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Insect Biochemistry and Molecular Biology 54 (2014) 42e52
Contents lists avai
Insect Biochemistry and Molecular Biology
journal homepage: www.elsevier .com/locate/ ibmb
Expression and characterization of an epoxide hydrolase
fromAnopheles gambiae with high activity on epoxy fatty acids
Jiawen Xu, Christophe Morisseau, Bruce D. Hammock*
Department of Entomology and Nematology, UC Davis Comprehensive
Cancer Center, University of California, One Shields Avenue, Davis,
CA 95616, USA
a r t i c l e i n f o
Article history:Received 19 July 2014Received in revised form8
August 2014Accepted 15 August 2014Available online 27 August
2014
Keywords:CharacterizationEpoxide hydrolaseAnopheles gambiaeEpoxy
fatty acids
Abbreviations: EH, epoxide hydrolase; JH, juveepoxide hydrolase;
mEH, microsomal epoxide hydrolepoxide hydrolase; AgEH, EH from
Anopheles gambiapiens; MsEH, sEH from Mus musculus; RsEH, sEH
frosEH1 from Strongylocentrotus purpuratus; SpEH2, spurpuratus;
AtEH, sEH from Arabidopsis thaliana; GsEHsEH from Solanum
tuberosum; CeEH1, sEH1 from CasEH2 from Caenorhabditis elegans;
HmEH, mEH fromfrom Rattus norvegicus; DmEH, mEH from Drosophilafrom
Manduca sexta; BmJHEH-r1, JHEH-r1 from Bomoxide; t-SO,
trans-stilbene oxide; t-DPPO, trans-depoxyeicosatrienoic acids;
EpOME, epoxy octadecadamantan-1-yl-ureido) dodecanoic
acid;trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-bentrifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl*
Corresponding author. Tel.: þ1 530 752 7519; fax
E-mail address: [email protected] (B.D. H
http://dx.doi.org/10.1016/j.ibmb.2014.08.0040965-1748/© 2014
Elsevier Ltd. All rights reserved.
a b s t r a c t
In insects, epoxide hydrolases (EHs) play critical roles in the
metabolism of xenobiotic epoxides from thefood resources and in the
regulation of endogenous chemical mediators, such as juvenile
hormones.Using the baculovirus expression system, we expressed and
characterized an epoxide hydrolase fromAnopheles gambiae (AgEH)
that is distinct in evolutionary history from insect juvenile
hormone epoxidehydrolases (JHEHs). We partially purified the enzyme
by ion exchange chromatography and isoelectricfocusing. The
experimentally determined molecular weight and pI were estimated to
be 35 kD and 6.3respectively, different than the theoretical ones.
The AgEH had the greatest activity on long chain epoxyfatty acids
such as 14,15-epoxyeicosatrienoic acids (14,15-EET) and
9,10-epoxy-12Z-octadecenoic acids(9,10-EpOME or leukotoxin) among
the substrates evaluated. Juvenile hormone III, a terpenoid
insectgrowth regulator, was the next best substrate tested. The
AgEH showed kinetics comparable to themammalian soluble epoxide
hydrolases, and the activity could be inhibited by AUDA
[12-(3-adamantan-1-yl-ureido) dodecanoic acid], a urea-based
inhibitor designed to inhibit the mammalian soluble
epoxidehydrolases. The rabbit serum generated against the soluble
epoxide hydrolase of Mus musculus can bothcross-react with natural
and denatured forms of the AgEH, suggesting immunologically they
are similar.The study suggests there are mammalian sEH homologs in
insects, and epoxy fatty acids may beimportant chemical mediators
in insects.
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
Anopheles gambiae mosquitoes are the most important vectorsof
malaria, which is one of the most severe insect-borne
diseases.Approximately 3.3 billion people worldwide are at risk
from
nile hormone; sEH, solublease; JHEH, juvenile hormonee; HsEH,
sEH from Homo sa-m Rattus norvegicus; SpEH1,EH2 from
Strongylocentrotus, sEH from Glycine max; StEH,enorhabditis
elegans; CeEH2,Homo sapiens; RmEH, mEHmelanogaster; MsJHEH, JHEHbyx
mori; c-SO, cis-stilbeneiphenylpropene oxide; EET,enoic acids;
AUDA, 12-(3-t-TUCB, trans-4-{4-[3-(4-zoic acid; TPPU, 1-) urea.: þ1
530 752 1537.ammock).
malaria, and it caused an estimated 627,000 deaths in 2012
(WHO,2012). In order to understand the blood feeding behavior and
theunique interactions between mosquitoes and their hosts,
recentstudies have found a variety of blood-derived factors that
areingested by female mosquitoes, and are still biologically active
inthe midgut. These blood components include some cytokines
(TGF-b1), growth factors (insulin and insulin-like growth factors),
path-ogen derived molecules (glycosylphosphatidylinositols and
hemo-zoin of Plasmodium falciparum) and others (Akman-Anderson et
al.,2007; Beier et al., 1994; Lim et al., 2005; Surachetpong et
al., 2009).These blood-derived molecules can trigger the conserved
signalingpathways in mosquitoes to affect mosquito physiology, like
aging,reproduction, immune responses and disease transmission
pat-terns (Pakpour et al., 2013), which can critically affect the
capacityof mosquitoes as disease vectors. To fully comprehend the
in-teractions between mosquitoes and their hosts, additional
bloodfactors need to be identified and their functions studied.
Epoxy fatty acids and their corresponding diols are autocrineand
paracrine signaling molecules. Epoxyeicosatrienoic acids(EETs) are
epoxygenated metabolites of C-20 arachidonic acid, andEETs belong
to a group of potent chemical mediators termed
Delta:1_given nameDelta:1_surnameDelta:1_given
namemailto:[email protected]://crossmark.crossref.org/dialog/?doi=10.1016/j.ibmb.2014.08.004&domain=pdfwww.sciencedirect.com/science/journal/09651748http://www.elsevier.com/locate/ibmbhttp://dx.doi.org/10.1016/j.ibmb.2014.08.004http://dx.doi.org/10.1016/j.ibmb.2014.08.004http://dx.doi.org/10.1016/j.ibmb.2014.08.004
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J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e52 43
eicosanoids. Together with prostaglandins and leukotrienes,
theseeicosanoids have been extensively studied in the mammalian
sys-tems in the context of human health and drug
development(Morisseau and Hammock, 2013; Tapiero et al., 2002). In
insects it isknown that eicosanoids are also involved in insect
physiology suchas ion transport, reproduction and immunity
(Stanley, 2006;Stanley and Kim, 2014; Stanley and Miller,
2006).
