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Mosquito NADPH-cytochrome P450 oxidoreductasekinetics and role of phenylalanine amino acid substitutions at leu86 and leu219 in CYP6AA3-mediated deltamethrin metabolism.

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A r t i c l e
MOSQUITO NADPHCYTOCHROME P450
OXIDOREDUCTASE: KINETICS AND
ROLE OF PHENYLALANINE
AMINO ACID SUBSTITUTIONS AT
LEU86 AND LEU219 IN
CYP6AA3-MEDIATED
DELTAMETHRIN METABOLISM
Songklod Sarapusit, Sirikun Pethuan, and
Pornpimol Rongnoparut
Department of Biochemistry, Faculty of Science, Mahidol University,
Bangkok, Thailand
The NADPH-cytochrome P450 oxidoreductase (CYPOR) enzyme is a
membrane-bound protein and contains both FAD and FMN cofactors.
The enzyme transfers two electrons, one at a time, from NADPH to
cytochrome P450 enzymes to function in the enzymatic reactions. We
previously expressed in Escherichia coli the membrane-bound CYPOR
(flAnCYPOR) from Anopheles minimus mosquito. We demonstrated the
ability of flAnCYPOR to support the An. minimus CYP6AA3 enzyme
activity in deltamethrin degradation in vitro. The present study revealed
that the flAnCYPOR purified enzyme, analyzed by a fluorometric method,
readily lost its flavin cofactors. When supplemented with exogenous
flavin cofactors, the activity of flAnCYPOR-mediated cytochrome c
reduction was increased. Mutant enzymes containing phenylalanine
substitutions at leucine residues 86 and 219 were constructed and found
Grant sponsors (from Thailand): National Center for Genetic Engineering and Biotechnology; National Science
and Technology Development Agency; Commission on Higher Education Staff Development Project; Royal
Golden Jubilee Program.
Additional Supporting Information may be found in the online version of this article.
Songklod Sarapusit’s present address is Department of Biochemistry, Faculty of Science, Burapha University,
Chonburi 20131, Thailand.
Correspondence to: Pornpimol Rongnoparut, Ph.D., Department of Biochemistry, Faculty of Science,
Mahidol University, Rama 6 Rd, Phyatai, Bangkok 10400, Thailand. E-mail: scprn@mahidol.ac.th
ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 73, No. 4, 232–244 (2010)
Published online in Wiley InterScience (www.interscience.wiley.com).
& 2010 Wiley Periodicals, Inc. DOI: 10.1002/arch.20354
Mosquito NADPH-Cytochrome P450 Oxidoreductase
233
to increase retention of FMN cofactor in the flAnCYPOR enzymes.
Kinetic study by measuring cytochrome c–reducing activity indicated that
the wild-type and mutant flAnCYPORs followed a non-classical two-site
Ping-Pong mechanism, similar to rat CYPOR. The single mutant (L86F
or L219F) and double mutant (L86F/L219F) flAnCYPOR enzymes,
upon reconstitution with the An. minimus cytochrome P450 CYP6AA3
and a NADPH-regenerating system, increased CYP6AA3-mediated
deltamethrin degradation compared to the wild-type flAnCYPOR
enzyme. The increased enzyme activity could illustrate a more efficient
electron transfer of AnCYPOR to CYP6AA3 cytochrome P450 enzyme.
Addition of extra flavin cofactors could increase CYP6AA3-mediated
activity supported by wild-type and mutant flAnCYPOR enzymes. Thus,
both leucine to phenylalanine substitutions are essential for flAnCYPOR
C 2010 Wiley
enzyme in supporting CYP6AA3-mediated metabolism. Periodicals, Inc.
Keywords: Anopheles minimus; CYPOR; CYP6AA3; deltamethrin; flavin
cofactors; kinetic study
INTRODUCTION
Pyrethroid insecticide detoxification mediated by cytochrome P450 monooxygenase
(CYP or P450), a family of enzymes metabolizing various endogenous and exogenous
compounds, has been reported in insects (Feyereisen, 1999). Catalysis by P450
enzymes requires an electron supplement from NADPH-cytochrome P450 oxidoreductase enzyme (CYPOR), a membrane-bound di-flavin enzyme. The CYPOR
functions to transfer electrons, one by one, from NADPH through FAD and FMN
cofactors to cytochrome P450 enzymes (Wang et al., 1997; Murataliev et al., 2004;
Iyanagi, 2005; Ortiz de Montellano, 2005).
