Peroxisomal Plant 3-Ketoacyl-CoA Thiolase Structure and Activity Are Regulated by a Sensitive ...

12 Pages·2010·1.83 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.285,NO.31,pp.24078–24088,July30,2010
©2010byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Peroxisomal Plant 3-Ketoacyl-CoA Thiolase Structure and
Activity Are Regulated by a Sensitive Redox Switch*□S
Receivedforpublication,January20,2010,andinrevisedform,April12,2010Published,JBCPapersinPress,May12,2010,DOI10.1074/jbc.M110.106013
ValerieE.Pye‡1,CasparE.Christensen‡,JamesH.Dyer§,SusanArent‡2,andAnetteHenriksen‡3
Fromthe‡ProteinChemistryGroup,CarlsbergLaboratory,GamleCarlsbergVej10,DK-2500Valby,Denmarkandthe§Department
ofChemistryandBiochemistry,MontclairStateUniversity,UpperMontclair,NewJersey07043
Thebreakdownoffattyacids,performedbythe(cid:1)-oxidation bidopsis thaliana seeds deficient in (cid:1)-oxidation enzymes are
cycle,iscrucialforplantgerminationandsustainability.(cid:1)-Ox- unable to germinate without an external sugar source; they
idation involves four enzymatic reactions. The final step, in havelargeandunusualperoxisomesandaccumulateC16–C20
whichatwo-carbonunitiscleavedfromthefattyacid,isper- fattyacids(5,6).(cid:1)-Oxidationisalsoresponsibleforthesynthe-
formed by a 3-ketoacyl-CoA thiolase (KAT). The shortened sisofjasmonicacid(7)andindole-3-aceticacid(auxin)viacon-
fattyacidmaythenpassthroughthecycleagain(untilreaching version from indole-3-butyric acid (8), which serve as crucial
acetoacetyl-CoA)orbedirectedtoadifferentcellularfunction. planthormonesregulatingplantdevelopmentandresponsesto
CrystalstructuresofKATfromArabidopsisthalianaandHeli- biotic and abiotic stress. Hydrogen peroxide, produced as a
anthus annuus have been solved to 1.5 and 1.8 A˚ resolution, byproduct during (cid:1)-oxidation, is used by catalase to oxidize
respectively. Their dimeric structures are very similar and different toxins (e.g. alcohols) and plays an important role in
exhibitatypicalthiolase-likefold;dimerformationandactive cellularsignaling(9).
site conformation appear in an open, active, reduced state. (cid:1)-Oxidation comprises four reactions. First, the CoA-acti-
Usinganinterdisciplinaryapproach,weconfirmedthepotential vatedacylchainisoxidizedto2-trans-enoyl-CoAbyanacyl-
ofplantKATstoberegulatedbytheredoxenvironmentinthe CoAoxidase(10),producingH O asabyproduct.Thedouble
2 2
peroxisomewithinaphysiologicalrange.Inaddition,co-immu- bondisthenreducedby2-trans-enoylhydrataseformingL-3-
noprecipitation studies suggest an interaction between KAT hydroxyacyl-CoAfollowedbyoxidationbyNAD(cid:1)-dependent
andthemultifunctionalproteinthatisresponsibleforthepre- L-3-hydroxyacyl-CoAdehydrogenase.Boththehydrataseand
cedingtwostepsin(cid:1)-oxidation,whichwouldallowaroutefor dehydrogenase activities are performed by a multifunctional
substrate channeling. We suggest a model for this complex protein(MFP)4(11,12).3-Ketoacyl-CoAthiolase(KAT)per-
basedonthebacterialsystem. forms the last step of (cid:1)-oxidation, thiolytically cleaving a
two-carbon unit from 3-ketoacyl-CoA (13). The shortened
fatty acyl-CoA can then be subjected to further rounds of
Fattyacidsarefundamentalbiomoleculesthatareabundant (cid:1)-oxidation or directed to other pathways. In mammals
inalllifeforms.Withtheirenormousvariationinchainlength there are two distinct pathways for (cid:1)-oxidation; very long
anddegreeofsaturation,theyareessentialforenergystorage, chainfattyacidsandbileacidintermediatesareshortenedin
formstructuralentitiesinbiomembranes,andserveassignal- peroxisomes, whereas medium to long chain mono- and
ingmolecules.Fattyacidsarebrokendowninacyclicmanner, dicarboxylic fatty acids, methyl-branched fatty acids, and
twocarbonsatatime,togeneratearangeofproductsbythe prostaglandins are broken down in mitochondria (14). In
processknownas(cid:1)-oxidation(1).Inhigherplants(2)andyeast bothmitochondrialandprokaryotic(cid:1)-oxidationsystemstri-
(3)(cid:1)-oxidationofallformsoffattyacidoccursintheperoxi- functional multi-enzyme complexes (comprising MFP and
somes.Inplants,(cid:1)-oxidationisessentialforaplethoraofphys- KAT activities) exist allowing for metabolite channeling
iological roles including responses to senescence and starva- (15–18).ThecrystalstructureofthePseudomonasfragitri-
tion, fatty acid turnover, and the regulation of plant lipid functional enzyme complex is known and consists of two
composition.Germinatingseedsdependon(cid:1)-oxidationforthe (cid:2)-subunits(hostingMFPfunctions;hereafterreferredtoas
mobilizationandreleaseofenergystoredintheseed(4).Ara- PfMFP)andtwo(cid:1)-subunits(hostingKATfunctions;hereaf-
terreferredtoasPfKAT)inadynamiccomplex(16).Plants
*ThisworkwassupportedbytheDanishNaturalScienceResearchCouncil, containanumberofisozymesforeachstepin(cid:1)-oxidation;
whichprovidedapostdoctoralstipendthroughDANSYNC(toS.A.)and theacyl-CoAoxidasesarebestdefinedwithregardtohaving
synchrotronbeamtime.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplemental“Discussion,”Figs.S1andS2,TableS1,andamovie.
Theatomiccoordinatesandstructurefactors(codes2WU9and2WUA)havebeen 4The abbreviations used are: MFP, multifunctional protein; ACAT, aceto-
depositedintheProteinDataBank,ResearchCollaboratoryforStructural acetyl-CoAthiolase;KAT,3-ketoacyl-CoAthiolase;PDB,ProteinDataBank;
Bioinformatics,RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/). DTT,dithiothreitol;CSH,cysteamine;CSSC,cystamine;TLS,translation/
1Towhomcorrespondencemaybeaddressed:SchoolofBiochemistryand libration/screw; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-
Immunology,TrinityCollegeDublin,Dublin2,Ireland.Fax:[phone]; propane-1,3-diol;L-,loop-;r.m.s.d.,rootmeansquaredeviation;Pf,Pseudo-
E-mail:[email protected]. monasfragi;At,Arabidopsisthaliana;Ha,Helianthusannuus;Sc,Saccharo-
2Presentaddress:DaniscoLtd.,EdwinRahrsVej38,8220Brabrand,Denmark. mycescerevisiae;Hs,Homosapiens;Bn,Brassicanapus;YTmedium,16gof
3Towhomcorrespondencemaybeaddressed.Fax:[phone];E-mail: BactoTryptone,10gofBactoYeastExtract,5gofNaClperliterofdistilled
[email protected]. H2O,pHadjustedwith5MNaOH.
