0.1
55.7 ( 11.2
a
Concentration for 50% inhibition ( SE of methyl (2E,6E )-
farnesoate epoxidation. Substrate concentration 50 µM; P450
b
concentration 85 nM. Spectral dissociation constant ( SE cal-
culated from type II difference spectra. P450 concentration 50 nM.
Active Site Top ology of P 450 6A1. P450 6A1 was
titrated with phenyldiazene to form a phenyl-iron
complex. The absorbance spectra showed that the loss
of the Soret band at 416 nm was accompanied by the
appearance of a band at 481 nm, with a saturation at
0.8 mM phenyldiazene (Figure 7). Treatment with
ferricyanide caused the 481-nm band to disappear,
indicating complete shift of the phenyl group to the
pyrrole nitrogens. Extraction and HPLC separation of
the N-phenylprotoporphyrin IX adducts showed that all
four isomers were formed with P450 6A1. Validation of
the procedure and identification of the isomers by HPLC
was done by comparison to standards obtained in a
similar fashion by reaction of phenyldiazene with horse
heart myoglobin. Table 5 shows that the adducts (NB:
NA:NC:ND isomers as eluted by HPLC) were formed in
P450 6A1 in a 17:25:33:24 ratio. This indicated that the
space over the pyrrole rings of the active site heme is
not selectively occluded in P450 6A1 and is only slightly
more accessible over pyrrole ring C.
strate), while with the (2Z,6E) isomers, both the 6,7- and
10,11-monoepoxides were epoxidized at similar rates
(Table 3). The reaction rates for the monoepoxides
continued to increase at substrate concentrations above
300 µM, at which point the incubation mixtures became
visibly cloudy (Figure 5). Although small amounts of a
second product were observed in the metabolism of the
10,11-epoxide of methyl (2E,6E)-farnesoate (see metabo-
lism of J H III above), this product was not seen with the
other three monoepoxides tested here.
In h ibition of P 450 6A1. The ability of two substi-
tuted imidazole inhibitors of juvenile hormone biosyn-
thesis to inhibit P450 6A1 was examined. The imidazole
compound TH27 (Figure 6) is a very potent inhibitor of
the methyl farnesoate epoxidase from the corpora allata
of the cockroach D. punctata, with an IC50 of ∼10 nM
(20). We found here that this compound is also an
effective inhibitor of P450 6A1 giving a concentration for
50% inhibition of activity (IC50) of ∼2 µM in the conver-
sion of methyl (2E,6E)-farnesoate to products (Table 4).
A second compound, TH76, has an imidazole moiety with
a 1-isobutyl subsituent like TH27 but the 5-(benzyloxy)-
phenyl moiety is replaced with an isobutenyl substituent
(Figure 6). This inhibitor was ∼25-fold less effective than
Discu ssion
The oxidative metabolism of juvenile hormone I sug-
gested to occur in Diptera (21) in addition to the more

162 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Andersen et al.
commonly seen juvenile hormone esterase and epoxide
hydrolase pathways was shown to be a 6,7-epoxidation
in house fly microsomes (22, 23). Epoxidation of juvenile
hormone I to the diepoxide was inducible by phenobar-
bital and was elevated in insecticide-resistant strains
(23). Furthermore, juvenile hormone I was shown to
inhibit aldrin epoxidation and to produce a type I binding
spectrum in microsomes of phenobarbital-induced resis-
tant flies (24). The metabolism of juvenile hormone I to
its diepoxide by P450 6A1, a major phenobarbital-
inducible P450 of insecticide-resistant flies (25), is en-
tirely consistent with these observations.
the amount of diepoxide produced also increases (Figure
3). The rate of increase in diepoxide formed is consistent
with the release of monoepoxide from the enzyme fol-
lowed by competition with methyl farnesoate for binding.