The kinetic mechanism of CYPOR is varied among different organisms, for
instance a Ping-Pong mechanism is shown for CYPORs of pig liver (Masters et al.,
1965), pig kidney (Fan and Masters, 1974), house fly (Mayer and Durrant, 1979), and
yeast (Lamb et al., 2001); random sequential mechanism for CYPORs of house fly
(Murataliev et al., 1999) and white rot fungus (Warrilow et al., 2002); and a two-site
Ping-Pong mechanism for rat CYPOR (Sem and Kasper, 1994). Recently, CYPOR at a
high expression level is implicated in protecting Anopheles gambiae mosquito against
permethrin insecticide (Lycett et al., 2006).
The mosquito An. minimus, one of the primary malaria vectors in Thailand, is
shown to have an increased level of CYP6AA3 and CYP6P7 transcripts during
selection for deltamethrin resistance (Rongnoparut et al., 2003; Rodpradit et al.,
2005). The membrane-bound full-length An. minimus CYPOR (flAnCYPOR) cDNA has
been isolated and expressed in Escherichia coli (Kaewpa et al., 2007). The purified
flAnCYPOR enzyme could support baculovirus-expressed CYP6AA3 to metabolize
deltamethrin insecticide in vitro (Kaewpa et al., 2007; Boonsuepsakul et al., 2008).
Prior studies in D55AnCYPOR, a membrane-deleted An. minimus CYPOR,
indicated that the enzyme followed a two-site Ping-Pong mechanism similar to rat
CYPOR enzyme (Sarapusit et al., 2008). However, for the D55AnCYPOR enzyme it is
easy to lose flavin cofactors compared to rat CYPOR. In addition, two phenylalanine
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substitutions at leucine 86 and leucine 219 in the FMN-binding domain of
D55AnCYPOR could improve FMN binding and protein stability resulting in an
increased turnover number without a change of the kinetic mechanism, NADPH Km,
and cytochrome c Km. Initial results of the membrane-deleted D55AnCYPOR enzyme
revealed that it could not support CYP6AA3-mediated deltamethrin degradation in
vitro, while the double mutant L86F/L219FD55AnCYPOR could slightly support
CYP6AA3-mediated reaction with much less activity than the full-length flAnCYPOR
(unreported data). This has prompted us to investigate the role of phenylalanine
replacement in the membrane-bound full-length flAnCYPOR enzyme. In the present
study, we show that the catalytically active and membrane-bound flAnCYPOR enzyme
is prone to lose flavin cofactors. The two-site Ping-Pong kinetic mechanism of
flAnCYPOR is more similar to rat CYPOR than the housefly insect. Two phenylalanine
replacements in the FMN domain of the flAnCYPOR could rescue the loss of FMN
cofactor without changing its kinetic mechanism and substrate-binding constants.
Significantly, a higher turnover number in the phenylalanine-replaced mutant
flAnCYPOR enzymes could increase CYP6AA3 activity in deltamethrin degradation
in vitro. Addition of exogenous flavins contributed to a greater increase in CYP6AA3mediated activity supported by the wild-type and mutant flAnCYPOR enzymes.
MATERIALS AND METHODS
Materials
Flavin mono-nucleotide (FMN), flavin-adenosine di-nucleotide (FAD), cytochrome c,
nicotinamide adenosine diphosphate (NADP1), nicotinamide adenosine diphosphate
reduced form (NADPH), phenylmethylsulphonyl fluoride (PMSF), glucose-6-phosphate (G6P), glucose-6-phosphate dehydrogenase (G6PDH), 1,2-didodecanoylrac-glycero-3-phosphocholine (DLPC), and deltamethrin were purchased from
Sigma-Aldrich (St. Louis, MO). Bioallethrin was obtained from ChemService (West
Chester, PA), Isopropyl-b-D-thiogalactopyranoside (IPTG) from USB (Cleveland,
OH), Ni21-NTA affinity column from Qiagen (Valencia, CA), and Bio-Rad protein
assay kit from Bio-Rad (Hercules, CA). All restriction enzymes and ligase from
New England Biolabs (Beverly, MA) were used according to the manufacturer’s
instructions.