This is an Open Access article under the CC BY license.
24078 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER31•JULY30,2010
RedoxControlofPeroxisomalThiolase
differentbutoverlappingsubstratespecificities.Communi- BODIPY-PTS1isoxidizedintheperoxisomesofratfibroblasts
cationbetweentheisoformsviaaprotein-proteincomplex, upon incubation with eicosanoic and phytanic acids suggests
similartothatfoundinP.fragi,coulddirectandcontrolsthe that the redox state of this organelle is unstable despite the
flowofmetabolitesthrough(cid:1)-oxidation,buttodatenosuch redoxbufferingeffectsoftheantioxidantdefensesystemspres-
complexinplantshasbeenreported. entinperoxisomes(29).Stressingtheneedforregulationeven
There are two distinct forms of 3-ketoacyl-CoA thiolases. further, catalase itself is inhibited at prolonged exposure to
Type I is 3-ketoacyl-CoA thiolase (EC 2.3.1.16), a catabolic H O generatedat2nmol/(mlmin)particularlyintheabsence
2 2
enzymeperformingthereverseClaisencondensationreaction of NADPH (30–34). Additionally, peroxisomes have been
involvedin,forexample,the(cid:1)-oxidationcycle.TypeIIthiolase showntochangetheirmorphologydramaticallyinresponseto
isacetoacetyl-CoAthiolase(ACAT;EC2.3.1.9)andisinvolved H O stresses(35).TheinactivationofcatalasebyH O would
2 2 2 2
in the anabolic mevalonate pathway performing Claisen con- lead to a breakdown of the peroxisomal antioxidant defense
densation.ArabidopsishasbeenshowntopossessthreeKAT system giving rise to toxic conditions, oxidative damage, and
genesthatencodethreeperoxisomalKATisoforms(1,2,and eventuallycelldeath.Thereforeastableandbalancedintracel-
5.2)andonecytosolicisoformlackingtheN-terminal43resi- lularredoxenvironmentiscrucialforallcells;acontrolpointin
dues (5.1) and two ACAT genes that encode four cytosolic the(cid:1)-oxidationcyclewouldaidthecontroloftheredoxcondi-
ACAT isoforms (1.1, 1.2, 2.1, and 2.2) and one peroxisomal tionsintheperoxisome.
isoform (1.3) (13). In Arabidopsis, KAT2 is the major type I Herewepresentresultsdemonstratingthattheredoxstate
thiolase isoform present during germination; it seems to be andactivityofAtKAT2areaffectedunderbiologicallyrelevant
important for triacylglycerol synthesis during seed develop- conditions as well as the crystal structures of two plant thio-
ment as well (6). KAT1 and KAT5 are not abundant in seed- lases, AtKAT2 and sunflower HaKAT (Helianthus annuus
lings, and their roles are less well defined; KAT5 is possibly (HaKAT)),inareduced,open,activeconformationtohighres-
involvedinflavonoidbiosynthesis(13).KAT2isreportedtobe olution.Wediscussthepotentialredoxcontrolof(cid:1)-oxidation
activeforthefullrangeoffattyacidlengthsfromacetoacetyl-
and as initiated from our co-immunoprecipitation pulldown
CoA upward (6). Thus, in contrast to the first step involving
assays,wesuggestapossiblecomplexformationwithMFP.For
substrate-specificacyl-CoAoxidases,thelaststepof(cid:1)-oxida-
completenesswehaveincludedinthesupplementalmateriala
tion is seemingly unspecific. The first structure of KAT was
comparisonoftheKATstructurespresentedherewithother
solved from Saccharomyces cerevisiae (ScKAT) (19, 20). This
availableperoxisomaltypeIKATstructuresatastructuraland
structurehasatypicalthiolase-likefold,isahomodimer,and
substratebindinglevel.
presumablyisinanactivestate.ThestructureofKAT2from
Arabidopsis (AtKAT2) was solved later and also shows a
EXPERIMENTALPROCEDURES
homodimer with a thiolase-like fold (21). However, the
Cloning, Expression, and Protein Purification—The full-
AtKAT2 structure is noticeably different from the ScKAT
length HaKAT construct (36) was recloned to remove the
structure;adisulfidebridgeisformedinvolvingthepresumed
N-terminalsignalingsequenceandinsertaC-terminalHis tag.
activesitenucleophile,causingtheactivesitetobeinaccessible 6
TheconstructforHaKAT(17–449)wasmadeusingforward
tosubstrateandresultinginachangeintheloopstructurethat
(5(cid:2)-GGAATTCCATATGCCCTCTTCTACTTCTTCATCC-
capstheactivesiteandadifferentdimerarrangement.Aregu-
lation of (cid:1)-oxidation by redox potential was suggested; how- TTGG-3(cid:2)) and reverse (5(cid:2)-GAATTGCGGCGTAGTGAT-
ever,theydidnotproduceeitherastructureofAtKAT2inan GGTGATGGTGATGTTTAGCTTAATTGCAC-3(cid:2)) primers.
active, reduced state or any results that suggested oxidation TheresultingPCRfragmentwasligatedintoapSTblue-1vec-
taking place within a biologically relevant redox range. torusingthePerfectlyBluntligationkit(Novagen).Thepuri-
Recently, a human peroxisomal KAT (HsKAT) structure was fiedplasmidwascutwithrestrictionenzymesNdeIandNotI
deposited in the PDB database by the Structural Genomics (NewEnglandBiolabs),andthesesiteswereusedtoinsertthe
Consortium (PDB code 2IIK). HsKAT is also in the active, PCR product into the pET24b expression vector (Novagen)
reducedstate. followedbytransformationofsupercompetentXL1-Bluecells
Peroxisomesprovideachallengingenvironmentforenzyme (Stratagene). Protein expression was carried out using
stability.Theredoxenvironmentoftheperoxisometradition- BL21(DE3)cells.AnexpressionconstructencodingAtKAT2-
allywasconsideredstableandreducingduetothepresenceof (36–462)withanN-terminalenterokinaseexcisableHis tag
6
catalase and its role in the breakdown of H O derived from was prepared in pET46 Ek/LIC from clone pda03502 (Riken)
2 2
photorespiration,oxidativeprocesses,andthedecomposition usingligationindependentcloningtechnology(Novagen)and
ofradicaloxygenspecies(22,23).However,H O isnowrec- theprimers5(cid:2)-GACGACGACAAGATGGCTGGGGACAGT-
2 2
ognizedasanimportantsignalingmoleculeinbothplantsand GCTGCATAT-3(cid:2) and 5(cid:2)-GAGGAGAAGCCCGGTCAGGC-
animals (for a review see Ref. 9), and plant peroxisomes have GTCCTTGGACAA-3(cid:2). Successful cloning was verified by
been shown to be net producers of H O , releasing it to the colony PCR and sequencing, and the construct was used to
2 2
cytosol(24,25)(forareviewseeRef.26).Thesubcellulardistri- transform RosettaGami2(DE3) cells by electroporation.