Methyl farnesoate monoepoxides derived from the
(2E,6E) and (2Z,6E) isomers are converted efficiently to
diepoxides regardless of the position of the substrate
epoxy group. Epoxidation of the internal 6,7-double bond
of the 10,11-epoxide of methyl (2E,6E)-farnesoate occurs
at a lower rate than epoxidation of the 10,11-double bond
of the 6,7-epoxide (Table 3). This indicates again that
the most favorable substrate orientation has the ω-ter-
minus in the vicinity of the heme. However, variation
in the geometry of the conjugated 2,3-double bond from
E to Z appears to reduce the access of the 10,11-double
bond to the activated oxygen (Table 3). The metabolism
of methyl farnesoate shows that a substrate can be
metabolized at different positions by the same P450
protein. CYP2C11 and CYP2C3 metabolize benzphet-
amine at three positions and several such examples have
been described for xenobiotic-metabolizing P450 proteins
(30).
In addition to binding different geometric isomers, the
substrate-binding pocket of P450 6A1 is capable of
accommodating methyl farnesoate analogs of varying
chain length and with different functionalization at the
acyl terminus. The enzyme epoxidizes methyl geranate
(the monoterpene homolog of methyl farnesoate), juvenile
hormone I [an 18-carbon analog of the 10,11-epoxide of
methyl (2E,6E)-farnesoate] and the aldehyde analog of
methyl (2E,6E)-farnesoate, suggesting that the distance
between the carboxyl and ω-termini is not critical and
that the methyl ester functionality is not required for
productive substrate binding (Table 1). No epoxides were
seen with (2E,6E)-farnesol or farnesoic acid as a sub-
strate, however, indicating that the increase in polarity
of the alcohol or carboxylic acid over the ester or aldehyde
may act to exclude the substrate from the binding pocket
(Table 1).
The presence in both adults and larvae of the house
fly of phenobarbital-inducible microsomal P450 enzyme-
(s) capable of metabolizing the insect growth regulators
methoprene and hydroprene was first shown by Terriere
and Yu (26). These authors also reported that these
compounds were modest inhibitors of heptachlor epoxi-
dation and that hydroprene and methoprene metabolism
was higher in microsomes from two insecticide-resistant
strains. Interestingly, phenobarbital treatment did not
induce the oxidative metabolism of four closely related
insect growth regulators (27). O-Demethylation was
shown to be the principal oxidative pathway of metho-
prene in vivo and in microsomes (22, 28), whereas
epoxidation at the 4,5-unsaturation was the major NAD-
PH-dependent reaction for hydroprene (29). Neither of
the two synthetic insect growth regulators was converted
to detectable products by the P450 6A1/NADPH-cyto-
chrome P450 reductase reconstituted system (Table 1).
Methoprene and hydroprene are not unsaturated at the
6- or 10-positions, but rather have a 2,4-conjugated
system. The lack of any epoxidized products suggests
that an unsaturation at the 4-position may be too distant
from the ω-terminus to be brought into proximity with
the active site or that the conjugated dienoate has
inherently less chemical reactivity at that position.
Interestingly, the O-methyl group located at the 11-
position of the growth regulator methoprene is not
attacked by P450 6A1 under our experimental conditions,
despite occupying a position close to the 10,11-unsatura-
tion of methyl farnesoate. Furthermore, there is no
hydroxylation of hydroprene at positions homologous to
the 10,11-unsaturation of methyl farnesoate. Hydro-
prene and methoprene were also not metabolized2 in a
reconstituted system of P450 6A1, NADPH-cytochrome
P450 reductase, and house fly cytochrome b5. Thus, the
reported epoxidation of hydroprene and O-demethylation
of methoprene by house fly microsomes is due to a
different microsomal form of cytochrome P450.
The presence of a large 5-(benzyloxy)phenyl substitu-
ent in the imidazole inhibitor TH27 increased the efficacy
of this compound by a factor of ∼25 over TH76, which
has a smaller 5-isobutenyl substituent. This suggests
that either steric stabilization or a greater opportunity
for hydrophobic binding near the active site heme con-
tributes to the potency of this compound. Hydrophobic
interactions of 1-alkylimidazoles with microsomal P450
have been well documented (31).