Expression and Purification of flAnCYPOR
Protein expression and purification of flAnCYPOR enzyme was performed as
previously described by Kaewpa et al. (2007) with slight modification. The pTrc-wtflAnCYPOR plasmid containing a membrane-bound form of An. minimus CYPOR
(flAnCYPOR) cDNA with an extra 6-kDa N-terminal 6 his-tag was transformed into
XL-1 blue E. coli cells. The cells were grown in TB medium for 64 h at 281C after
induction of protein expression with 0.2 mM IPTG when an OD600 of culture reached
0.8–1. The cells were harvested by centrifugation at 5,000 g for 10 min and cells
resuspended in buffer A (50 mM Na2HPO4, pH 8.0, 0.1 M NaCl, and 10 mM
imidazole) containing 0.2 mM PMSF. The cells were lysed by means of sonication and
cell debris was removed by centrifugation at 5,000 rpm. The supernatant was further
centrifuged at 100 Kg for 60 min. The cytosol was removed and the yellow-membrane
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fraction was solubilized with 0.5% CHAPS on ice for 2 h, then centrifuged at 100 Kg for
60 min. The supernatant containing flAnCYPOR enzyme was applied to a previously
equilibrated Ni21-column. The protein was eluted by increasing the imidazole
concentration up to 150 mM, the protein was concentrated and the purity visualized
by SDS-PAGE. For flavin-reconstitution enzyme, the purified enzyme was incubated
with 10-fold excess flavin cofactors on ice for 15 min and applied to G25 superdex
column to remove unbound cofactors. The yellow protein fraction of reconstituted
flAnCYPOR enzyme was concentrated and the protein concentration determined
using BSA as a standard.
Mutant flAnCYPOR Construction, Expression, and Purification
The mutant flAnCYPOR enzymes were constructed using previously constructed
mutant D55AnCYPOR cDNAs subcloned into pET28a plasmid as a template
(Sarapusit et al., 2008). The XmnI-SacI DNA fragment containing the L86F mutation
and the SacI-HindIII DNA fragment containing the L219F mutation were each excised
from their original D55AnCYPOR single mutant plasmids. The DNA fragments were
purified and replaced the corresponding fragment of the pTrc-wt-flAnCYPOR
plasmid by ligation, producing the L86F-flAnCYPOR and L219F-flAnCYPOR
mutants, respectively. For the L86F/L219F-flAnCYPOR double mutant, the SacIHindIII DNA fragment containing the L219F-mutation was ligated in place of the
corresponding fragment of pTrc-L86F-flAnCYPOR plasmid. Upon DNA sequencing
on both DNA strands to verify the mutated sites of the clones, each construct was
transformed, expressed, and purified using a similar protocol as for the wild-type
enzyme.
Measurement of Flavin Content
FAD and FMN content of each sample was measured using a fluorometric method as
described (Aliverti et al., 1999). Commercial FMN and FAD were used for establishing
the standard curve and for cofactor supplementation experiments. The concentrations
of standard FAD and FMN solution were determined spectrophotometrically at
450 nm using extinction coefficients of 11.3 and 12.2 mM1cm1 (Aliverti et al., 1999),
respectively. The protein concentration was determined using BSA as standard.
Spectrophotometric Methods
All spectrophotometric measurements were performed using an Alligent 8453
spectrophotometer. The oxidized enzyme was diluted in 0.3 M potassium phosphate
buffer, pH 7.7, to a final concentration of 5–10 mM, and the spectrum was recorded.
The stable-semiquinone spectrum was obtained after addition of NADPH (100 mM
final concentration) under aerobic conditions.
The CYPOR-mediated cytochrome c reduction was carried out in 0.3 M potassium
phosphate buffer, pH 7.7 (Shen et al., 1989, 1991; Shen and Kasper, 1996; Döhr et al.,
2001; Sarapusit et al., 2008). Briefly, the purified enzyme was pre-incubated in buffer
containing 2.0 mM FAD, 2.0 mM FMN, and 50 mM cytochrome c at 251C for 1 min and
the reaction was initiated by addition of 50 mM NADPH. The NADPH-dependent
cytochrome c reduction was followed by a change in the absorbance at 550 nm with an
extinction coefficient of 21 mM1cm1 (Shen et al., 1989; Sem and Kasper, 1994).