bution of H O is unknown, but the total concentration of Bothproteinswereexpressedandpurifiedinthesameman-
2 2
H O in unstressed leaves has been measured as high as 3.6 ner.1literof2(cid:3)YTmediumsupplementedwiththeappro-
2 2
(cid:3)mol/g(freshweight)translatingtoanaverageof3.6mM(27, priateantibioticswasinoculatedwitha1/50overnightculture
28). The finding that the diene containing fluorescent probe andgrowntoA (cid:4) 0.5 at 37°C. Cells were then cooled to
600
JULY30,2010•VOLUME285•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 24079
RedoxControlofPeroxisomalThiolase
(cid:1) (cid:1) (cid:2)(cid:2)
25°C before induction with 1 mM isopropyl 1-thio-(cid:1)-D-ga- (cid:5)(cid:2) (cid:6)(cid:1)(cid:2)x(cid:6)(cid:6) (cid:5)(cid:2) (cid:6)(cid:1)(cid:2)x(cid:6)(cid:2)exp m(cid:5)x(cid:7)c50%(cid:6)
lactopyranoside and incubation (shaking) at 25°C for 18 h. 0 0 1 1 R(cid:2)T
Cell pellets were resuspended in a prechilled solution of 50 f(cid:5)x(cid:6)(cid:5) (cid:1)m(cid:5)x(cid:7)c (cid:6)(cid:2)
mM Tris-HCl, 200 mM NaCl, pH 7.7, and lysed with Bug- 1(cid:6)exp R(cid:2)T 50%
Buster (Sigma; 1/10 dilution) and Benzonase nuclease
(Novagen;1/1000dilution)for1hatroomtemperature.The (Eq.1)
lysedcellpelletswerecentrifugedat18,000(cid:3)gfor1h,and
thesupernatantwasappliedtoa5-mlNi2(cid:1)affinitycolumn Here(cid:2) and(cid:1) representtheyaxisinterceptandslopeofthe
n n
(Amersham Biosciences). After washing, the protein was first(n(cid:4)0)andsecond(n(cid:4)1)linearpartsofthecurve,Ris
eluted by an imidazole gradient. Protein fractions were thegasconstant,Tisthetemperatureandc andmrepresent
50%
assessed by SDS-PAGE, concentrated using a Vivaspin20 themidpointoftransitionanditsslope,respectively.Themid-
30,000molecularweightcut-offfilterdevice(Sartorius),and point of transition was determined and used with the Nernst
appliedtoaHiLoad26/60Superdex200prepgrade(Amer- equation and a standard electrode potential of CSH of (cid:7)260
sham Biosciences) size exclusion column pre-equilibrated mV (39) to estimate the midpoint electrode potential of
AtKAT2.Itwasfoundtobe(cid:7)372mVatpH8.5and(cid:7)283mV
andrunwith50mMTris-HCland200mMNaCl,pH7.7,at
atpH7.0.TheelectrodepotentialofAtKAT2wasassumedto
4°C. Protein fractions were again assessed by SDS-PAGE
follow the (cid:7)59 mV/pH unit correlation identified previously
andthendialyzedovernightagainst20mMTris-HCl,150mM
NaCl,2mMEDTA,5%(v/v)glycerol,and2mMDTT,pH9.0. forthioredoxins(40,41).FarUVCDspectraof5(cid:3)MAtKAT2
Thiolase Activity Assay—The activity of AtKAT2 was incubatedwith10mMCSHor5mMCSSCwererecordedinthe
same buffer as described above at 25°C in a Jasco J-810 CD
determinedspectroscopicallybymonitoringtheabsorbance
spectrometer with a 1-mm light path quartz cuvette at 20
increase at 232 nm due to the formation of new acyl-CoA
nm/minand0.1-nmpitch,averaging6scans.
ester bonds (37). All buffers were deoxygenated by purging
inananaerobicgloveboxfor1h.Thereactions,consistingof Crystallization—HaKAT at 10 mg/ml in 20 mM Tris-HCl,
50(cid:3)Macetoacetyl-CoAand100(cid:3)MCoAin35mMTris-HCl 150mMNaCl,2mMEDTA,5%(v/v)glycerol,and2mMDTT,
pH 9.0, was mixed in a 1:1 ratio with 20% (w/v) polyethylene
and100mMNaCl,pH8.5,werestartedbytheadditionof5
glycol 3350, 0.2 M Li(CH COO) , and 0.002% (v/v) ethylene
nM AtKAT2 preincubated with 20 mM cysteamine (CSH; 3 2
glycol and crystallized by vapor diffusion (hanging drop)
Sigma) or 10 mM cystamine (CSSC; Sigma) at 25°C. The
against a reservoir containing 20% (w/v) polyethylene glycol
enzyme preparations were diluted 2500(cid:3) before sampling,
3350,0.2MLi(CH COO) ,and0.01Mtaurine.Thecrystalswere
resultinginfinalconcentrationsofCSHandCSSCof8and4 3 2
flash-frozeninliquidnitrogenusing30%(v/v)ethyleneglycol
(cid:3)M, respectively. The absorbance change at 232 nm was
asacryoprotectant.AtKAT2at11.8mg/mlin20mMTris-HCl,
recorded in a quartz microcuvette with a lambda-45 UV-
150mMNaCl,2mMEDTA,5%(v/v)glycerol,and2mMDTT,
visible spectrophotometer equipped with a Peltier thermal
pH 9.0, was mixed in a 1:1 ratio with 20% (w/v) polyethylene
control element set to 25°C (PerkinElmer Life Sciences).
glycol3350,0.2MLi(CH COO) ,0.002%(v/v)ethyleneglycol,
Background reactions containing the buffer and CSH or 3 2
and2mMC12-CoAandcrystallizedbyvapordiffusion(hang-
CSSC were recorded and used to normalize the data. The
ingdrop)againstareservoircontaining20%(w/v)polyethylene
half-livesweredeterminedbyfittingtheequationforexpo-
glycol3350and0.2MLi(CH COO) .Thecrystalswereflash-
nential decay to the data. Acetoacetyl-CoA and CoA were 3 2
frozen in liquid nitrogen using 30% (v/v) ethylene glycol as
prepared immediately before mixing, and concentrations
cryoprotectant.Inbothcasescrystalsgrewwithin1week.
werecheckedbyUVspectroscopyusing(cid:4)257(cid:4)16,840/Mcm X-rayDataCollection,StructureSolution, and Refinement—
for CoA and (cid:4)260 (cid:4) 16,000/M cm for acetoacetyl-CoA with X-raydatacollectionandrefinementdetailsaregiveninTable
(cid:4) /(cid:4) (cid:4)1.89(38).