Epoxy fatty acids are endogenous substrates in mosquitoes.
Likemammals, mosquitoes may synthesize epoxides of unsaturatedfatty
acids by a variety of cytochrome p450 enzymes. In addition,epoxy
fatty acids are regular components of mammalian blood(Imig, 2012;
Jiang et al., 2012, 2005), and may be xenobiotic sub-strates for
the female mosquitoes during blood feeding.
In mammals, EETs are short-lived lipid signaling molecules,
andare mainly hydrolyzed by the soluble epoxide hydrolase
(sEH),which was discovered while studying the mammalian
metabolismof insect juvenile hormone and its analogs (Gill et al.,
1974, 1972).The sEH turned out to be a therapeutic target for a
variety ofmammalian diseases (Imig, 2005; Imig and Hammock,
2009;Schmelzer et al., 2005; Zhang et al., 2007). In insects,
epoxide hy-drolases with activities on juvenile hormones (JHEHs)
are the bestcharacterized EHs (Anspaugh and Roe, 2005; Keiser et
al., 2002;Khalil et al., 2006; Seino et al., 2010; Tsubota et al.,
2010; Zhanget al., 2005). These EHs are believed to be involved in
the meta-bolic degradation of juvenile hormones in vivo (Li et al.,
2004;Prestwich et al., 1996), which are key developmental and
repro-ductive hormones (Goodman and Cusson, 2011). So far, the
insectmEHs and JHEHs characterized are homologous to
mammalianmicrosomal epoxide hydrolases (Newman et al., 2005;
Prestwichet al., 1996). The homologs of mammalian soluble epoxide
hydro-lases in insects have not been studied to our knowledge,
althoughthe sEH homologs had been reported in the Caenorhabditis
elegans(Harris et al., 2008). The AgEH characterized here shows
evolu-tionary, biochemical, and immunological similarities to
mamma-lian sEHs, suggesting there are sEH homologs in insects, and
epoxyfatty acids may be important chemical mediators for insects.
Thebiochemical characterization from this study provides
knowledgeand tools to pave the road for investigating whether epoxy
fattyacids (such as EETs, known for biomedical studies from
mammals)play a profound role in mosquito biology.
2. Materials and methods
2.1. Phylogeny analysis
Protein sequences of previously reported epoxide hydrolasesand
putative mosquito EH sequences were obtained from thedatabase in
the National Center for Biotechnology. Sequences werealigned and
compared by ClustalWOmega. The phylogeny tree wasgenerated using
MEGA Version 5.2.1 (Tamura et al., 2011) with theNeighbor-Joining
method (Saitou and Nei, 1987). 26 EH sequenceswere employed to
infer the bootstrap consensus tree from 1000replicates
(Felsenstein, 1985). The percentage of replicate trees inwhich the
associated taxa clustered together in the bootstrap test(1000
replicates) is shown next to the branches. The
evolutionarydistances were computed using the Poisson correction
method.
2.2. Generation of recombinant virus
Many epoxide hydrolases have been successfully expressed inthe
baculovirus system by insect cells. We also chose to express
theAgEH with this eukaryotic expression system. The sf-9 cell lines
areof insect origin, and we did not detect significant
backgroundepoxide hydrolase activities with the substrates used
under theassay conditions. The open reading frame sequence (AGAP
011972)
was purchased from GenScript (Piscataway, NJ). Primers
weredesigned to add Bgl II and EcoR I endonuclease-cutting sites at
theN-terminal and C-terminal end, respectively. There were no
tagsadded. The insert was cloned into the transfer vector pAcUW
21(Weyer et al., 1990) by T4 DNA ligase (New England Biolabs,
MA).Recombinant baculoviruses were generated by co-transfection
ofinsect Sf9 cells with Bsu 36 I-digested BacPak 6 viral DNA
(Clontech,CA) and the transfer vector pAcUW 21. Recombinant viruses
wereamplified and isolated by three consecutive plaque assays to
ensureconsistency. The titer of final recombinant viruses was
determinedby plaque assays, which was 5.4 � 108 pfu/ml.
2.3. Baculovirus expression and differential centrifugation
Control recombinant CpJHE baculoviruses and recombinantAgEH
baculoviruses were used to infect insect Sf-9 cells, whichwere
grown to a density of 1 � 106 cells/ml in Ex-cell 420 serumfree
medium (SigmaeAldrich, MO) with 1% Pen/Step
antibiotics(SigmaeAldrich, MO) in a 50 ml shaker. The two
recombinant vi-ruses were generated in the same way, except that
the insertedgenes were different. Cells infected by CpJHE will
express anesterase (Kamita et al., 2011) instead of an epoxide
hydrolase. Virus(10 M.O.I) was added, and cells were harvested two
days postinfection.
All of the following operations were carried out at 4 �C or
lower.Infected cells were pelleted at 100 � g for 10 min and
resuspendedin pH 8, 50 mM Tris buffer with 1 mM EDTA and 1 mM PMSF.
APolytron homogenizer (6000 rpm for 60 s) was used to break
thecells. The crude homogenates were centrifuged at 800 � g for10
min to remove cell debris, and the supernatant was centrifugedat
17,000 � g for 20 min to spin down mitochondria and peroxi-some
fractions. Then the supernatant was subjected to 100,000 � gfor 60
min. The resulting supernatant was collected as the
cytosolicfraction, and the pellet resuspended in Tris buffer
containing 0.02%CHAPS as the microsomal fraction. For each step,
the pellets werewashed once by Tris buffer before any further
processing. Proteinconcentrations were determined by the BCA
protein assay (Pierce,IL) throughout this study with BSA as the
standard.
2.4. Optimal pHs for enzyme activity and stability
In order to investigate the effects of pH on enzyme activity
andstability, cells were disrupted in different buffers. Sodium
acetate(pH 5 and 6), BiseTris (pH 6 and 7), phosphoric buffer (pH 7
and 8),Tris (pH 8 and 9), borate (pH 9 and 10), CAPS (pH 10 and 11)
werechosen to cover the pH range between 5 and 11. All buffers
were50 mM in concentration and sodium chloride was added
accord-ingly to make constant ionic strength at 100 mM for each
buffer.Enzyme activity was detected with t-DPPO as the
substrate.