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Kinetic Study of flAnCYPOR Enzyme-Substrate
The substrate saturation experiments for cytochrome c reduction of flAnCYPOR
enzymes were performed in the presence of fixed 2 mM each of FAD and FMN
co-factors, with a varying cytochrome c concentration at a fixed 50 mM NADPH and
vice versa. Kinetic parameters were obtained by non-linear regression of data using
Graph Pad Prism 4 (GraphPad Software Inc., San Diego, CA).
The steady-state kinetics for cytochrome c reduction of flAnCYPORs was performed at
251C in a final volume of 0.8 ml. The experiments were performed by varying cytochrome c
concentrations at four different fixed NADPH concentrations (3, 6, 12, and 30 mM). The
reaction was started by addition of 40 nM enzyme supplemented with each of flavin co-factors
at a 2-mM final concentration. Kinetic parameters were obtained by non-linear regression of
data using a GraFit 6 software package (Erithacus Software Limited, Surrey, UK). The kinetic
mechanism of enzyme for flavin cofactor binding was studied in the fixed concentration of
FAD with four different concentrations of NADPH or in the fixed concentration of FMN with
four different concentrations of cytochrome c. After addition of an appropriate amount of
enzyme, the reaction was measured by the increase in absorption at 550 nm and double
reciprocal plots were generated using Graphpad Prism 4.0. For inhibition studies, the
inhibitors used were NADP1 (20, 40, and 80 mM) or cytochrome c21 (25, 50, and 100 mM).
Inhibition constants and double reciprocal plots were derived by GraphPad prism 4.
CYP6AA3-flAnCYPOR Reconstitution System
The membrane fraction containing full-length An. minimus CYP6AA3 enzyme was purified
from CYP6AA3 expressing Sf9 cells and the enzymatic assays were performed as
previously described with minor modification (Kaewpa et al., 2007; Boonsuepsakul et al.,
2008). The CYP6AA3 membrane fraction (10 mg) and flAnCYPOR enzymes (1 pmol) were
preincubated with 80 mM deltamethrin insecticide substrates in the presence of DLPC. The
CYP6AA3-mediated deltamethrin metabolism was initiated with the addition of the
NADPH-regenerating system in a final volume of 250 ml and incubated at 301C for 30 min.
The reaction was terminated by addition of HCl. The remaining deltamethrin insecticide
was extracted with ethyl acetate containing bioallethrin as an internal standard and the
extract was dried under a stream of N2 gas. The dried samples were dissolved with
acetonitrile and subjected to high-performance liquid chromatography (HPLC) analysis
using a C18 column (Waters, Milford, MA). Each of the reconstitution experiments was
performed in three independent repetitions. The enzymatic activities were detected as
substrate disappearance by subtracting the deltamethrin substrate in the test reaction from
substrate at zero time. The proportion of remaining deltamethrin substrate was obtained,
and the proportion of deltamethrin substrate metabolized was calculated. The bioallethrin
internal standard was used for normalization among reactions as previously described
(Kaewpa et al., 2007; Boonsuepsakul et al., 2008). The percent extraction recovery of the
bioallelthrin internal standard was 93.270.4.
RESULTS
Expression and Purification of flAnCYPOR Enzymes
We previously reported that leucine residues at positions 86 and 219 in the
FMN-binding domain are important for the binding of flavin cofactors in the
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An. minimus AnCYPOR protein lacking the first N-terminal 55 residues (D55AnCYPOR). In this study, we employed the membrane-bound enzymes, wild-type (wt)
flAnCYPOR, and mutants with phenylalanine substitutions at leucine positions 86 and
219, for further kinetic characterization. Three mutant constructs containing two
single mutations, namely L86F-flAnCYPOR and L219F-flAnCYPOR, and one double
mutation, namely L86F/L219F-flAnCYPOR, were generated. The membrane-bound
flAnCYPOR wild-type and mutants, upon expression, were successfully purified into a
major protein band at 83 kDa on SDS-PAGE, comprising 6 kDa N-terminal His-tag
sequence and 77 kDa flAnCYPOR protein as previously shown in Kaewpa et al. (2007).