260 232 1;5%ofthereflectionsflaggedforR werechoseninresolu-
free
Determination of Reduction Potential of AtKAT2—Tryp-
tion shells using SFTOOL because of assumed non-crystallo-
tophanfluorescenceof5(cid:3)MAtKAT2in35mMTris-HCland graphicsymmetry(NCS).ApreliminaryHaKATdatasetwas
100 mM NaCl, pH 8.5, was recorded after a 15-min incuba- collectedin-houseto2.5Åresolution(datanotshown),andthe
tionat25°CwithvaryingratiosofCSHtoCSSCkeepingthe AtKAT2 (21) monomer was used as a search model for two
totalconcentrationofsulfurconstant.50-(cid:3)lreactionswere molecules/asymmetric unit using PHASER (42). One clear
preparedinhalf-areaUV96-wellmicroplates(Corning3679, solutionresulted;thisstructurewasusedforrefinementagainst
CorningInc.),andwavelengthscanswererecordedinafluo- the higher resolution HaKAT (data reported here; the same
rescenceplatereader(SpectraMAXEM/Gemini,Molecular R set was applied and extended). Initial rigid body refine-
free
Devices) exciting with 285 nm UV-light and reading emis- ment was performed treating each monomer as a rigid body.
sion from 325 to 450 nm with a 325-nm cut-off filter and Cyclesofrestrainedrefinementincludingmotionsfortransla-
photomultiplier sensitivity set to medium. Control experi- tion/libration/screw(TLS)(foursegments/chain,definedusing
ments with 4 mM H O or 1 mM DTT were included. The the TLS server (43) and selected on B-factor distribution,
2 2
emission reading at 333 nm was plotted against [CSH]2/ resultinginonegroupfortheN-terminaldomain,twogroups
[CSSC], and the midpoint of transition was found to be fortheL-domain,andonegroupfortheC-terminaldomain)in
0.17Mbyfittingatwo-statefunctionwithvariableslopesto Refmac (44, 45), the use of TLS lowered the R and R
free work
thedata(Equation1). values.MediumNCSrestraints(chainsAandB)wereusedin
24080 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER31•JULY30,2010
RedoxControlofPeroxisomalThiolase
the initial rounds of refinement; these were loosened for the oxisomaleukaryoticAtKAT2,theirsequencesweresubjected
restoftherefinement.Themaindeviationsbetweenthemole- to a BLAST (53) search, and then unique sequences (56 for
culesoccurredinthepositioningofthe(cid:1)-hairpinintheL-do- PfKAT (sequence identity 57–99%) and 52 for AtKAT2
main.Watermoleculeassignmentandmanualrebuildingwas (sequence identity 43–96%)) were compiled and aligned in
performed in COOT (46) It was noticed that the B-factors ClustalW(54).Thesemultiplesequencealignmentswerethen
assigned to the water molecules during this TLS refinement usedwiththeirrespectivestructuresinConSurf(55),theout-
werelargelyunexpected,whichwasalsoreflectedintheelec- put of which allows coloring of the PDB files with respect to
trondensity,withB-factorseithersuspiciouslyhighwithposi- conservation, from blue (conserved) through the rainbow to
tive density or suspiciously low with negative density. This is red (unconserved). PfKAT and AtKAT2 structures were not
reportedasapossible“bug”inthecurrentversionofRefmac used alone for ConSurf, as many unrelated thiolases were
(version 5.5.0035). Therefore, the final rounds of refinement selected by the server. Instead, we opted to select a range of
were performed in PHENIX (47) using the default settings, prokaryoticKATsforthePfKATandperoxisomaleukaryotic
which resulted in more realistic B-factors and density for the KATsfortheAtKAT2tokeepthecomparisonfocusedonthe
water molecules. The HaKAT dimer model was used as the (cid:1)-oxidationpathway.ThePfMFPandAtMFPstructureswere
searchmodelfortheAtKAT2datausingPHASERandresulted subjectedtothebasicConSurf50-sequenceselectionwitha
inoneclearsolution.RefinementproceededaspertheHaKAT PSI-BLASTE-valuecut-offof0.001.Analysisoftheprotein-
structure. PDB coordinates and structure factors have been protein interactions was aided by the use of PDBsum and
depositedwithaccessioncodes2WUAand2WU9(deposited PROTORP (56). Gap volume index values were calculated
viatheProteinDataBankinEurope(PDBe)). with PROTORP and NOXclass (57), where these values
Co-ImmunoprecipitationStudies—Enrichedperoxisomally- variedslightly(anaveragehasbeenreported).Figs.1,4,and
satewasisolatedinthefollowingmanner.Seedlings(Brassica 5 were prepared using PyMOL. For clarity, for the online
napus cv.) were grown in the dark for 7 days; the cotyledons supplementalmovie,amissingloopcontainingTrp175inthe
werethenharvested,homogenizedin10mlof0.25Msucrose, AtKAT2 structure has been modeled using Modeler
inactive
0.1MTris-HCl,pH7.4,1mMEDTA,1mMMgCl ,0.1%ethanol, 7v7(58).
2
and complete EDTA-free protease inhibitor (Roche Applied
RESULTS
Science), and filtered through two layers of Myra cloth. Cell
debris was removed by centrifugation at 600 (cid:3) g for 5 min. Crystal Structures of AtKAT2 and HaKAT—The crystal
Peroxisomesweresedimentedbycentrifugation(15,000(cid:3)g,20 structuresofArabidopsisandHelianthus3-ketoacyl-CoAthio-
min), resuspended in 1 ml of 0.1 M Tris-HCl, pH 7.4, 1 mM lase have been solved in their dimeric, open, reduced, active
EDTA,1mMMgCl ,150mMNaCl,andcompleteEDTA-free statetohighresolution(1.5and1.8Å,respectively).AtKAT2
2
proteaseinhibitor(RocheAppliedScience),andincubatedon consistsofresidues46–448(A)and47–448(B),andHaKAT
icefor20mintolysetheperoxisomalmembranes.Thelysate consistsofresidues47–438(A)and45–438(B).Datacollec-
wasclearedtwicebycentrifugationat15,000(cid:3)gfor20min. tion,refinement,andvalidationdetailsaregiveninTable1.To
Theclearedlysatewasthenusedforco-immunoprecipitation facilitateaneasyreferencesystemforthesecondarystructure
studies using the Seize X protein A kit (Pierce) following the elements,thesequencesofAtKAT2andHaKATwerealigned
manufacturer’s guidelines, immobilizing antibodies against withsequencesofotherknownKAT2structures,namelyfrom
eitherKATorMFPonthebeads.Theelutedantigenswererun A.thaliana in the inactive form (AtKAT2 ; PDB code
inactive
in duplicate on SDS-PAGE (4–12% bis-Tris, Invitrogen) and 2C7Y(21)),Homosapiens(HsKAT;2IIK),S.cerevisiae(ScKAT;
blotted onto nitrocellulose membranes. Western blots were 1AFW(19)),andP.fragi(PfKAT;1WDK,chainsCandD(16)).