The degree of stereospecificity observed in P450-
mediated epoxidation reactions has been examined in a
Methyl farnesoate isomers are sequentially converted
by P450 6A1 to either 6,7- or 10,11-monoepoxides and
then to 6,7,10,11-diepoxide (Table 1, Figure 2). All four
possible geometric isomers of methyl farnesoate were
epoxidized, demonstrating that the binding pocket is
spacious enough to accommodate the numerous configu-
rations that would be represented by these isomers (Table
2). The (2E,6E), (2Z,6E), and (2Z,6Z) isomers of methyl
farnesoate are converted predominantly to the 10,11-
epoxide, indicating that in its most favorable orientation
the substrate has its ω-terminus held in the vicinity of
the heme iron. For the (2E,6Z) isomer, the orientation
allowing ω-epoxidation is still predominant but to a lesser
degree than for the other isomers. As the concentration
of the substrate methyl (2E,6E)-farnesoate is increased,
number of instances. P450BM-3
(CYP102) from the
bacterium Bacillus megaterium will epoxidize styrene
derivatives with degrees of stereospecificity varying from
40:60 (S:R) to 27:73 (S:R), whereas P450terp (CYP108)
shows much higher degree of stereospecificity with the
same set of styrene analogs (32). The crystal structures
of these two enzymes have been determined, and the
stereospecificity of the epoxidation reactions has been
rationalized in part by steric constraints imposed by the
geometry of the substrate-binding pocket (33, 34). The
pocket of P450BM-3 is comparatively spacious, while the
pocket of P450terp is constrained by impinging amino
acid side chains. Stereoselectivity in the 10,11-epoxida-
tion of methyl (2E,6E)-farnesoate by P450 6A1 is ob-
served with a S:R ratio of ∼3:1 at the 10-position. This
degree of stereospecificity suggests that the binding
pocket of P450 6A1 is spacious and allows multiple
2
V. Guzov and R. Feyereisen, unpublished results.

P450 6A1 Metabolism of Terpenoids
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 163
orientations of the 10,11-unsaturation as opposed to the
enzyme that synthesizes juvenile hormones (see below).
mone III) or its homoisoprenoid congeners (40). In
cockroaches and locusts, a microsomal cytochrome P450
enzyme is responsible for the epoxidation of methyl
farnesoate in the corpora allata, the glands that secrete
the hormone (19, 41). In higher Diptera (flies), however,
the primary secreted product of the corpus allatum is
reported to be the 6,7,10,11-diepoxide (12, 42). Does the
metabolism of methyl farnesoate and juvenile hormone
III to a diepoxide by house fly P450 6A1 provide a model
for juvenile hormone biosynthesis in higher Diptera? It
is not known if the two epoxidations occurring in the
dipteran corpus allatum are catalyzed by a single enzyme
or a pair of enzymes. A single glandular epoxidase would
have to perform two regiochemically distinct epoxidations
while maintaining absolute stereospecificity (17) in each.
The chemistry of the sequential conversion of methyl
farnesoate to mono- and diepoxide by P450 6A1 would
be identical to the likely path taken by a single allatal
epoxidase. However, P450 6A1 is not completely ste-
reospecific and produces juvenile hormone III in a 3:1
ratio of the (10S) and (10R) enantiomers. Furthermore,
Moshitsky and Applebaum (43) have claimed that the
allatal epoxidase(s) of Drosophila melanogaster utilize
farnesoic acid rather than methyl farnesoate as substrate
for the epoxidations. Thus, the allatal epoxidase(s) of
higher Diptera probably bind their sesquiterpenoid sub-
strate in a manner significantly different from P450 6A1.
The fate of juvenile hormone III and its diepoxide in
Diptera has been shown to involve specific epoxide
hydrolases (44).
The complex resulting from the interaction of phenyl-
diazene with P450cam was shown by X-ray crystal-
lography to be a σ-bonded phenyl-iron complex (35).