The yield of the purified wild-type and double mutant flAnCYPOR were 3 and 8 mg
protein/liter of culture, respectively. Upon NADPH reduction under aerobic condition,
the typical absorbance spectra of oxidized flAnCYPOR enzymes were decreased and
there was formation of an air-stable semiquinone species with a characteristic broad
peak around 500–650 nm as reported (Sarapusit et al., 2008), indicating the presence
of flavin co-factors in the purified active flAnCYPOR wild-type and mutant enzymes
(unreported data).
Activities and Flavin Content of flAnCYPOR Enzymes
The wt-flAnCYPOR showed NADPH-dependent cytochrome c reduction activity
similar to that previously reported (Kaewpa et al., 2007) and it could be increased
upon supplementation with both flavin co-factors (Fig. 1). The single and double
phenylalanine replacements in flAnCYPOR resulted in increased cytochrome
c–reducing activity (Fig. 1). Supplementation of exogenous flavin cofactors could
further elevate cytochrome c–reduction activity of mutant flAnCYPOR enzymes, with
highest activity in the double mutant L86F/L219F-flAnCYPOR enzyme (Fig. 1). Since
addition of exogenous cofactors could increase flAnCYPOR enzyme activity, this could
indicate a loss of flavin cofactors in the purified enzymes. As shown in Table 1, flavin
content analysis using BSA as standard showed that all flAnCYPORs readily lost flavin
cofactors compared to rat CYPOR measured previously (Sarapusit et al., 2008). The
replacement by two phenylalanine residues in the single and double mutants and the
membrane-deletion of the AnCYPORs resulted in a 2–6-fold enhanced FMN binding
compared to wt-flAnCYPOR (Table 1). Phenylalanine replacement in the FMNbinding domain did not affect enzyme binding to the FAD cofactor, except that
Figure 1. Specific activity of flAnCYPOR enzymes with cytochrome c substrate, with and without addition
of FAD and FMN cofactors. The specific activity is expressed as nmol of substrate reduced/min/mg of
protein. The dot bar is wt-flAnCYPOR, the diagonal-line bar is L86F-mutant, the horizontal-line bar is
L219F-mutant, and the vertical-line bar is L86F/L219F-mutant. The data shown are the average of triplicate
measurements. Protein concentration was determined using Bio-Rad protein assay reagent and BSA as
standard.
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Table 1. Flavin Contents of flAnCYPOR and D55AnCYPOR Enzymes
Enzyme
Rat CYPORb
Wild-type flAnCYPOR
L86F- flAnCYPOR
L219F- flAnCYPOR
L86F/L219F- flAnCYPOR
Wild-type D55AnCYPOR
L86F- D55AnCYPOR
L219F- D55AnCYPOR
L86F/L219F- D55AnCYPOR
FMNa
FADa
0.9270.01
0.1170.02
0.2870.01
0.3370.03
0.5870.07
0.2470.02
0.5070.06
0.4670.01
0.7370.02
1.0570.05
0.2270.06
0.1970.02
0.2270.04
0.2870.05
0.3770.02
0.3370.05
0.2870.04
0.4170.01
a
The flavin content is expressed as mean7SD for mol of flavins per mol of protein in triplicate experiments. The
protein concentration was determined by Bio-Rad protein assay reagent using BSA as standard.
b
Sarapusit et al. (2008).
Table 2. Kinetic Constants for Cytochrome c Reduction by wt-flAnCYPOR and L86F/L219FflAnCYPOR Double Mutant Enzyme
Kinetic constanta
1
Vmax, s
Km for NADPH, mM
Km for cytochrome c, mM
Ki for NADP1, mM
Km for NADH, mM
wt-flAnCYPOR
Double mutant-flAnCYPOR
56.770.6
12.570.9
19.071.1
11.570.6
4.070.7
100.971.4
11.670.9
23.971.9
10.970.6
4.370.8
a
The values are obtained from steady-state kinetic studies as described in Materials and Methods and are
means7SD from triplicate experiments.
membrane-deleted D55AnCYPOR had an approximately half-fold increased FAD
content compared to wt-flAnCYPOR.