2.5. Effect of ionic strength and inhibitors on enzyme
activity
Sodium chloride solutions (0 mMe3000 mM) were preparedand added
into 50mM Tris buffers, pH 8 to adjust the ionic strengthbetween 50
mM and 2000 mM. The effects of ionic strength(50 mM �2000 mM) on
enzyme activity were detected with t-DPPO, juvenile hormone III and
14,15-EET as the substrate(Morisseau, 2007).
Six small molecule inhibitors of different structural classes
wereused to study the inhibition patterns of the recombinant AgEH.
Thesynthesis, chemical and physical properties of these
compoundsare described somewhere else (Morisseau et al., 1999,
2002;Severson et al., 2002). Inhibitors were prepared in DMSO. 1 ml
ofinhibitors at the appropriate concentration was added into 100
mlenzyme solutions before addition of substrates (t-DPPO, JH III
or
-
J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e5244
14,15-EET). 1 ml of DMSO was added into enzyme solutions as
acontrol, although inhibition by DMSO was not observed at 1%
(v/v)concentration. Enzymes from the microsomal fractions were
used,and the incubation time was 5 min.
2.6. Solubilization of AgEH activity from the membrane
Enzymes were obtained by disrupting insect Sf-9 cells two
daysafter infection by baculoviruses at an M.O.I of 10. Microsomes
wereprepared as described in Section 2.3. Multiple conditions
wereevaluated to release the enzyme from the membrane,
includinghigh salt buffer (sodium chloride, 0 Me3 M), urea (1 M),
sonication(3 � 30 s) and detergents (Triton-X100, CHAPS at varied
concen-trations). The microsomes were treated and incubated for 1 h
at4 �C. The solution was centrifuged again at 100,000 � g for 1 h.
Theenzyme activity in the supernatant and pellet wasmeasuredwith
t-DPPO as the substrate.
2.7. Enzyme assays and determination of kinetics
c-SO, t-SO, t-DPPO and JH III were tritium-labeled
epoxidehydrolase substrates. The enzyme activity was determined
bypartition assays as previously described (Borhan et al., 1995;
Gillet al., 1983a; Mumby and Hammock, 1979). Briefly 1 ml of 5
mMc-SO, t-SO, t-DPPO, 14,15-EET, 9,10-EpOME or 0.5 mM JH III
sub-strates in DMSO were added into 100 ml of the
appropriatelydiluted enzyme solutions with 0.1 mg/ml BSA. For
9,10-EpOMEand 14,15-EET, the enzyme incubation was similar to
tritium-labeled compounds, but the products were analyzed by
LC-MS/MS (Morisseau, 2007).
The enzyme kinetics was studied using t-DPPO, JH III and
14,15-EETas substrates. Awide range of substrate concentrations was
firstemployed to determine the approximate range of the Km. Then
fivemore specific substrate concentrations covering the range Km/5
to5 � Km were used. Enzymes were diluted accordingly, and
incu-bated for 5 min, 10 min and 30 min at 30 �C in a water bath.
Eachassay was run in triplicate, and all the data within the linear
rangewere included in subsequent calculations (Morisseau, 2007).
Thekinetics was determined by three independent experiments. As
aresult, for each substrate concentration, at least 9 datum
pointswere available for the software SigmaPlot (Systat Software,
CA) tofit the MichaeliseMenton equation.
2.8. Partial purification of AgEH by ion exchange
chromatographyand isoelectric focusing
Solubilized AgEH fractions following treatment with 0.3%CHAPS
were used as the starting materials, and was assigned as100%
activity. Prepacked Q-Sepharose columns (GE healthcare, CA)were
washed by 10 column volumes of starting buffer, whichcontains 20
mM, pH 8 TriseHCl, 0.3% CHAPS and 10% glycerol.Solubilized AgEH
fractions were loadedwith a syringe, and the flowthrough was
collected. The column was then washed by 5 columnvolumes of
starting buffer, and then NaCl gradients (0.2 Me1 M).The flow rate
was controlled at 1 ml/min.
The NaCl eluate with the highest enzyme specific activity
wasdesalted by ultrafiltration (Amicon Ultra, 10K NMWL), and
recon-stituted with pure water to the final volume of 60 ml, which
con-tained 2% (w/v) pH 3-10 ampholytes (Bio-Rad, CA), 0.3% CHAPS
and10% glycerol. In a cold room with temperature set at 4 �C,
sampleswere loaded on the Rotofor cell (Bio-Rad, CA), and focusing
was runat 10 W constant power with a cooling circulator set at 4 �C
for 4 h.The initial conditions were 500 V and 20 mA. At equilibrium
thevalues were 2000 V and 5 mA. 20 Rotofor fractions were
collectedby vacuum aspiration. The pH of each fractionwasmeasured
at 4 �C
using a Corning 430 pH meter. The pH was adjusted to 8 by
adding100 ml of fraction solutions to 900 ml of 50 mM, pH 8 Tris
buffer. Thespecific activity of each fractionwas then measured with
t-DPPO asthe substrate.
2.9. SDS-PAGE and western blot analysis of purification
fractions
Solubilized enzymes (S), 0.2 M NaCl Q-Sepharose eluate and
aselection of Rotofor fractions (#3-#13) were loaded on a
4e20%Triseglycine gel (Life Technologies, CA), and stained by
Sypro®
Ruby Red stain (Life Technologies, CA). The proteins were
alsotransferred to a nitrocellulose membrane by the Pierce G2
FastBlotter (Thermo Scientific, IL). The membrane was blocked by
1%milk in Tris-buffered saline with 0.1% tween 20 (TBST) at
roomtemperature for one hour, and blotted against a 1/10,000
dilutedpolyclonal mouse sEH antibody (Imig et al., 2002) in TBST at
4 �Covernight. The antibody was screened and found to have
cross-reactivity with the AgEH (Fig. S4). The membrane was
thenwashed in TBST and incubated with the 1/10,000 diluted
goatanti-rabbit secondary antibody conjugated to horseradish
perox-idase (Abcam, UK). Detection was achieved by incubating
themembrane with SuperSignal® West PicoChemiluminescent Sub-strate
(Thermo Scientific, IL) and exposing blot to an X-ray film for1
min.
2.10. Immunoprecipitation of the AgEH activity
20 mL of Pierce Protein A/G Plus Agarose slurry (10 mL of
settledresin) was added into a microcentrifuge tube, and washed by
1Xcoupling buffer (Pierce, IL). Absorption at 280 nm was used to
es-timate the protein concentration of the rabbit anti-mouse
sEHserum (Imig et al., 2002). The serum varying from 30 mg to 1200
mgtotal IgG was added into the tube. The agarose-antibody
mixturewas incubated on a rotator at room temperature for 60 min.