Treating this complex with ferricyanide can induce the
migration of the phenyl group from the iron to the
porphyrin nitrogens, with the direction of the phenyl shift
being controlled by the structure of the active site. Thus,
the pattern of N-phenylprotoporphyrin IX isomers formed,
specific for each P450 protein, provides information on
the regions above the heme group that are occluded by
the P450 protein residues (36). This technique revealed
a relatively open active site above the heme in P450 6A1
(Table 5) or at least a relatively similar level of protein
occlusion above each of the four pyrrole rings.
P450 6A1 and CYP102 (P450BM-3) are the only P450
proteins studied so far in which the B pyrrole ring is
exposed and in which all four rings are labeled to some
extent. Biosynthetic P450s, such as CYP11A (P450scc
)
and CYP51 (lanosterol 14R-demethylase), or the highly
stereospecific bacterial CYP108 (P450terp) form only a
single N-phenylprotoporphyrin isomer under similar
conditions. An attempt to correlate the ratio of isomers
formed with the position of the P450 in a phylogenetic
tree indicates that there is no discernible evolutionary
constraint on the occlusion of the heme group.3 Thus,
relatively few amino acid changes may be needed to
modify the topology of the active site, as suggested also
by site-directed mutagenesis studies (37-39). However,
only a dozen P450 proteins have been analyzed by this
method to date, and the mutagenesis studies have been
directed at highly conserved residues.
Ack n ow led gm en t. We thank Dr. E. Kuwano (Ky-
ushu University, Fukuoka, J apan) for the gift of the
imidazole inhibitors, Drs. V. Guzov and M. Murataliev
for critical reading of the manuscript, and anonymous
reviewers for helpful comments. J .K.W. was supported
in part by the University of Arizona Undergraduate
Biology Research program. The work was supported in
part by NSF Grant BIR9419402, NIH Grants GM39014
and DK34549, and Center Grant ES 06694.
In summary, the lack of geometric specificity, the
regiospecificity of epoxidation and enantioselectivity, and
the ratio of formation of N-phenylporphyrin adducts
suggest that the binding pocket of P450 6A1 is spacious
and allows a number of different substrate orientations.
Additionally, the accommodation of substrates with chain
lengths shorter than methyl farnesoate, acceptance of the
aldehyde as a substrate, and the epoxidation of nonoxy-
genated substrates such as cyclodiene insecticides sug-
gest a lack of specific protein interactions with the methyl
ester function. The increased efficacy of an imidazole
inhibitor having a large hydrophobic substituent at the
5-position over one with a shorter substituent indicates
that the enzyme possesses a large hydrophobic binding
surface, possibly similar to that of the substrate access
channel seen in the crystal structure of P450BM-3 (33). It
is likely that P450 6A1 can metabolize a large number
of unrelated xenobiotics. Overexpression of this P450 in
insecticide-resistant strains (1, 2) therefore may confer
cross-resistance to several classes of insecticides. The
fitness costs associated with this adaptation remain to
be elucidated. Reduced fecundity and altered develop-
mental rates in resistant strains might be related to the
altered metabolism of endogenous substrates or to the
overproduction of reactive oxygen species by a P450
whose broad substrate specificity does not allow tight
coupling of substrate oxygenation and consumption of
NADPH and molecular oxygen. This aspect of P450 6A1
biochemistry is currently under investigation.
Refer en ces
(1) Carin˜o, F., Koener, J . F., Plapp, F. W., J r., and Feyereisen, R.
(1992) Expression of the cytochrome P450 gene CYP6A1 in the
house fly, Musca domestica. ACS Symp. Ser. 505, 31-40.
(2) Carin˜o, F. A., Koener, J . F., Plapp, F. W., J r., and Feyereisen, R.
(1994) Constitutive overexpression of the cytochrome P450 gene
CYP6A1 in
a house fly strain with metabolic resistance to
insecticides. Insect Biochem. Mol. Biol. 24, 411-418.