Kinetic Characterization of flAnCYPOR Enzymes
The wt-flAnCYPOR enzyme catalyzed the NADPH-dependent cytochrome c reduction
following Michaelis-Menten kinetics with respect to both cytochrome c and NADPH
substrates and the kinetic constants are shown in Table 2. The best fit of initial velocity
data to the equation of the double reciprocal plot (Fig. 2A) indicated that the
flAnCYPOR kinetic mechanism is classical Ping-Pong. In this mechanism, the NADPH
(electron donor) must leave before NADP1 (product) binding, thus the NADP1 should
not competitively inhibit the binding of the NADPH substrate (Segel, 1975). But the
results indicated that NADP1 could competitively inhibit NADPH and cyt c21 could
competitively inhibit cyt c31 (Fig. 2B and C), suggesting that flAnCYPOR followed a
non-classical two-site Ping-Pong mechanism in which the enzyme possesses two
independent substrate-binding sites, one for electron donor (NADPH) and one for
electron acceptor (cyt c31) to allow for the formation of ternary complex as a catalysis
intermediate (Segel, 1975; Cleland, 1977). In addition, the best fit to the equation and
the intersection of double reciprocal lines of initial velocity data (Fig. 2D and E)
demonstrated that FAD and NADPH bind at the electron donor site while FMN and
cytochrome c bind the other. Kinetic studies in the double mutant flAnCYPOR enzyme
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Figure 2. Initial velocity patterns obtained from a substrate saturation experiment. Lineweaver-Burk plots
of cytochrome c reduction were produced with wt-flAnCYPOR using variable cytochrome c concentrations at
four different NADPH concentrations (A), the NADP1 inhibition study of flAnCYPOR (B), and the cyt c21
inhibition study of flAnCYPOR (C). Flavin-dependent initial velocity flAnCYPOR using variable FAD
concentrations at different fixed NADPH concentrations (D) and variable FMN concentrations at different
fixed cytochrome c concentrations (E) are shown. All data points are means of triplicate measurements.
revealed that the mutant enzyme followed the non-classical two-site Ping-Pong
mechanism (unreported data) with substrate-binding constants similar to the wild-type
enzyme; however, there was a 2-fold increased turnover number (Table 2).
Furthermore, the apparent Km values for NADH of the wild-type and double mutant
enzymes are about 1,000 fold higher than NADPH (Table 2), thus demonstrating the
importance of 20 -phosphate of NADPH in interaction with AnCYPOR.
Effect of L86F and L219F flAnCYPOR Mutation on CYP6AA3-Mediated Deltamethrin
Degradation
The in vitro P450-CYPOR reconstitution system was performed to evaluate the effect
of flAnCYPOR mutations on CYP6AA3-mediated deltamethrin metabolism. The wtflAnCYPOR could support CYP6AA3 to metabolize deltamethrin substrates in vitro,
with an increased CYP6AA3 activity when flAnCYPOR is supplemented with extra
flavin cofactors (Table 3). The mutant flAnCYPOR enzymes could increase CYP6AA3
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Table 3. Specific Activity of CYP6AA3-Mediated Deltamethrin Degradation Reconstituted with
flAnCYPOR Enzymes
Deltamethrin metabolizeda
Enzyme
() flavins
(1) flavins
Without AnCYPOR flAnCYPOR
-wt
-L86F
-L219F
-L86F/L219F
0.8570.62
3.3170.75
4.8770.15
7.8170.38
9.9570.18
0.9570.51
4.6670.38
9.4470.39
8.3770.28
11.1470.29
a
The CYP6AA3-mediated deltamethrin metabolism activity was measured in the absence and presence of extra
flavins with variant flAnCYPORs. The specific activity is expressed as pmol of deltamethrin metabolized/min/mg
protein. The data shown are the average of triplicate measurements.
enzymatic activity towards deltamethrin substrates by 2–3-fold, with a greater increase
in CYP6AA3 activity supported by the L86F/L219F-flAnCYPOR double mutant
(Table 3). The results thus consistently support the role of phenylalanine replacement
in enzymatic activation, and co-factor binding of the membrane-bound flAnCYPOR
enzymes.
DISCUSSION
In this study, the flAnCYPOR enzymes showed optimal activity (Table 2) when assayed
under high ionic strength (0.3 M Kpi, pH7.7), and had low activity at low ionic
strength conditions (0.1 M Tris pH 7.5, see Supp. Table S1, which is available online).