Thetubewas subject to centrifugation at 1000� g for 2 min at 4 �C,
andthe supernatant was discarded. The resinwas thenwashed twice
by1X coupling buffer. The solubilized AgEH solution was
pre-clearedby incubating 500 mL of the solubilized AgEH with the
controlagarose resin (Pierce, IL) at 4 �C for 60 min with gentle
end-over-end mixing. The pre-cleared solution was then added to
theagarose resin coupled with mouse sEH serum. The mixture
wasincubated overnight at 4 �C. After the incubation, the tube
wascentrifuged, and the supernatant was collect for measuring
theAgEH activity with t-DPPO as the substrate.
3. Results
3.1. Phylogeny analysis of the epoxide hydrolase from A.
gambiae(AgEH)
Most epoxide hydrolases studied belong to the a/b
hydrolasefamily, which share similar three-dimensional structures
andenzymatic mechanism (Morisseau and Hammock, 2005; Newmanet al.,
2005). Based on such structural and enzymatic similarities,in
silico studies of the genome of A. gambiae revealed one
putativeinsect epoxide hydrolase (AGAP 011972) that was distinct
frominsect mEHs and JHEHs in homology. The resulting sequence
(AGAP011972, showed in Fig. S1) contained conserved catalytic triad
(D-159, H-319, D-291), oxyanion hole motif (HGXP, residues
90e93)and two tyrosines (Y-206, Y-261), which are all signature
elementsfor epoxide hydrolases to function.
In the phylogenic analysis (Fig. 1), the AgEH was clustered
withputative EH sequences from the other two medically
importantmosquitoes, Aedes aegypti (2 sequences, prefixed with
‘AAEL’) andCulex quinquefasciatus (5 sequences, prefixed with
‘CPIJ’). Except for
-
Fig. 1. Phylogeny tree of the AgEH and other epoxide hydrolases
from plants, insects, nematodes, sea urchins, chickens and mammals.
The tree was generated by MEGA 5.2.1(Tamura et al., 2011). The full
names of abbreviations are detailed in the paper. The accession
number of amino acid sequences is shown in the parenthesis. The
percentage ofreplicate trees in which the associated taxa clustered
together in the bootstrap test (1000 replicates) is shown next to
the branches.
J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e52 45
the orthologs in mosquitoes, the AgEH was most close to
solubleepoxide hydrolases from C. elegans (CeEH 1 and 2), EH 3 and
EH 4from Homo sapiens. It was also homologous to soluble
epoxidehydrolase from plants (AtEH, from cress Arabidopsis
thaliana; StEH,from potato Solanum tuberosum and GsEH, from soybean
Glycinemax) to a lesser extent. These epoxide hydrolases contained
theconserved C-terminal epoxide hydrolase domain of sEHs
frommammals (Arahira et al., 2000; Morisseau et al., 2000), but
lackedthe N-terminal phosphatase domain. Among all the
sequencesanalyzed, the AgEH was remotely related to the reported
micro-somal EH homologs, including mammalian microsomal EHs(RmEH,
from rat Rattus norvegicus; HmEH, from human H. sapiens),mainly
known for their role in detoxification (Morisseau andHammock,
2008), a microsomal EH (DmEH, from fruit flyDrosophila
melanogaster) that cannot hydrolyze juvenile hormones(Taniai et
al., 2003), and also insect JHEHs (MsJHEH, from the to-bacco horn
worm Manduca sexta; BmJHEH-r1, from silkwormBombyx mori) that have
a high hydrolytic activity on juvenile hor-mones (Seino et al.,
2010; Touhara et al., 1994). There are also threeputative JHEH
sequences in the tree (prefixed with AGAP), and theyall clustered
with previously reported insect mEHs and JHEHs.
3.2. Substrate selectivity of AgEH
We moved on to test the hypothesis that the sequence AGAP011972
did code for a catalytically active epoxide hydrolase. Theenzyme
was expressed in insect Sf-9 cells infected by 10 M.O.I
re-combinant AgEH viruses or recombinant CpJHE viruses as a
control.The reported data were corrected for non-enzymatic
hydration inthe assays (Fig. S2). Epoxide hydrolase activity
detected in celllysate from the CpJHE virus infection was about
700e1000 timeslower than the cell lysate from the AgEH virus
infection (Fig. S2).
The structures of epoxide substrates are shown (Fig. 2).
Therewas no activity greater than the non-enzymatic hydrolysis of
c-SOand t-SO in cells infected by the recombinant AgEH
viruses(Table 1). Although a typical substrate for many mammalian
andinsect microsomal EHs (Gill et al., 1983b; Kamita et al.,
2013;Morisseau and Hammock, 2005; Taniai et al., 2003), c-SO was
nota substrate for the AgEH. However, the AgEH was
catalyticallyactive on JH III (98 nmol diol formed � min�1 � mg1
protein), t-DPPO (564 nmol diol formed � min�1 � mg�1 protein),
14,15-EET(550 nmol diol formed � min�1 � mg�1 protein) and
9,10-EpOME(360 nmol diol formed � min�1 � mg�1 protein). t-DPPO is
a
-
Fig. 2. Epoxide containing substrates evaluated in the study.
The full names of thesubstrates are detailed in the text.
Table 1Substrate selectivity of the AgEH.
Substrate Specific activity (nmol diolformed/(min � mg
protein))
c-SO
-
Fig. 3. Effects of pH and buffer composition on enzyme activity.
Enzyme activity wasmeasured with t-DPPO as the substrate with
triplicate assays. Data representmean ± SD (n ¼ 3) except when SD
is smaller than the datum point.
Fig. 4. Enzyme stability in different buffers and pHs. The
enzyme was stored in a bucket of crwith triplicate assays. Data
represent the percentage of activity remaining (activity at day
1
Fig. 5. Inhibitors of the sEH (1e3) and mEH (4e6) used in the
study. AUDA, t-TUCB andElaidamide (#4) is a potent microsomal EH
inhibitor (Morisseau et al., 2008). #5 and #6 ar
J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e52 47
3.5 fold and 11 fold higher than those of 9,10-EpOME, JH III and
t-DPPO respectively.