(3) Andersen, J . F., Utermohlen, J . G., and Feyereisen, R. (1994)
Expression of house fly CYP6A1 and NADPH-cytochrome P450
reductase in E. coli and reconstitution of an insecticide-metaboliz-
ing P450 system. Biochemistry 33, 2171-2177.
(4) Plapp, F. W., J r., and Vinson, S. B. (1973) J uvenile hormone
analogs: toxicity and resistance in the housefly. Pestic. Biochem.
Physiol. 5, 233-241.
(5) Hammock, B. D., and Quistad, G. B. (1981) Metabolism and mode
of action of juvenile hormone, juvenoids and other insect growth
regulators. In Progress in Pesticide Biochemistry (Hutson, D. H.,
and Roberts, T. R., Eds.) Vol. 1, pp 1-83, J ohn Wiley and Sons,
New York.
(6) Forman, B. M., Goode, E., Chen, J ., Oro, A. E., Bradley, D. J .,
Perlmann, T., Noonan, D. J ., Burka, L. T., McMorris, T. Lamph,
W. W., Evans, R. M., and Weinberger, C. (1995) Identification of
a nuclear receptor that is activated by farnesol metabolites. Cell
81, 687-693.
(7) Harmon, M. A., Boehm, M. F., Heyman, R. A., and Mangelsdorf,
D. J . (1995) Activation of mammalian retinoid X receptors by the
insect growth regulator methoprene. Proc. Natl. Acad. Sci. U.S.A.
92, 6157-6160.
(8) Koener, J . F., Carin˜o, F. A., and Feyereisen, R. (1993) The cDNA
and deduced protein sequence of house fly NADPH-cytochrome
P450 reductase. Insect Biochem. Mol. Biol. 23, 439-447.
In most insect species, juvenile hormone is the 10R,-
11-epoxide of methyl-(2E,6E)-farnesoate (juvenile hor-
3
R. Feyereisen, unpublished results.

164 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Andersen et al.
(9) Omura, T., and Sato, R. (1964) The carbon monoxide-binding
pigment of liver microsomes I. Evidence for its hemeprotein
nature. J . Biol. Chem. 239, 2370-2378.
(29) Yu, S. J ., and Terriere, L. C. (1977) Metabolism of [14C]hydroprene
(ethyl 3,7,11-trimethyl-2,4-dodecadienoate) by microsomal oxi-
dases and esterases from three species of Diptera. J . Agric. Food
Chem. 25, 1076-1080.
(30) Oguri, K., Yamada, H., and Yoshimura, H. (1994) Regiochemistry
of cytochrome P450 isozymes. Annu. Rev. Pharmacol. Toxicol. 34,
251-279.
(31) Wilkinson, C. F., Hetnarski, K., Cantwell, G. P., and DiCarlo, F.
J . (1974) Structure-activity relationships in the effects of 1-alkyl-
imidazoles on microsomal oxidation in vitro and in vivo. Biochem.
Pharmacol. 23, 2377-2386.
(32) Fruetel, J . A., Mackman, R. L., Peterson, J . A., and Ortiz de
Montellano, P. R. (1994) Relationship of active site topology to
substrate specificity for cytochrome P450terp (CYP108). J . Biol.
Chem. 269, 28815-28821.
(33) Ravichandran, K. G., Bodupalli, S. S.,Hasemann, C. A., Peterson,
J . A., and Deisenhofer, J . (1993) Crystal structure of hemoprotein
domain of P450BM-3, a prototype for microsomal P450’s. Science
261, 731-736.
(34) Hasemann, C. A., Ravichandran, K. G., Peterson, J . A., and
Deisenhofer, J . (1994) Crystal structure and refinement of cyto-
chrome P450terp at 2.3 Å resolution. J . Mol. Biol. 236, 1169-
1185.
(35) Raag, R., Swanson, B. A., Poulos, T. L., and Ortiz de Montellano,
P. R. (1990) Formation, crystal structure, and rearrangement of
a cytochrome P-450cam iron-phenyl complex. Biochemistry 29,
8119-8126.