The flAnCYPOR kinetic mechanism carried out under high ionic strength is a nonclassical two-site Ping-Pong mechanism, suggesting that the NADPH-CYPORcytochrome c ternary complex is an important intermediate species for fast electron
transfer during mosquito CYPOR catalysis. The kinetic mechanism is similar to that of
rat CYPOR in which NADP1 competitively inhibits NADPH, while cyt c21
competitively inhibits cyt c31 (Fig. 2B). Consistent with the kinetic mechanism of rat
CYPOR, NADP1 un-competitively inhibits cyt c31, and cyt c21 non-competitively
inhibits NADPH (see Supp. Fig. S1) (Sem and Kasper, 1994, 1995). The mosquito
CYPOR kinetic mechanism is different from housefly and fungus, and yeast CYPORs,
of those enzymes, follow the classical Ping-Pong mechanism at high ionic strength
(Mayer and Durrant, 1979; Murataliev et al., 1999; Lamb et al., 2001; Mokovec and
Breskvar, 2002). Surprisingly, the flAnCYPOR kinetic mechanism was random Bi-Bi at
low ionic strength (see Supp. Fig. S2), similar to that observed for CYPORs of
rat, housefly, fungus, and yeast. It is suggested for rat CYPOR that the electron donor
site follows the Ping-Pong mechanism, but the electron acceptor site is changed
from Ping-Pong at high ionic strength to random Bi-Bi at low ionic strength. These
sites are accommodated with 2 separate domains shown in the crystal structure of
rat CYPOR, including an FMN-binding domain for cytochrome c binding and an
FAD/NADPH-binding domain for NADPH binding (Sem and Kasper, 1994, 1995;
Wang et al., 1997). Therefore, the enzymatic activity and kinetic mechanism of the
An. minimus CYPOR is ionic strength dependent, similar to those CYPOR enzymes
from rat (Rattus norvegicus), housefly (Musca domestica), fungi (Rhizopus nigricans), and
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241
yeast (Saccharomyces cerevisiae) (Shen et al., 1991; Sem and Kasper, 1995; Murataliev
et al., 1999; Mokovec and Breskvar, 2002).
Although there is no structural information for insect CYPOR enzymes, a higher
trypsin sensitivity of AnCYPOR over rat CYPOR (Sarapusit et al., 2008) may indicate
more open conformation of flAnCYPOR enzyme compared to rat CYPOR structure
(Wang et al., 1997; Hubbard et al., 2001). As a result, the open structure could make
flAnCYPOR loosely bind flavin cofactors, and, unlike rat and housefly CYPORs, could
not be reconstituted with exogenous flavins in vitro (Mayer and Durrant, 1979; Shen
et al., 1989). However, an increase in cytochrome c reduction activity upon addition of
exogenous flavins indicated free access of exogenous flavins into a large and flexible
binding site during enzymatic reaction. The absence of cytochrome c–reduction
activity in the absence of flAnCYPOR enzyme excludes the possibility of an external
electron transfer pathway to cytochrome c by exogenous flavins (unreported data).
The results of kinetic studies further indicated that FAD cofactor and NADPH
(or NADP1) are bound at site 1 (the FAD/NADPH domain) while FMN and
cytochrome c bound at site 2 (the FMN domain) of the flAnCYPOR enzyme. In
Penicillium chrysogenum fungus, nitrate reductase (NR), an enzyme that catalyzes the
NADPH-dependent reduction of inorganic nitrite to nitrate, follows a non-classical
two-site Ping-Pong mechanism and requires exogenous FAD coenzyme for its activity.
The NR enzyme has site 1 for random binding by NADPH (or NADP1) and FAD,
while site 2 for NO
3 (or NO2 ). It is then the FAD that may mediate the transfer of
electrons of both sites 1 and 2 of the NR enzyme, or act as a shuttle for electron flows
between the two sites (Renosto et al., 1981, 1982). A requirement for exogenous flavins
to fulfill the reduction activity might be a regulatory system of the An. minimus
mosquito CYPOR in vivo to increase electron transfer to their target P450 enzymes,
thus regulating the mosquito P450 metabolizing activity by CYPOR activity.
