Mitochondrial NAD+-dependent malic enzyme from Anopheles stephensi:a possible novel target for malaria mosquito control

Pon Jennifer, Napoli Eleonora, Luckhart Shirley, Giulivi, Giulivi Cecilia

Description

Anopheles stephensi mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized A. stephensi (ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development. Methods To characterize the mitochondrial ME from immortalized ASE cells (Mos. 43; ASE), mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference. Results The encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from Anopheles gambiae (98%) and 59% homologous to the mitochondrial NADP + -dependent ME isoform from Homo sapiens . Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that ( i ) V max with NAD + was 3-fold higher than that with NADP + , ( ii ) addition of Mg 2+ or Mn 2+ increased the V max by 9- to 21-fold, with Mn 2+ 2.3-fold more effective than Mg 2+ , ( iii ) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, ( iv ) among the analogs of L-malate tested as inhibitors of the NAD + -dependent ME catalyzed reaction, small (2- to 3-carbons) organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity (e.g., oxaloacetate, tartronic acid and oxalate). Conclusions The biochemical characterization of Anopheles stephensi ME is of critical relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development.

Sujets

• Malaria
• Mitocondria
• Bioenergetics
• Metabolism
• Inhibitor
• Mosquitoes

Informations

• Publié par: biomed
• Publié le: 01 janvier 2011
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Mitochondrial

NAD+-dependent

camil

enzyme

fromAnophelesstephensi:apossiblenovel

targetformalariamosquitocontrol

Pon
etal
.

Pon
etal
.
MalariaJournal
2011,
10
:318
http://www.malariajournal.com/content/10/1/318(26October2011)

Pon
etal
.
MalariaJournal
2011,
10
:318
http://www.malariajournal.com/content/10/1/318

RESEARCHOpenAccess
MitochondrialNAD
+
-dependentmalicenzyme
from
Anophelesstephensi:
apossiblenoveltarget
formalariamosquitocontrol
JenniferPon
1
,EleonoraNapoli
1
,ShirleyLuckhart
3
andCeciliaGiulivi
1,2*

Abstract
Background:
Anophelesstephensi
mitochondrialmalicenzyme(ME)emergedashavingarelevantroleinthe
provisionofpyruvatefortheKrebs
’
cyclebecauseinhibitionofthisenzymeresultsinthecompleteabrogationof
oxygenuptakebymitochondria.Therefore,theidentificationofMEinmitochondriafromimmortalized
A.stephensi
(ASE)cellsandtheinvestigationofthestereoselectivityofmalateanaloguesarerelevantinunderstandingthe
physiologicalroleofMEincellsofthisimportantmalariaparasitevectoranditspotentialasapossiblenoveltarget
forinsecticidedevelopment.
Methods:
TocharacterizethemitochondrialMEfromimmortalizedASEcells(Mos.43;ASE),massspectrometry
analysesoftrypsinfragmentsofME,genomicsequenceanalysisandbiochemicalassayswereperformedto
identifytheenzymeandevaluateitsactivityintermsofcofactordependencyandinhibitorpreference.
Results:
TheencodinggenesequenceandprimarysequencesofseveralpeptidesfrommitochondrialMEwere
foundtobehighlyhomologoustothemitochondrialMEfrom
Anophelesgambiae
(98%)and59%homologousto
+themitochondrialNADP-dependentMEisoformfrom
Homosapiens
.MeasurementsofMEactivityinmosquito
++mitochondriais
2
o
+
latedfr
2
o
+
mASEcellsshowedthat(
i
)
V
max
withNADw
2
a
+
s3-foldhigherthanthatwithNA
2
D
+
P,(
ii
)
additionofMgorMnincreasedthe
V
max
by9-to21-fold,withMn2.3-foldmoreeffectivethanMg,(
iii
)
succinateandfumarateincreasedtheactivityby2-and5-fold,respectively,atsub-saturatingconcentrationsof
malate,(
iv
)amongtheanalogsofL-malatetestedasinhibitorsoftheNAD
+
-dependentMEcatalyzedreaction,
small(2-to3-carbons)organicdiacidscarryinga2-hydroxyl/ketogroupbehavedasthemostpotentinhibitorsof
MEactivity(e.g.,oxaloacetate,tartronicacidandoxalate).
Conclusions:
Thebiochemicalcharacterizationof
Anophelesstephensi
MEisofcriticalrelevancegivenitsimportant
roleinbioenergetics,suggestingthatitisasuitabletargetforinsecticidedevelopment.
Keywords:
malaria,mitochondria,bioenergetics,metabolism,inhibitors,mosquitoes