(36) Ortiz de Montellano, P. R., and Graham-Lorence, S. E. (1993)
Structure of cytochrome P450: heme binding site and heme
reactivity. In Cytochrome P450 (Schenkman, J . B., and Greim,
H., Eds.) pp 169-181, Springer-Verlag, Berlin.
(10) Anderson, R. J ., Henrick, C. A., Siddall, J . B., and Zurfluh, R.
(1972) Stereoselective synthesis of the racemic C-17 juvenile
hormone of Cecropia. J . Am. Chem. Soc. 94, 5379-5386.
(11) Nilles, G. P., Zabik, M. J ., Connin, R. V., and Schuetz, R. D. (1973)
Synthesis of bioactive compounds: J uvenile hormone mimetics
affecting insect diapause. J . Agric. Food Chem. 21, 342-347.
(12) Richard, D. S., Applebaum, S. W., Sliter, T. J ., Baker, F. C.,
Schooley, D. A., Reuter, C. C., Henrich, V. C., and L. I. Gilbert,
L. I. (1989) J uvenile hormone bisepoxide biosynthesis in vitro by
the ring gland of Drosophila melanogaster: A putative juvenile
hormone in the higher Diptera. Proc. Natl. Acad. Sci. U.S.A. 86,
1421-1425.
(13) Feyereisen, R. (1985) Radiochemical assay for juvenile hormone
III biosynthesis in vitro. Methods Enzymol. 111, 530-539.
(14) Schooley, D. A. (1977) Analysis of the naturally occurring juvenile
hormonesstheir isolation, identification, and titer determination
at physiological levels. In Analytical biochemistry of insects.
(Turner, R. B., Ed.) Elsevier Scientific Publishing, Amsterdam.
(15) Tuck, S. F., Peterson, J . A., and Ortiz de Montellano, P. R. (1992)
Active sites of cytochrome P450101 (P450cam), P450108 (P450terp),
and P450102 (P450BM3). In-situ rearrangement of their phenyl-
iron complexes. J . Biol. Chem. 267, 5614-5620.
(16) Liedtke, R. J ., and Djerassi, C. (1972) Mass spectrometry in
structural and stereochemical problems. CCXVII. The electron
impact induced behavior of terpenoid esters of the juvenile
hormone class. J . Org. Chem. 37, 2111-2118.
(17) Herlt, A. J ., Rickards, R. W., Thomas, R. D., and East P. D. (1993)
The absolute configuration of juvenile hormone III bisepoxide. J .
Chem. Soc., Chem. Commun. 1497-1498.
(18) J udy, K. J ., Schooley, D. A., Dunham, L. L., Hall, M. S., Bergot,
B. J ., and Siddall, J . B. (1973) Isolation, structure and absolute
configuration of a new natural insect juvenile hormone from
Manduca sexta. Proc. Natl. Acad. Sci. U.S.A. 70, 1509-1513.
(19) Feyereisen, R., Pratt, G. E., and Hamnett, A. F. (1981) Enzymic
synthesis of juvenile hormone in locust corpora allata. Evidence
for a microsomal cytochrome P-450 linked methyl farnesoate
epoxidase. Eur. J . Biochem. 118, 231-238.
(20) Andersen, J . F., Ceruso, M., Unnithan, G. C., Kuwano, E.,
Prestwich, G. D., and Feyereisen, R. (1995) Photoaffinity labeling
of methyl farnesoate epoxidase in cockroach corpora allata. Insect
Biochem. Mol. Biol. 25, 713-719.
(21) Ajami, A. M., and Riddiford, L. M. (1973) Comparative metabolism
of the cecropia juvenile hormone. J . Insect Physiol. 19, 635-645
(22) Hammock, B. D., Mumby, S. M., and Lee, P. W. (1977) Mecha-
nisms of resistance to the juvenoid methoprene in the house fly,
Musca domestica. Pest. Biochem. Physiol. 7, 261-272.