The kinetics of AgEH on 14,15-EET and 9,10-EpOME is compa-rable
to the soluble epoxide hydrolase from H. sapiens, to whichepoxy
fatty acids are considered the endogenous substrates (Yuet al.,
2000; Zeldin et al., 1993). The Vmax/Km of AgEH on 14, 15-EET and
9,10-EpOME were slightly higher (2.3 and 1.6 timesrespectively)
than that of sEH fromH. sapiens, even considering thata crude
microsomal fraction was used to determine the kinetics.The AgEH and
the sEH from H. sapiens can both hydrolyze juvenilehormone III, at
a lower Vmax/Km ratio than on epoxy fatty acids. TheJHEH
fromManduca sexta hydrolyzed JH III with a low Km (0.28 mM)and a
low Vmax (0.095 mmol � min�1 � mg�1 proteins) while AgEHhydrolyzed
JH III with a high Km (9.8 mM) and a high Vmax(1.3 mmol � min�1 �
mg�1 proteins). For t-DPPO, the Vmax/Km ofAgEH was 8 times lower
than that of human sEH, but 46 timeshigher than that of MsJHEH.
ushed ice at a 4 �C freezer. Enzyme activity was measured with
t-DPPO as the substrateis assigned 100%).
TPPU (#1 to #3) are urea-based mammalian sEH inhibitors
(Morisseau et al., 1999).e two potent inhibitors for the JHEH from
Manduca sexta (Severson et al., 2002).
-
Table 3Inhibition of the AgEH by sEH, mEH or JHEH
inhibitors.
Inhibitors t-DPPO JH III 14,15-EET
5 nM 50 nM 500 nM 5 nM 500 nM 50 nM 5 nM 50 nM 500 nM
1 44.9 9.5 N.D. 57.2 11.2 4.7 79.0 34.7 7.12 90.4 57.3 12.5 96.3
85.2 34.3 98.4 60.3 91.03 99.5 94.0 81.8 100 98.9 100 100 100 1004
98.6 79.2 60.6 100 98.8 96.4 100 100 95.45 20.4 8.7 N.D. 70.3 45.9
22.6 100 86.3 32.36 50.6 28.7 8.6 94.7 70.2 50.7 100 100 64.0
Values are % of activity remaining with the presence of
inhibitors. Inhibitors in DMSO were added, and incubated for 5 min
before substrates were added into enzyme so-lutions. Enzymes from
the microsomal fraction were used. Inhibition assays were done with
triplicate assays. The SDs (n ¼ 3) are all within 10% of the mean
value, and are notshown in the table. The enzyme assays were
performed in 50 mM TriseHCl, pH 8.0 containing 50 mM substrates (5
mM for JH III), inhibitors, 1% (v:v) DMSO and 0.1 mg/ml BSAat 30
�C.
Fig. 6. IC50 of AUDA (Compound 1 in Fig. 5) on the AgEH's
activity on 14,15-EET. The 4parameter logistic model describes the
sigmoid-shaped response was used to calculatethe IC50 (SigmaPlot,
C.A).
Table 5Attempts to release the AgEH activity from the microsomal
membrane by treat-ments of salts, urea and sonication.
Treatment % Of activity recoveredfrom treatment
% Of activity remaining inthe microsomes
Washed microsomes 100 86 ± 5þ1M NaCl 94 ± 2 92 ± 4þ1M Urea 88 ±
2 81 ± 2þ3 � 30s Sonication 90 ± 1 85 ± 6
Microsomes were prepared and subjected by the corresponding
treatments above.They were then repelleted and washed once with
Tris buffer before t-DPPO activitywas measured in the resulting
pellets and supernatant. Values are means ± SD(n ¼ 3).
J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e5248
3.7. Solubilization of AgEH activity from the membrane
In order to solubilize the AgEH from the membrane, high
saltbuffers, urea, sonication were first evaluated to release the
enzymefrom the membrane. High salt buffer (sodium chloride at 0e3
M),urea (1 M) and sonication (3 � 30 s) did not release a
significantamount of enzyme activity from the membrane (Table 5),
whichsuggested that the enzyme was not loosely bound with
themembrane.
Table 4Enzyme kinetics of the AgEH on four epoxide hydrolase
substrates.
Substrate Kinetic parameter
t-DPPO Km (mM)Vmax (mmol � min�1 � mg�1)Vmax/Km(L � min�1 �
mg�1)
9,10-EpOME Km (mM)Vmax (mmol � min�1 � mg�1)Vmax/Km(L � min�1 �
mg�1)
JH III Km (mM)Vmax (mmol � min�1 � mg�1)Vmax/Km(L � min�1 �
mg�1)
14,15-EET Km (mM)Vmax (mmol � min�1 � mg�1)Vmax/Km(L � min�1 �
mg�1)
Enzymes from the microsomal fractions were used for kinetics.a
The kinetics of MsJHEH on t-DPPO and JH III are from Severson and
Touhara respective
the substrate.b The kinetics of human sEH on t-DPPO, 9,10-EpOME
and 14,15-EET are from Morisse
Then we tried to solubilize the enzyme with two
detergents(Triton X-100 and CHAPS), and the result is shown (Table
6). Theaddition of 0.3% CHAPS was detrimental to the activity (49%
re-covery from resuspended microsomes), but could solubilize 75%
ofrecovered enzyme activity to the supernatant, while in
lowerconcentrations (0.01%e0.1%), the majority of activity was
still in thepellets. Triton X-100 was not as efficient as CHAPS in
solubilizingAgEH activity in terms of recovery and solubilized
activity (33%maximum).
3.8. Partial purification of the AgEH and analysis of
purificationfractions by SDS-PAGE and western blot
The starting material was 40 ml solubilized enzyme fractionswith
a total activity of 52 U (1 U ¼ 1 mmol/min) and a specific
AgEH MsJHEHa hsEH
30.5 ± 5.0 65.6 6.2 ± 0.6b1.2 ± 0.1 0.059 2.1 ± 0.10.041 0.0009
0.34
7.0 ± 0.6 2.6 ± 0.4b
1.5 ± 0.4 N.A. 0.35 ± 0.030.21 0.13
9.8 ± 2.0 0.28 1.5 ± 0.61.3 ± 0.1 0.095 0.067 ± 0.0070.13 0.34
0.04
3.0 ± 0.3 7.0 ± 0.3b
1.4 ± 0.03 N.A 1.4 ± 0.050.46 0.20
ly (Severson et al., 2002; Touhara et al., 1994). N.A. indicates
data are not available for
au (Morisseau et al., 2000, 2010).
-
Fig. 7. pH gradient and specific activity of Rotofor fractions.
pH of fractions weremeasured, and 100 ml of each fraction was
diluted with 900 ml 50 mM TriseHCl, pH 8buffer before activity was
measured. Specific activity (mmol diols/(min � mg protein))was
measured with t-DPPO as the substrate. The pI determined was
6.3.