Background
forflightmetabolisminthetsetsefly[3],themosquito
Recently,severalpathwaysforenergyproductionhave
Aedesaegypti
[4]aswellasotherinsects[5].About20%
beenidentifiedinmitochondriafrom
Anophelesste-
oftheglutamateproducedbyprolineoxidationisin
phensi
[1],awell-studied
Anopheles
speciesintheinves-turnoxidizedbyglutamatedehydrogenase[6],whereas
tigationofmalariatransmission[2].Themitochondria-theremainderundergoestransaminationbyreaction
dependentenergypathwaysmainlyuseproline,pyru-withpyruvateandtheresultingalanineaccumulatesas
vate,
a
-glycerophosphate,andacyl-carnitinederivativestheprolineisutilized.The2-oxoglutarateformedby
assuitablesubstrates.ProlineisalsothemainsubstratetransaminationisfurthermetabolizedbytheKrebs
’
cycle.Originallypyruvatewasthoughttobeproduced
*Correspondence:[e-mail]
fromoxaloacetatebyanoxaloacetatedecarboxylase[7],
1
DepartmentofMolecularBiosciences,SchoolofVeterinaryMedicine,
butthisenzymewaslaterlocalizedinthecytoplasm
UniversityofCaliforniaDavis,Davis,CA95616,USA
whereasprolineoxidationandsubsequentreactionsall
Fulllistofauthorinformationisavailableattheendofthearticle
©2011Ponetal;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommons
AttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,andreproductionin
anymedium,providedtheoriginalworkisproperlycited.

Pon
etal
.
MalariaJournal
2011,
10
:318
http://www.malariajournal.com/content/10/1/318

takeplaceinthemitochondria[6],consistentwithpre-
viousstudies[1].Mitochondriaofculturedcells[ASE
cellline(
A.stephensi
Mos.43cellline)]from
A
.
ste-
phensi
,aswellasflightmusclemitochondriaofabeetle
(
Popilliajaponica
),whichalsohavetheabilitytooxidize
prolineatahighrate,havebeenshowntocontainan
unusuallyactivemalicenzyme[8].Thelatterspecies
utilizesNAD
+
preferentiallyasacoenzymeandpresum-
ablyproducespyruvatebytheoxidativedecarboxylation
ofmalate[8].Thismitochondrialenzymeininsectsmay
haveacriticalroleinthereplenishmentofpyruvatefor
eithertransaminationorKrebs
’
cycle.
Malicenzyme(ME;EC1.1.1.39)catalysesthereversi-
bleoxidativedecarboxylationof
L
-malatetopyruvate
andCO
2
withtheconcomitantreductionofthecofac-
torNAD
+
orNADP
+
[9-11].Theenzymerequires
divalentcations(Mg
2+
,Mn
2+
,orothers)inthecataly-
sisofthisreaction.MEactivitywasfirstisolatedfrom
pigeonliver[12]andhassincebeenfoundinmostliv-
ingorganisms,frombacteriatohumans.MostMEsare
homotetramers,withmonomerscontaining550amino
acidsandhavingmolecularweightsof60kDa.The
aminoacidsequencesofMEsarehighlyconserved
acrossallstudiedorganisms,buttheylackrecognizable
homologytootherproteins,includingotheroxidative
decarboxylases.ThewidedistributionofMEactivityin
natureandthehighdegreeofsequenceconservation
areconsistentwiththeimportantbiologicalfunctions
oftheseenzymes,suchasphotosynthesisinC4plants
andevensomeC3plants[13]andbiosynthesisoffatty
acidsandsteroidsinliverandadiposetissuesinani-
mals.Inmammals,threeisoformsofMEhavebeen
identified
–
cytosolicNADP
+
-dependentME(ME-1;
[14]),mitochondrialNADP
+
-dependentME(ME-3;
[15]),andmitochondrialNAD(P)
+
-dependentME
(ME-2;[10]),whichcanuseeitherNAD
+
andNADP
+
asacofactor(dualspecificity),butprefersNAD
+
underphysiologicalconditions.Ininvertebrates,andin
particularininsects,unusuallyhighactivityofNAD
+
-linkedmalicenzymehasbeenreportedinflightmus-
clemitochondriaofthebeetle
Popilliajaponica
[8]
andfromthetsetseflyandotherinsects[16].
Basedonpreviousreports[1],ASEmitochondrialME
emergedashavingarelevantroleintheprovisionof
pyruvatefortheKrebs
’
cyclebecausethechemicalinhi-
bitionofthisenzymeresultedinthecompleteabroga-
tionofoxygenuptakebymitochondria.Therefore,the
identificationofMEinASEmitochondriaandtheinves-
tigationofthestereoselectivityofmalateanaloguesare
relevantinunderstandingthephysiologicalroleofME
incellsofthisimportantmalariaparasitevectorandits
potentialasapossiblenoveltargetforinsecticide
development.