thendevelopedusingpolyclonalantibodiesagainsteitherMFP (cid:1)-Strandsandhelices((cid:2)and3 )weredefinedusingPDBsum
10
orKATasprimaryantibodies.TheMFPantibodieswereraised (50), incorporated, and numbered according to domains
against recombinant AtMFP2, and the KAT antibodies were (supplementalFig.S1online).TheAtKAT2andHaKATstruc-
raisedagainstrecombinantB.napusKAT2(48).TheAtMFP2 tures reported here are very similar and exhibit the expected
antibodieshavealsobeenshowntocross-reactwithrecombi- thiolase-likefolddescribedfortheScKATstructure(19).Each
nantAtAIM1.Acontrolpulldownwasperformedusingimmo- subunitconsistsofthreedomains(Fig.1A);theN-domainand
bilizedantibodiesagainstHistagswiththeperoxisomallysate. C-domainarerelatedbyapseudo2-foldaxisandhavethesame
Structure Analysis and Modeling—Secondary structure (cid:2)/(cid:1)tertiary fold consisting of a central five-stranded (cid:1)-sheet
assignment and protein-protein interactions were defined where the fifth strand is anti-parallel to strands 1–4. Two
using PROMOTIF (49) via the PDBsum server (50). To con- (cid:2)-helicescovertheoutersideofthe(cid:1)-sheets,andone(cid:2)-helix
structtheArabidopsis(cid:1)-oxidationcomplexmodelbasedonthe from each domain is buried between the sheets. The loop
bacterialmulti-enzyme(cid:1)-oxidationcomplex,wesimplysuper- domain(L-domain)iscomposedoffour(cid:2)-helices(H2,H3,H4,
imposedtheAtMFP2structure(51)ontothePfMFP(chainsA andH5),two3 -helices(H1andH6a),anda(cid:1)-hairpin(S1and
10
andB)andtheAtKAT2dimerontothePfKAT(chainsCandD; S2;(cid:1)-hairpin1)thatextendsawayfromtheglobularproteinby
PDBcode1WDK)andthenperformedasimulatedannealing (cid:8)25Å.Thedimer(consistingofsubunitsAandB)isformedby
energyminimizationusingthecrystallographyandNMRsys- interactionsinvolvingmainlytheN-domains.
tem(CNS)(52)tominimizeclashesbetweensidechainsofthe SubunitAsuperimposesontosubunitBwithanr.m.s.d.of
twoproteins;backboneatomsremainedunchanged.Tolookat 0.3Åover403C(cid:2)atomsintheAtKAT2structureandwithan
theaminoacidconservationoftheprokaryoticPfKATandper- r.m.s.d.of0.4Åover391C(cid:2)atomsintheHaKATstructure.As
JULY30,2010•VOLUME285•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 24081
RedoxControlofPeroxisomalThiolase
TABLE1
X-raydatacollection,processing,andrefinementstatistics
Dataset
Statistics
HaKAT AtKAT2
Datacollectionandprocessing
Collectionsite ID23,ESRF,France X12,DESY,Germany
Wavelength(Å) 0.9762 1.0
Spacegroup P222 P2
Unitcelldimensions a(cid:4)17181.6Å;b(cid:4)100.7Å;c(cid:4)105.3Å a(cid:4)160.8Å;b(cid:4)86.7Å;c(cid:4)72.7Å;(cid:1)(cid:4)106.7o
Resolution(Å) 78.6-1.8(1.9-1.8)a 34.6-1.5(1.58-1.5)a
R (%) 8.6(51.9)a 7.8(50.0)a
merge
R b% 3.8(26.9)a 3.1(23.7)a
pim
Mean(I)/S.D.mean(I) 18.0(2.6)a 15.2(2.6)a
Completeness(%) 99.2(94.8)a 97.2(81.2)a
No.ofuniquereflections 77,995 112,318
Multiplicity 8.2(5.4)a 5.1(4.3)a
WilsonBfactorc(Å2) 28.44 12.00
Refinement
No.ofnon-hydrogenproteinatoms 5,875 6,151
No.ofwatermolecules 709 1,134
No.ofethyleneglycolmolecules 0 9
R d(%) 18.5(24.4)e 14.9(20.9)e
work
R d(%) 21.6(28.6)e 17.7(21.9)e
free
S.D.d
Bondangles(o) 1.020 1.088
Bondlengths(Å) 0.006 0.006
MeanBd(Å2) 33.4 14.3
Mainchain(Å2) 29.8 9.9
Sidechains(Å2) 34.3 14.0
Solvent(Å2) 44.6 27.5
Ramachandranplotf
Mostfavored(%) 91.8 93.4
Additionallyallowed(%) 7.4 6.0
Generouslyallowed(%) 0.5g 0.3g
Disallowed(%) 0.3h 0.3h
aNumbersinparenthesesrefertoouterresolutionbin.
bMultiplicity-weightedR (66).
merge
cFromTruncateanalysis(67).
dFromphenix.refine(47).
eHighestresolutionbin.
fFromProCheck(68).
gHaKATGln135andAtKAT2Gln137adoptstrainedconformationinactivesite.
hHaKATLys64andAtKAT2Lys66adoptstrainedconformationinwelldefineddensityintightturn.
expectedtherearedeviationsinthepositionandqualityofelec- betweenthesetwoproteins,afewchangesinmainchainposi-
tron density at the very extremities of the N and C termini. tions and side chain orientations can be observed, mainly in
Thereissomedeviationintheorientationof(cid:1)-hairpin1,byas solvent-exposedareasofthestructures.Fig.1Bshowsasuper-
muchas5ÅinAtKAT2,andsmalldifferences((cid:8)1Å)inthe positionofalltypeIthiolasestructuresavailable;thesearecom-
(cid:2)-helicalregionoftheL-domain.Electrondensityisofacon- paredinmoredetailinsupplementalTableS1andFig.S1.
tinuoushighqualitywithonlyafewexceptions;theloopfrom AtKAT2 Redox Potential and Activity—Sundaramoorthy et
theN-domaintotheL-domainandtheextremitiesof(cid:1)-hairpin al.(21)suggestthattheoxidizedformfoundintheirAtKAT2
1 are of poorer quality, and here side chains are defined only crystals represents an inactive state of a biologically relevant
marginally.AmutationwasalsonoticedintheHaKATelectron redoxswitchregulatingperoxisomal(cid:1)-oxidationinArabidop-
densityatposition223;thiswasconfirmedbysequencingtobe sis;however,nodataarepresentedbridgingtheformfoundin
athreonineinsteadofanalanine.ThisresidueissituatedonH4 their crystal structure to activity. Our activity data (Fig. 2A)
in the L-domain, and the mutation is not thought to have showthatAtKAT2wasinactivated,withahalf-lifeof0.6h,in
affectedtheoverallstructurebecausetheAtKAT2andHaKAT thepresenceoftheoxidantCSSC(10mM),whereasinthepres-
structures are almost identical as expected from their 85% enceofthereductantCSH(20mM)fullactivitywasretainedfor
sequenceidentity(dimersuperimposeswithanr.m.s.d.of0.57 morethan12h(datanotshown).IntheabsenceofbothCSSC
Åover780C(cid:2)atoms).AtKAT2andHaKATexhibitonlysmall andCSH,AtKAT2wasspontaneouslyinactivatedwithahalf-
differencesintwoloops;amovementofthe(cid:1)-hairpin1insub- lifeof2.6h,indicatingaverylowreductionpotential.Toinves-
unitBthatresultsinaC(cid:2)differenceinpositionof(cid:8)7Åatthe tigatethisfurther,tryptophanfluorescencewasrecordedwhile
tipoftheloop(Lys247 versusLys249 ).Thisdiffer- titratingtheenzymewithCSH.Trp175istheonlytryptophan
AtKAT2 HaKAT
enceisonly(cid:8)1ÅintheAsubunits,suggestingacrystalpacking residueinAtKAT2.Buriedatthesurfaceneartheactivesiteand
influence.Toamuchlesserextentthereisalsoavariationinthe thesubunitinterface,itisoptimallypositionedtoreportfluo-
loop between the N-domain and the L-domain. There is an rescentlyontheredox-coupledrearrangementofthemolecule
additional(cid:2)-helix(H6a)intheC-domainofAtKAT2thatisnot (supplementalmovieonline).Thetryptophanfluorescenceof
observedinHaKATorinanyoftheothertypeIthiolasestruc- AtKAT2increaseswithincreasing[CSH]2/[CSSC]ratios(Fig.