Table 6Solubilization of the AgEH activity by CHAPS and Triton
X-100.
Activity(mmol diol/min)in the pellet
Activity(mmol diol/min)in the supernatant
Recovery ofactivity aftertreatment (%)
% Of activityin thesupernatant
CHAPS percentage0.01% 0.08 ± 0.003 0.002 ± 0.0001 100 20.02%
0.07 ± 0.001 0.006 ± 0.0003 93 80.07% 0.06 ± 0.007 0.009 ± 0.0008
84 130.1% 0.06 ± 0.002 0.01 ± 0.003 85 140.2% 0.04 ± 0.001 0.015 ±
0.002 67 270.3% 0.01 ± 0.004 0.03 ± 0.002 49 750.4% 0.007 ± 0.001
0.015 ± 0.004 27 680.5% N.D. N.D. 0 0
Triton X-100 percentage0.01% 0.08 ± 0.003 0.003 ± 0.0001 100
40.02% 0.06 ± 0.002 0.015 ± 0.0004 90 200.07% 0.05 ± 0.004 0.018 ±
0.0005 82 260.1% 0.04 ± 0.001 0.02 ± 0.003 72 330.2% 0.03 ± 0.003
0.009 ± 0.0001 47 230.3% 0.009 ± 0.0008 0.001 ± 0.0001 12 10
The activity in microsomes with 0.01% detergent was assigned
100% activity. Thepellets were incubated with detergent at 4 �C for
1 h and re-pelleted. The criticalmicelle concentration (CMC) of
CHAPS and Triton X-100 is 8e10 mM(0.4920e0.6150%, w/v) and
0.22e0.24 mM (0.013%e0.015%, w/v) respectively.
J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e52 49
activity of 0.18 U/mg protein (Table 7). The 0.2 M NaCl
Q-Sepharoseeluate contained 67% of total activity but only achieved
1.7 fold ofpurification, and lower specific activities were also
detected inother fractions (Fig. S3). Ionic strength from 0.05 M to
2 M did nothave a significant effect on enzyme activity when t-DPPO
was usedas the substrate (Table S1). The #7 Rotofor fraction had a
total ac-tivity of 5 U and a specific activity of 3.13 U/mg
proteins. Thus, thepurification factor was 17 fold with 10%
recovery (Table 7).
The cDNA of the AgEH (AGAP 011972) is 1492 bp long with adeduced
340 amino acid sequence (Fig. S1). The predicted molec-ular mass
and pI are 40.9 kD and 9.2 respectively (Artimo et al.,2012). The
Rotofor determined pI of the AgEH was 6.3. High spe-cific
activities were detected in fraction #4, 5, 6, 7, 8, and the
highestactivity was found in fraction #7, which had a pH of 6.3
(Fig. 7).When proteins were loaded on a 4e20% gradient Triseglycine
SDS-PAGE gel (Life Technologies, CA), and the PageRuler
UnstainedProtein ladder (Thermo Scientific, MA) was used as the
marker, aband approximate 35 kD (Fig. 8a) was found to correlate
well withthe enzyme activity detected in different fractions. The
band wasalso recognized by a rabbit serum against mouse soluble
epoxidehydrolase (Fig. 8b). The rabbit serum also recognized a band
ap-proximates 35 kD in the crude lysate of insect cells (Fig. S4)
infectedby the recombinant AgEH baculoviruses, but not lysate of
the CpJHEinfected cells (Fig. S4), which expresses a recombinant
juvenilehormone esterase from C. quinquefasciatus. The CpJHE
viruses weregenerated in the sameway as the AgEH, except for that
the insertedgene was different (Kamita et al., 2011). The band was
also cut forprotein sequencing (UC Davis Proteomics Core), which
was diges-ted by trypsin. The data were analyzed by Scaffold
version 4.3.2(Proteome Software Inc., OR) based on peptide and
protein iden-tifications. The protein sequence of the AgEH was
identified with
Table 7Partial purification of the recombinant AgEH.
Volume (mL) Total protein (mg) Total activity (mmo
Solubilized fraction 40 282 52Q-Sepharose 20 114 35Rotofor
fraction#7 3 1.6 5
t-DPPO was used as the substrate. 1 U is 1 mmol/min 20 fractions
were collected from R
100% probability to a false discover rate less than 0.1% and
1.0% forpeptide and protein identifications (Nesvizhskii et al.,
2003)respectively.
3.9. Immunoprecipitation of the AgEH activity
The result of immunoprecipitation study is shown (Fig. 9).Whena
constant amount of the solubilized AgEH was incubated with avarying
amount of rabbit anti-mouse sEH serum, the AgEH activitywas
precipitated in a dose-dependent manner, indicating the nat-ural
form of the AgEH also cross-reacted with the rabbit serum.Elution
of the AgEH by low pH and high salt buffer had not beensuccessful,
and elution by SDS loading buffer resulted in a largecontamination
of antibodies and other proteins. Therefore, we havenot obtained a
homogenous and catalytically active AgEH.
4. Discussion
The AgEH has a different evolutionary history from insect
mEHsand JHEHs. They share the same subcellular location, but
havecomplementary and overlapping substrate selectivities. As a
result,EH activities detected from a specific subcellular location
cannot besimply assigned to one enzyme.
The AgEH orthologs are also found in the genome of A. aegyptiand
C. quinquefasciatus, two medically important mosquitoes aswell as
A. gambiae. Interestingly, wewere not able to find orthologsand
activities on EETs in D. melanogaster. The orthologs all share
anevolution different than the previously characterized insect
JHEHsand mEHs. The catalytic triad (Asp-Asp-His) present in the
ortho-logs is more commonly seen in sEHs. It is tempting to
characterizethe orthologs in A. aegypti and C. quinquefasciatus,
and determinewhether the orthologs are EHs, whether they can
hydrolyze epoxyfatty acids or juvenile hormones, and whether the
inhibitor andantibody described in this study can be useful
tools.
l/min) Specific activity (U/mg protein) Yield (%) Purification
factor
0.18 100 10.31 67 1.73.13 10 17
otofor cell and fraction #7 is shown because it has the highest
specific activity.
-
Fig. 8. SDS-PAGE (a) and western blot analysis (b) of Rotorfor
fractions.1 mg of solubilized enzymes (S), Q-Sepharose eluate (Q)
and Rotofor fractions #3 e #13 were loaded on a4e20% Triseglycine
gel (a). The proteins were also transferred to a nitrocellulose
membrane and blotted against the rabbit serum against mouse sEH
(b). The membrane wasexposed to an X-ray film in a dark room for 1
min.