Page2of12

Methods
Chemicals
OrganicacidswerepurchasedfromSigmaChemicalCo.
(St.Louis,USA).Allreagentswereofanalyticalgrade.
Cellmaintenance
Theimmortalized
A.stephensi
ASEcelllinewasgrown
inmodifiedEagle
’
sminimalessentialmedium("E5
”
)
supplementedwithglucose,L-glutamine,vitaminsolu-
tion,nonessentialaminoacids,penicillinandstreptomy-
cin,and5%heat-inactivatedfetalbovineserumat28°C
with5%CO
2
asdescribed[1].Thepopulationdoubling
timeofthesecellsisapproximately18-20h.Thecells
weresplit1:10intoE5mediumandgrownin50mlcul-
tureflasksuntilconfluent.Theseflaskswereusedto
seed500-mlcultureflaskstoprepare~2billioncellsfor
mitochondriapreparation.Cellsharvestedformitochon-
driapreparationweregentlypipetted,resuspendedin
themedium,andtransferredto50-mltubes.Cellswere
pelletedbycentrifugationat800gfor5min.Thesuper-
natantwasremoved,andthecellswereresuspendedin
asmallamountofmediumbygentlepipettingand
transferredtoasterileholdingtubeonice.Thiscycle
wasrepeated,withcollectionoftheconcentratedcells
intoonetube,untilallflaskswereprocessed.
Isolationofmitochondria
Cellswerecentrifugedfor1minat500gat4°Cand
mitochondriawereisolatedfrompelletedcellsas
described[1].ThepelletwasweighedandMSHEbuffer
wasaddedataratioof3mlofMSHEbuffer(220mM
mannitol,70mMsucrose,0.5mMEGTA,0.1%fatty
acid-freebovinealbumin,and2mMHEPES,pH7.4)
per1gofcells.Thecellsweredisruptedbygentle
homogenization,centrifugedat600gfor5minat4°C,
thepelletwasdiscarded,andthesupernatantwascentri-
fugedat10,300gfor10minat4°C.Thepellet,whichis
richinmitochondria,wasresuspendedinasmall
volumeofMSHE.Usingthisproceduretheyieldwas
7.5±0.5
μ
gmitochondrialprotein/10
6
cells.Protein
concentrationwasdeterminedbyusingtheBCAProtein
Assay(Pierce).
Enzymaticassays
TheMEenzymaticassaywasperformedusingamethod
outlinedby[17]withthefollowingmodifications.Mos-
quitomitochondriawerehomogenizedinMSHEcon-
taining2mMmercaptoethanol.Ina1mlcuvette,2-
μ
g/
mlantimycin,1mML-malate,0.3mMNAD
+
,50mM
HEPES(pH7.8),and3mMofMnCl
2
(unlessindicated
otherwise)wereadded.Ther