tures. In addition to the side chains, which are different 2B). The increase is accompanied by a small red shift of the
24082 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER31•JULY30,2010
RedoxControlofPeroxisomalThiolase
FIGURE1.ThestructureofKAT.A,schematicofAtKAT2monomer:N-domain
isshownindarkblue,C-domainingreen,andL-domainincyan.Thesecondary
structureelementsarelabeled.B,superpositionofKATsinopenconforma-
tion. The r.m.s.d. values and sequence identities are given in supple-
FIGURE2.Characterizationofredoxstatedependentactivity.A,AtKAT2is
mentalTableS1.Adetailedcomparisonofthesestructuresisalsoincludedin
inactiveinthepresenceofCSSCandactiveinthepresenceofCSH.Therates
thesupplementalmaterial(DiscussionFig.S1,andTableS1).
ofacetoacetyl-CoAcleavingbyAtKAT2weredeterminedasafunctionoftime
andincubationwithoxidantorreductant.12.5(cid:3)Menzymepreparationswere
incubatedat25°Cin35mMTris(pH8.5),0.1MNaCland20mMCSH(F),10mM
emission (Fig. 2C) in accord with a slightly higher degree of CSSC((cid:1))orbuffer(f).TheeffectsofCSH(E)andCSSC(ƒ)onthesubstrate
solvation of Trp175 in the open, active conformation. Adding areincludedforreference.Theincubationsweresampledatthegiventime
(cid:1)-mercaptoethanol to the solution resulted in a similar pointsbyrapidmixingand2500(cid:3)dilutionwith100(cid:3)MCoA,50(cid:3)Maceto-
acetyl-CoAinamicrocuvettewithabsorbancerecordedat233nm.Thefinal
increase,whereasaddingH2O2resultedinadecrease.Compar- concentrationsofAtKAT2,CSH,andCSSCinthereactionswere5,8,and4nM,
ingtheoverallsecondarystructurecontentofAtKAT2 and respectively.B,AtKAT2changestertiarystructureuponincubationwithCSSC.
active AtKAT2wasincubatedwithdifferentratiosof[CSH]2/[CSSC]at25°Cfor10
theoxidizedAtKAT2inactive,thereisamere2.4%differencein min,andtryptophanfluorescencewasrecordedat333nmwithexcitation
the (cid:2)-helical content in favor of the AtKAT2 and 2.5% wavelength285nm.ThetotalconcentrationofCSHandCSSCwaskeptcon-
more(cid:1)-structureintheAtKAT2 .TheCDascptiveectraofoxi- stantat125mg/liter.Atwo-stateequationwasfittedtothenormalized(solid
inactive line)dataandthemidpointoftransitiondetermined.C,Fluorescencespectra
dizedandreducedAtKAT2differedinsignificantly,supporting ofreduced(solidline)andoxidized(dottedline)AtKAT2at1and0.03M[CSH]2/
the interpretation that the fluorescence signals represent [CSSC],respectively.ConditionsasinBexceptemissionrecordedfrom325to
450nm.
the local rearrangements observed in the AtKAT2 and
inactive
AtKAT2 crystal structures (see supplemental movie
active
and“Discussion”online).Thetransitionmidpointbetweenthe threeenzymaticstepsof(cid:1)-oxidationinbacteriaandmitochon-
twostatesis0.17(cid:9)0.02M,whichbyusingtheNernstequation drialedustoinvestigatewhetherasimilartrifunctionalcom-
givesanE atpH8.5of(cid:7)372mVequaltoE of(cid:7)283mVatpH plex exists in plants. We employed pulldown assays from
m 0
7.0 (assuming linear proportionality with pH with a slope of enriched peroxisomal lysate isolated from B.napus cv. seed-
(cid:7)59mVinthispHrange(40,41)),indicatingthatthisredox lingsandfocusedonapossibleinteractionbetweenMFP2and
potential falls within a physiological range (see “Discussion” KAT2.BrassicaandArabidopsisbothbelongtothemonophy-
below). letic family of the Brassicaceae and share a recent common
PotentialofKATtoFormaMulti-enzymeComplexwithMFP— ancestryendingabout20millionyearsago(59).TheBnKAT2
The existence of multifunctional enzymes harboring the last andAtKAT2sequencesare96%identicalandtheBnMFP2and
JULY30,2010•VOLUME285•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 24083
RedoxControlofPeroxisomalThiolase
inlivingplantcellshasbeendeterminedtobeabout(cid:7)320mV
inthecytosolatpH7.2and(cid:7)360mVinthemitochondriaatpH
8.0 (60). Assuming that the peroxisomes can attain a redox
potentialsimilartothatofthemitochondria,theratiobetween
oxidizedandreducedAtKAT2atthermodynamicequilibrium
wouldbe0.24atpH8.5,whereasat(cid:7)340mVitwouldbe99%
oxidized.Otherdithiolshavebeensuggestedtoberedoxsen-
sors; the disulfides of Orp1 and the redox-sensing domain of
thecytosolicYap1havepotentialsaslowas(cid:7)315and(cid:7)330mV
atpH7.0(61),whereasthepotentialoftheactivecysteinesof
wild-type plant cytosolic thioredoxins varies from (cid:7)280 to
(cid:7)304 mV at pH 7.0 (62). Yap1 is thought to be kept in the
reduced state by the non-equilibrium ratios of 102 to 104
reduced:oxidizedthioredoxinexistinginthecells.Althougha
recent proteomics study identified two putative thioredoxins
and a glutathione reductase in Arabidopsis peroxisomes (63),
thereislittleknowledgeaboutdisulfidereducingagentsinthe
peroxisomes.Overall,itislikelythatAtKAT2representsavery
sensitiveredoxswitchcapableofregulating(cid:1)-oxidationevenat
minor perturbations of the peroxisomal redox environment.
Interestingly,ithasbeenobservedthattheactivityofKATis
compromised by 0.1 mM H O in catalase-inhibited enzyme
2 2
preparations from peroxisomes of rat liver (64). It therefore
seemslikelythattheoxidizedandreducedformsofAtKAT2,
crystallized under different conditions, represent biologically
relevantformsofAtKAT2.