(23) Yu, S. J ., and Terriere, L. C. (1978) Metabolism of juvenile
hormone I by microsomal oxidase, esterase, and epoxide hydrolase
of Musca domestica and some comparisons with Phormia regina
and Sarcophage bullata. Pest Biochem. Physiol. 9, 237-246.
(24) Fisher, C. W., and Mayer, R. T. (1982) Characterization of house
fly microsomal mixed function oxidases: inhibition by juvenile
hormone I and piperonyl butoxide. Toxicology 24, 15-31.
(25) Feyereisen, R., Koener, J . F., Farnsworth, D. E., and Nebert, D.
W. (1989) Isolation and sequence of cDNA encoding a cytochrome
P-450 from an insecticide-resistant strain of the house fly Musca
domestica. Proc. Natl. Acad. Sci. U.S.A. 86, 1465-1469.
(26) Terriere, L. C., and Yu, S. J . (1973) Insect juvenile hormones:
Induction of detoxifying enzymes in the housefly and detoxication
by housefly enzymes. Pestic. Biochem. Physiol. 3, 96-107.
(27) Yu, S. J ., and Terriere, L. C. (1975) Microsomal metabolism of
juvenile hormone analogs in the house fly, Musca domestica, L.
Pestic. Biochem. Physiol. 5, 418-430.
(37) Tuck, S. F., Graham-Lorence, S., Peterson, J . A., and Ortiz de
Montellano, P. R. (1993) Active sites of cytochrome P450cam
(CYP101) F87W and F87A mutants. Evidence for significant
structural rearrangemnt without alteration of catalytic regiospec-
ificity. J . Biol. Chem. 268, 269-275.
(38) Tuck, S. F., Hiroya, K., Shimuzu, M., Hatano, M., and Ortiz de
Montellano, P. R. (1993) The cytochrome P450 active site:
Topology and perturbations caused by glutamic acid-318 and
threonine-319 mutations. Biochemistry 32, 2548-2553.
(39) Pikuleva, I. A., Mackman, R. L., Kagawa, N., Waterman, M. R.,
and Ortiz de Montellano, P. R. (1995) Active-site topology of
bovine cholesterol side-chain cleavage cytochrome P450 (P450scc
)
and evidence for interaction of tyrosine 94 with the side chain of
cholesterol. Arch. Biochem. Biophys. 322, 189-197.
(40) Schooley, D. A., and Baker F. C. (1985) J uvenile hormone
biosynthesis. In Comprehensive Insect Physiology, Biochemistry
and Pharmacology (Kerkut, G. A., and Gilbert, L. I., Eds.) Vol. 7,
pp 363-389, Pergamon Press, Oxford, England.
(41) Hammock, B. D. (1975) NADPH-dependent epoxidation of methyl
farnesoate to juvenile hormone in the cockroach Blaberus gigan-
teus. L. Life Sci. 17, 323-328.
(42) Lefevere, K. S., Lacey, M. J ., Smith, P. H., and Roberts, B. 1993)
Identification and quantification of juvenile hormone biosynthe-
sized by larval and adult Australian sheep blowfly Lucilia cuprina
(Diptera: Calliphoridae). Insect Biochem. Mol. Biol. 23, 713-720.
(43) Moshitsky, P., and Applebaum, S. W. (1995) Pathway and
regulation of J H III bisepoxide biosynthesis in adult Drosophila
melanogaster corpus allatum. Arch. Insect Biochem. Physiol. 30,
225-237.
(44) Casas, J ., Harshman, L. G., Messeguer, A., Kuwano, E., and
Hammock, B. D. (1991) In vitro metabolism of juvenile hormone
III and juvenile hormone III bisepoxide by Drosophila melano-
gaster and mammalian epoxide hydrolase. Arch. Biochem. Bio-
phys. 286, 153-158.
(28) Quistad, G. B., Staiger, L. E., and Schooley, D. A. (1975)
Environmental degradation of the insect growth regulator me-
thoprene. Pestic. Biochem. Physiol. 5, 233-241.
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