Since microsomal P450 activities require electron supplement from CYPOR, any
change in CYPOR could affect the function of all P450 enzymes. The mutations could
affect electron transfer activity of CYPOR enzyme, resulting in decreased cytochrome
c–reduction activity and the P450-mediated oxygenase reaction in vitro. The loss of
NADPH, FAD, and FMN from its specific binding site is the major cause of diminished
P450 activity, especially when the mutation sites are located close to the corresponding binding sites. Among human CYPOR mutational sites, the R457H and
V492E mutations at the FAD-binding site have caused a total loss in CYP17A1
(17a-hydroxylase/17, 20-lyase) and CYP19A1 (aromatase) activities and impaired the
drug-metabolizing CYP1A2 enzyme activity in vitro (Marochic et al., 2006; Flú´ck et al.,
2007; Pandey et al., 2007; Kranendonk et al., 2008). Additionally, the R457H and
V492E mutant enzymes, when FAD reconstituted, could restore the cytochrome
c–reduction activity and the CYP1A2 activity (Kranendonk et al., 2008), emphasizing
structural integrity of CYPOR for exogenous flavins to incorporate into the CYPOR
enzyme during enzymatic catalysis. Mutations in other positions could also affect P450
activity, possibly through induction of CYPOR conformational change (Flú´ck et al.,
2004, 2007; Huang et al., 2005; Pandey et al., 2007).
We previously reported that the leu86 and leu219 play a crucial role in the folding
of An. minimus CYPOR (Sarapusit et al., 2008). Although these two leucine residues are
not directly involved in FMN binding determined based on rat CYPOR crystal
structure (Wang et al., 1997), they are buried in two hydrophobic cores (one formed by
F69, F83, F135, and F152; the other by F94, F171, F201, F216, and W219) of the FMN
domain and thus could influence FMN binding. This is shown by high flavin cofactor
Archives of Insect Biochemistry and Physiology
242
Archives of Insect Biochemistry and Physiology, April 2010
binding in flAnCYPOR mutant enzymes containing phenylalanine replacement at
leu86 and leu219 positions, accompanied by increased cytochrome c reduction activity
compared to the wild-type flAnCYPOR. However, the mutations do not cause any
change in the substrate-binding mode and kinetic mechanism, since the Km values for
NADPH and cytochrome c were similar to the wild-type flAnCYPOR. Further double
mutation by phenylalanine replacement at both leu86 and leu219 resulted in a higher
turnover number of enzyme, a more efficient electron transfer, and thus the support of
CYP6AA3-mediated deltamethrin degradation in vitro shown in this study.
The endogenous Sf9 CYPOR enzyme might also support CYP6AA3-mediated
reaction, but accounted for low CYP6AA3 enzymatic activity as shown in Table 3. The
membrane-deleted D55AnCYPOR did not support a CYP6AA3-mediated reaction
since the reconstitution reaction retained similar activity as seen for the control
reaction, possibly due to endogenous CYPOR (unreported data). The present study
thus revealed that the N-terminal membrane domain of mosquito CYPOR contributes
significantly to its enzymatic properties and catalysis. The results are in agreement with
prior reports that the N-terminal membrane region of CYPOR is required for P450
interaction and the CYPORs that contain N-terminal anchor deletion could retain
cytochrome c–reducing activity, but could no longer reduce cytochrome P450
(Andersen et al., 1994; Hayashi et al., 2003). In addition, the significant lower
cytochrome c Km in flAnCYPOR compared to the D55AnCYPOR enzyme (Sarapusit
et al., 2008) highlighted the important role of the membrane-binding domain in
enzyme properties and substrate binding of AnCYPOR compared to CYPORs of rat,
human, and fungus (Shen et al., 1989; Lamb et al., 2001; Elmore and Porter, 2002).
The AnCYPOR enzyme thus could be used as a mosquito CYPOR model for further
characterization of the role of the N-terminal domain in enzyme folding and possibly
the specific interaction with mosquito P450 enzymes.
ACKNOWLEDGMENTS
We thank Dr. Jung-Ja P. Kim and Dr. Chuanwu Xia for helpful suggestions during the
preparation of this manuscript.
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acid, substitution, phenylalanine, mosquitoes, amin, leu86, oxidoreductasekinetics, leu219, deltamethrin, mediated, cytochrome, cyp6aa3, p450, metabolico, nadph, role
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