When comparing the AtKAT2 (this study) and
active
AtKAT2 (PDB code 2C7Y (21)) structures, there are a
inactive
numberofdifferences.AtthemonomerlevelAsp38–Asp51in
theAtKAT2 structureforma(cid:1)-strand(S1a),whichaffil-
inactive
iateswiththeC-terminaldomainandpairsupwiththeequiv-
FIGURE3.Westernblotanalysisfromco-immunoprecipitationstudies. alent strand in the dimer formation (supplemental movie
Lanescontainedthefollowing:lane1,elution1fromMFPbeads;lane2,load online),resultinginanextendedcentral(cid:1)-sheet.(cid:1)-StrandS1a
ontoMFPbeads;lane3,peroxisomallysate;lane4,loadontoKATbeads;lane
5,elution3fromKATbeads;lane6,elution2fromKATbeads;lane7,elution1 isnotseenintheAtKAT2structuredescribedhere,anddespite
fromKATbeads;lane8,molecularweightmarker.A,Westernblotdeveloped theresiduesbeingpresentintheconstruct,theyareunseenin
withprimaryantibodyagainstKAT.B,Westernblotdevelopedwithprimary
antibodyagainstMFP.AbandisseenforMFPfromtheKAT2pulldownsug- theelectrondensity.Onereasonforthisdifferencecouldbethe
gesting a complex (black circle). Elution steps were performed only after presenceoftheN-terminalHis taginourconstruct;however,
extensivewashing. 6
wethinkthisisanunlikelyexplanation,astheequivalentresi-
AtMFP2 sequences are 90% identical. When BnKAT2 anti- duesarepresentintheHaKATconstructandarealsounseenin
bodies were immobilized for co-immunoprecipitation, both the structure where there is no N-terminal His tag. The
6
BnKAT2andAtMFP2antibodiesidentifiedbandsofanappro- (cid:1)-strand formation is therefore more likely the result of the
priatemolecularweightontheresultingWesternblot(seeFig. dimerreorientationintheoxidizedformofAtKAT2,causedby
3), suggesting that these proteins interact. However, when thedisulfidebridgeformation.Theinactiveconformationpro-
AtMFP2antibodieswereimmobilized,onlytheAtMFP2anti- videsadifferentinterfacethatallowsstabilizationoftheother-
bodiesidentifiedabandontheWesternblot,suggestingthat wiseflexiblechain,providingmoreinteraction.Theactivesite
KAT2 was not pulled down with MFP2. The inability of the loopintheinactiveform,between(cid:1)-strandS3and(cid:2)-helixH5
anti-AtMFP2toco-immunoprecipitateBnKAT2doesnotdis- intheN-domain,isflippedouttowardsubunitB,repositioning
credittheexistenceofthecomplex,astheantibodiesmaydis- the Cys138 C(cid:2)by 5.4 Å (superimposing A chains) or 5.8 Å
rupt the interaction. When a negative control using an unre- (superimposingBchains)awayfromthehelixdipole,whichis
lated antibody, anti-His, in place of the antibodies against importantfortheactivationofCys138(20).Theshort3 -helix
10
KAT2andMFP2forthepulldownwasperformedandWestern (N-domainH4)containingtheactivesitecysteine,presentinall
blotsweredevelopedusingantibodiesagainstKAT2andMFP2, of the active structures, is not observed in the AtKAT2
inactive
nobandswereidentified(datanotshown). structure.ThelocationofthisloopsegmentinAtKAT2
inactive
would cause steric clashes with(cid:1)-strand S3 in the N-domain
DISCUSSION
and the preceding loop on subunit B in the active dimer. As
RedoxRegulationofAtKAT2andComparisonofActiveand such,subunitBisrepositionedintheAtKAT2 dimerviaa
inactive
Inactive Structures—The redox potential of the peroxisomes rotationof(cid:8)30oandtranslationof(cid:8)10Å(supplementalmovie
hasnotbeendeterminedquantitatively,buttheredoxpotential online). The Met168–Pro195 region in AtKAT2 L-domain
active
24084 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER31•JULY30,2010
RedoxControlofPeroxisomalThiolase
capsovertheS2a-H1loopand(cid:2)-helixH3insubunitBN-do-
mainandcontainsashort3 -helix(H1)andan(cid:2)-helix(H2).In
10
the AtKAT2 structure, this loop is largely disordered,
inactive
with Met173–Lys184 not seen in the structure and residues
Met168–Pro172extendingouttowardwhere(cid:2)-helixH1inthe
L-domainispositionedinAtKAT2 .InAtKAT2 ,H2
active inactive
andH3intheL-domainformonecontinuous(cid:2)-helixviaaflip
of Pro195, which is then, remarkably, incorporated into the
(cid:2)-helixreorientingthepolypeptidesuchthatCys192isreposi-
tionedby(cid:8)9.5Å(supplementalmovieonline).Thelargecon-
formationalchangeofthisregion(CterminusofN-domainS4
toNterminusofC-domainH4),uponthedisulfidebridgefor-
mation,resultsinthedomainreorientation.(cid:1)-Hairpin1inthe
L-domain and S1-H1a in the C-domain have a difference in
position of (cid:8)6 Å, and (cid:1)-hairpin 2 has a difference of (cid:8)3 Å.
When superimposing the B subunits there is an alternative
movementin(cid:1)-hairpin1;Lys249C(cid:2)ispositioned2.8Åinthe
oppositedirectiontothe7Åmovementobservedinthisloopin
theHaKATstructuresuggestingthatthisishighlymobile.The
3 -helix,H6a(observedinAtKAT2 ,HaKAT,andPfKAT),
10 active
in the L-domain is not observed in the inactive form; it is
replaced with two (cid:1)-turns, as in the HsKAT and ScKAT
structures.
The AtKAT2 dimer is very different from all other
inactive
KATstructuresandhasroughlyhalftheamountofintersub-
unit contacts as the active dimers. Twenty-six residues from
each chain are involved, giving an interacting surface area of
1225Å2with11hydrogenbondsand168non-bondedcontacts
comparedwith54residuesfromeachchainwithaninteracting
surface area of 2500 Å2, 41 hydrogen bonds, and 408 non-
bonded contacts (values calculated in PDBsum) for the
AtKAT2 dimer. In addition, the gap volume index (this
active
givesanindicationofhowwellthetwoprotomersfittogether,
with a lower number indicating a better fit) is 3.9 Å for the
inactiveformand2.3Åfortheactiveform;andtheinterpro-
tomer salt bridges that are present in all of the active KAT FIGURE4.ConstructionoftheArabidopsisKAT2(cid:2)MFP2complexmodel
based on the prokaryotic multi-enzyme complex (PDB code 1WDK).
structures (Asp99–Arg136 and Arg132–Asp147 (AtKAT2 num-
A,PseudomonasstructurewithMFPinmediumgrayandKATinlightgray.
bering)) are not formed in the AtKAT2 conformation. Region1((cid:2)-helical)andregion2((cid:1)-hairpin1)arehighlightedinblack.B,Ara-
inactive
bidopsis model follows the same coloring scheme as A with AtMFP2 in
Thisstronglysuggeststhattheactive,reducedconformationis
mediumgrayandAtKAT2dimerinlightgray.