J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e5250
The substrate selectivity (structures showed in Fig. 2)
suggeststhe AgEH hydrolyze 1,2-disubstituted epoxides (t-DPPO,
14,15-EET,9,10-EpOME) better than tri-substituted epoxides (JH
III), and theAgEH does not hydrolyze epoxides that are sterically
hindered onboth sides by bulky groups (c-SO and t-SO). The kinetics
and inhi-bition patterns both show that epoxy fatty acids are
preferredsubstrates among those that tested, and sEH inhibitor AUDA
is themost potent inhibitors among the inhibitors evaluated.
However,we can not exclude the possibility that the AgEHmay involve
in themetabolism of juvenile hormone in certain conditions. While
ju-venile hormone esterases are secreted to the hemolymph
(Kamitaand Hammock, 2010), membrane-associated epoxide
hydrolasesmay have a significant kinetic advantage regulating
juvenile hor-mone titer within cells. Comparing the AgEH to the
well-studiedJHEH from Manduca sexta, there are enormous differences
in theKm and Vmax, while their catalytic efficiencies are within
the samerange (Table 4). When the titer of juvenile hormones
increases, the
Fig. 9. Immunoprecipitation of the AgEH activity. 100% activity
refers to the amount ofactivity in the solution from non-immune IgG
coupled agarose. Solubilized AgEH wassubjected to
immunoprecipitation with rabbit serum against mouse soluble
epoxidehydrolase. Different amount of IgG were bound to Pierce
protein A/G resin plusagarose. Enzyme solutions were added, and
incubated with gentle end-over-endmixing overnight at 4 �C before
activity in the solution was measured with t-DPPOas the
substrate.
capacity of the JHEH to regulate juvenile hormone
metabolismmaybe strongly limited as the titer surpasses the low Km
of the JHEH.Meanwhile, the contribution of the AgEH to juvenile
hormonemetabolism may be significant because it has a high Vmax,
and ahigh Km that lies at a point that juvenile hormone titer is
probablynot able to reach. As a result, the AgEHmay play a role in
regulatingjuvenile hormone titer under conditions that a high
juvenile hor-mone titer is present locally and need to be
dramatically down-regulated.
In our case, the experimentally determined molecular
weight(around 35 kD) and pI (6.3) were different from the
theoretical ones(41kD, 9.2). It is not uncommon that amino acid
sequences are usedto predict the molecular weight and pI in
biochemical studies, butamino acid sequences cannot be used to
predict the three-dimensional structure and the post-translational
modifications(cleavage of signal peptide, glycosylation, attachment
of lipid),which can lead to miscalculation of physical properties,
such asmolecular weight and pI. As a membrane-associated enzyme,
theAgEH is expected to contain a signal peptide that is cleaved
duringprotein folding and processing, which may be the reason a
smallermolecular weight was detected than the predicted
molecularweight.
The rabbit serum for the mouse sEH can both detect the
dena-tured and natural form of the AgEH, indicating immunologically
theAgEH is similar to the mouse sEH. Although the overall
homologybetween the AgEH andmammalian sEHs is relatively low
(20e30%)(Table S2), the AgEH may also share similar
three-dimensionalstructure with the mammalian enzymes. Proteins
with lowsequence homology but similar structures have been
reportedbefore (Dickerson and Geis, 1983; Olsen et al., 1975).
Epoxide hy-drolases belong to the a/b hydrolase fold, the members
of whichshares no or low sequence homology, but have rather
similarstructures (Ollis et al., 1992). The similarities between
the AgEH andmammalian sEHs in overall sequence homology, conserved
cata-lytic triad, biochemistry and immunology clearly suggest that
theyhave diverged from a common ancestor, and they have evolved
topreserve similar epoxide hydrolase activities.
In mosquitoes, the epoxy fatty acids may also be endogenouslipid
signaling molecules or xenobiotic blood factors. In mammals,epoxy
fatty acids are lipid signaling molecules and players in
-
J. Xu et al. / Insect Biochemistry and Molecular Biology 54
(2014) 42e52 51
immune responses. The EpOMEs (leukotoxin) and its correspond-ing
diols have been reported to be a strong mediator of acute
res-piratory distress syndrome (ARDS) (Moghaddam et al., 1997),
andEETs are anti-inflammatory molecules that exert its effect
byreducing the activity of NF-kB (Inceoglu et al., 2011; Liu et
al., 2005;Morin et al., 2010; Node et al., 1999). In mosquitoes,
the Toll andImd pathways are the major immune signaling pathways
that arestudied in the context of immunity and disease
transmission. Bothpathways are highly conserved and depend on the
NF-kB tran-scription factor to play crucial roles in anti-pathogen
defense(Silverman and Maniatis, 2001). Many immune genes were
re-ported to be regulated by NF-kB, such as diptericin,
cecropin,attacin, defensing as well as nitric oxide synthase (Dong
et al.,2006; Hillyer and Estevez-Lao, 2010; Luna et al., 2006;
Richmanet al., 1997; Vizioli et al., 2000). As prostaglandins and
metabolitesfrom the LOX pathway have been reported to mediate
insect im-munity, the inhibitor and the antibody described in the
study canbe used to investigate whether epoxy fatty acids are
players in in-sect immunity, how the immunity is regulated and how
the diseasetransmission patterns will be impacted.
Acknowledgment
This work is supported in part by National Institute of
Envi-ronmental Health Sciences (NIEHS) Grant ES002710,
NIEHSSuperfund Grant P42 ES004699, the UC Davis Jastro-Shields
Grad-uate Research Award and the China Scholarship Council.
Wegracefully thank Dr. Shizuo Kamita for reading the manuscript
andproviding tips on baculovirus expression system. We also thank
Dr.Ahmet Inceoglu for detailed discussions and helpful
suggestions.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ibmb.2014.08.004.
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-
ATGATCCAGTACTACGTCCGCGAGTCGATCCAGTTTGTGGTGTCGTACGCCCTGTGCCTG
-M--I--Q--Y--Y--V--R--E--S--I--Q--F--V--V--S--Y--A--L--C--L—
20
TTCTACAGCTGCCGGGTGCTGTTCGGTCTGCTGGTGCTGCTCGTCACCAAACCGCACACC
-F--Y--S--C--R--V--L--F--G--L--L--V--L--L--V--T--K--P--H--T-
40
AAATTTTGGGCCACAAAGGAGCGGCCCGTGCCGCCGGAATGTTTGCGCAATCACGAGTAC
-K--F--W--A--T--K--E--R--P--V--P--P--E--C--L--R--N--H--E--Y-
60
GGCACCGATAAGTACCAGAATGCGAACGGCATACGGATACATTTCGTGGAGAATGGAGAT
-G--T--D--K--Y--Q--N--A--N--G--I--R--I--H--F--V--E--N--G--D-
80
CGCAGCAAACCGCTCATGGTGCTCGTGCACGGCTTTCCCGAGTTTTGGTTCTCGTGGCGC
-R--S--K--P--L--M--V--L--V--H--G--F--P--E--F--W--F--S--W--R- 100
CATCAGCTGAAGGAGTTCGCCAAAGATTACTGGGTGGTGGCGTTGGATATGCGCGGGTAC
-H--Q--L--K--E--F--A--K--D--Y--W--V--V--A--L--D--M--R--G--Y-
120
GGTGACACCGAGAAGCCCCAGTACCAGTACGCCTATCGGATCGACAACATGACCGAGGAC
-G--D--T--E--K--P--Q--Y--Q--Y--A--Y--R--I--D--N--M--T--E--D-
140
ATCCGGTGCCTGGTGCGACAGTTAGGTCGTCAAAAGTTTACCCTCGTTGCGCACGACTGG
-I--R--C--L--V--R--Q--L--G--R--Q--K--F--T--L--V--A--H--D--W- 160
GGCGCAGTGATTGGATGGCACTTCATCACCAAACACATGGAGATGGTCGATCGGTACATC
-G--A--V--I--G--W--H--F--I--T--K--H--M--E--M--V--D--R--Y--I-
180
ATGATGGACGCACCCTCGCAGAAGATTGCCCGGAAGCTGTTCTCCACCAGCAAAACCCAG
-M--M--D--A--P--S--Q--K--I--A--R--K--L--F--S--T--S--K--T--Q-
200
TTCAAGATGTCCTGGTACATCTTCTTCTACCAAATGCCCTGGCTGCCGGAGTTCTTCGTG
-F--K--M--S--W--Y--I--F--F--Y--Q--M--P--W--L--P--E--F--F--V- 220
CGCCTGATGGACTTCCACCTGTTCGAGGTGGTGTTCCGCCACCACGGTGGGCCGGACGTG
-R--L--M--D--F--H--L--F--E--V--V--F--R--H--H--G--G--P--D--V-
240
ATCGAGGCGTTCAAGTACACGTTCTCCAAACCGCACGCCATGACGTACCCGATCAACTAC
-I--E--A--F--K--Y--T--F--S--K--P--H--A--M--T--Y--P--I--N--Y-
260
TATCGCCAGAATTTCCGCTTCTTCACGAGGCGGCAGATGCCACCGCGGCCGAAAACGTTC
-Y--R--Q--N--F--R--F--F--T--R--R--Q--M--P--P--R--P--K--T--F-
280
-
GCCCCCGGGCTGTACCTGATCGGCGAGAAGGATCTGTACATCTCGAAGGAGTCGGGACCG
-A--P--G--L--Y--L--I--G--E--K--D--L--Y--I--S--K--E--S--G--P- 300
CTGATGCAGCAGGAGTTTGAGAATCTGGAGTTCCGTGTCGTGCCCGGTGTCGATCACTTC
-L--M--Q--Q--E--F--E--N--L--E--F--R--V--V--P--G--V--D--H--F- 320
CTGCAGCAGCACAACCCGGAGCTGGTCAACCAGGTCATGCGAGAATTTCTGTCCAAGAGC
-L--Q--Q--H--N--P--E--L--V--N--Q--V--M--R--E--F--L--S--K--S-
340
-
00.050.10.150.20.250.30.35
Loaded flowthrough
Bufferwash
0.2MNaCl
0.4MNaCl
0.8MNaCl
1.0MNaCl
SpecificActivity
-
IonicStrength
(mM)t‐DPPOJHIII14,15‐EET
5010015030040050010002000
410±22122±7369±6384±50133±12310±32360±32131±18210±10400±22137±11233±22390±19129±7184±23393±3393±4175±18353±2082±7140±4380±3054±1123±2
Three substrates were tested with the addition of 0 mM to 3000
mM sodium chloride into 50 mM Tris-HCl buffer to adjust the ionic
strength. Data represent mean specific activity ± SD (n=3).
Table S1 Effects of ionic strength on enzyme activity.
-
Accession numbers of sequences included can be found in Fig. 1.
Protein sequences were used for the blast.
SequencesblastedagainstAGAP011972
aminoacid
sequencelength
Maxidentity
Scores
E‐value
sEHfromH.sapiensEH3fromH.sapiensEH4fromH.sapienssEHfromM.musculussEHfromC.eleganssEHfromA.thalianamEHfromH.sapiensmEHfromD.melanogasterJHEHfromM.sexta
555
360362488404321455463462
24%
37%
35%
28%
37%
29%
25%
26%
29%
95.5
211
208
101
203106
29.6
37.0
37.0
2e‐22
7e‐65
2e‐63
3e‐23
4e‐613e‐26
2.30.0100.013
Table S2 Protein BLAST results against the AgEH.
Expression and characterization of an epoxide hydrolase from
Anopheles gambiae with high activity on epoxy fatty acids1
Introduction2 Materials and methods2.1 Phylogeny analysis2.2
Generation of recombinant virus2.3 Baculovirus expression and
differential centrifugation2.4 Optimal pHs for enzyme activity and
stability2.5 Effect of ionic strength and inhibitors on enzyme
activity2.6 Solubilization of AgEH activity from the membrane2.7
Enzyme assays and determination of kinetics2.8 Partial purification
of AgEH by ion exchange chromatography and isoelectric focusing2.9
SDS-PAGE and western blot analysis of purification fractions2.10
Immunoprecipitation of the AgEH activity
3 Results3.1 Phylogeny analysis of the epoxide hydrolase from A.
gambiae (AgEH)3.2 Substrate selectivity of AgEH3.3 The subcellular
locations of AgEH3.4 Effects of pH on enzyme activity and
stability3.5 The inhibition patterns3.6 Enzyme kinetics3.7
Solubilization of AgEH activity from the membrane3.8 Partial
purification of the AgEH and analysis of purification fractions by
SDS-PAGE and western blot3.9 Immunoprecipitation of the AgEH
activity
4 DiscussionAcknowledgmentAppendix A Supplementary
dataReferences