themoststabledimerform.Ifwelookattheisoformspresentin
Arabidopsis,bothAtKAT1andAtKAT5possesstheCys192and 1WDM, and 2D3T (16)). The Pseudomonas complex is com-
Pro195 and so are also likely to be regulated via their redox posedoftwoMFPsubunits(1WDKsuperimposeswithAtMFP
environment.Incontrast,AtACAT1andAtACAT2(responsi- withanr.m.s.d.of1.9Åover1406residues;sequenceidentity
bleforthereversereactionandsharing34and36%sequence 31%)andtwoKATsubunits(Fig.4A)constitutingadimerthat
identitywithAtKAT2)donothavetheseresidues,suggesting isindistinguishablefromthatofthereducedplantAtKAT2
active
that the reverse reaction (Claisen condensation) is not regu- structure(1WDKsuperimposeswithanr.m.s.d.of1.2Åover
latedbyredox.Cys192andPro195areconservedinallnon-mi- 735 amino acids; sequence identity 42%). The interactions
tochondrialeukaryoticKATsinwhichtheyhavebeensought. between the two MFPs and between the MFP and KAT sub-
However,theseresiduesareabsentintheprokaryoticandmito- unitsinthePseudomonasstructurearequitesmallinsurface
chondrialKATs,thusrevealingaverydistinct,perhapsevolu- areabutcollectivelyformastablecomplexthatcanwithstand
tionary, difference (peroxisome versus mitochondrion) in the purificationviagelfiltration(16,65).Inaddition,substrate-de-
regulationof(cid:1)-oxidation. pendent conformational changes are seen within the Pseudo-
Model of Plant (cid:1)-Oxidation Multi-enzyme Complex—The monastri-complex,mainlyinvolvingtheMFPs(comparePDB
crystal structures of the plant KATs presented here and the 1WDK with 2D3T for extremities), but the protein-protein
crystal structure of AtMFP2 (presented in an accompanying interactions remain constant. Our co-immunoprecipitation
paper(Arentetal.(51))bearcloseresemblanceinstructureto experimentssuggestaninteractionbetweenMFPandKATin
the components of the bacterial (cid:1)-oxidation multi-enzyme plants,andthuswewereencouragedtoseewhetheracomplex
complex from Pseudomonas (PDB codes 1WDK, 1WDL, similar to the Pseudomonas multi-enzyme complex could be
JULY30,2010•VOLUME285•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 24085
RedoxControlofPeroxisomalThiolase
Pseudomonas. However, this posi-
tiveareaisnotatallconserved,and
inthestructuresavailablenohydro-
gen bond interactions were
observed. Asp135, in the loop pre-
cedingH1,isactuallysituatedneara
negativelychargedareaoftheMFP.
Overall, the interactions between
region 1 in the KAT with the MFP
are more substantial when com-
paredwithregion2withrespectto
surface area, conservation, and
complementaryshape.
In comparison, the complex
modeled for AtKAT2 and AtMFP2
is fairly similar. The calculated
interaction surface area overall in
the Arabidopsis model is actually
larger than in the Pseudomonas
structure,withregion1,theH1-H2
region, involved in an interface of
FIGURE 5. Comparison of prokaryotic multi-enzyme complex with model complex of AtKAT2 and (cid:8)750 Å2 and region 2, the (cid:1)-hair-
AtMFP2.AandC,Pseudomonasstructure.BandD,Arabidopsismodel.PfMFPandAtMFPareshownasan pin,involvedinaninterfaceof(cid:8)920
electrostaticsurfacecoloredaccordingtoelectrostaticpotential(fromred(mostelectronegative)towhiteto
blue(mostelectropositive)).PfKATandAtKAT2areshownasarainbow-coloredschematicaccordingtocon- Å2.However,theshapefitofregion
servationamongprokaryoticandplantKATs,respectively(fromblue(mostconserved)togreentoyellowtored 1 is not as favorable as that in the
(unconserved)).
Pseudomonas structure, with a gap
volume index of 4 Å. Region 2,
modeledfromourArabidopsisKAT2andMFP2crystalstruc- which is more extensive in the Arabidopsis model, has a gap
tures(see“ExperimentalProcedures”fordetailsandFig.4).A volumeof4.3Å,whichismoresustainableasacontactthanthis
complex between MFP2 and KAT2 would allow metabolite regioninthePseudomonasstructure(7Å);residuesaresimi-
channeling, facilitating the product of one enzyme action to larly conserved. It should be noted that upon actual complex
betransferreddirectlytothenextinthecyclewithouteitherthe formation,smallmovementsmayoccurprovidingadifferentfit
equilibrationoftheintermediatewiththebulksolventorthe thanoursimplemodel.(cid:1)-Hairpin1islongerinAtKAT2and
pursualofanalternativepathway.Additionally,thelackofsub- thushasthepotentialtomakealargerinterfacewithAtMFP2.
strate specificity at the final thiolytic step of the pathway is Lys244onS1ispositionednicelytoformanelectrostaticinter-
consistentwiththeideaofsubstratechannelingwhileallowing actionwiththeindentednegativesurfaceofMFP2(Asp185;Fig.
theredoxcontrolofthecycleasawholeinresponsetoH O 5D),andtheseresiduesareconservedinplantKAT/MFPs.In
2 2
levels. thecaseofArabidopsis,thereisevenapotentialforasaltbridge
InthePseudomonasstructures,bothMFPsubunitsinteract formation between Glu253 on S2 and His179 in AtMFP2.
withbothKATsubunits,althoughtheinteractingsurfaceareas Althoughtheseresiduesarenot100%conservedamongplant
aresmall(500–850Å2).TherearetworegionsintheL-domain MFP/KATs,thechargesaremoresowiththeglutamicacidwell
oftheKATsubunitsthatinteractwithMFPsubunits(Fig.5): conservedandhistidinesometimesreplacedbyarginine.
Region1iscomposedofH1-H2andhasaninteractionsurface There are a number of conserved proline residues in the
areaof(cid:8)800Å2andagapvolumeindexof2.5Å,indicatinga vicinityoftheinterfacethatcouldbekeytoaddingstructural
favorablecomplementaryshape.Region2involves(cid:1)-hairpin1 rigiditytotheloopsinvolvedintheinteractionwithMFP2(Fig.
from the KAT subunit and has an interaction surface area of 5, C and D). The potential electrostatic interaction for Lys146
(cid:8)550Å2andagapvolumeindexof7Å,indicatingaloose,less seeninthePseudomonasstructuredoesnotexistinthecaseof
complementaryshapedinteractionthanthatofRegion1.There the Arabidopsis model, as there is no charged residue at that
arefewhydrogenbonds,somefavorableelectrostaticinterac- positionandalsothesurfacepotentialofAtMFP2isnotnega-
tions,butnosaltbridgesatthisinterface.Thesizeandlimited tiveinthatarea.Indeed,theAtMFP2areaaroundH2islargely
specific interactions suggest that this complex could be fairly neutralinsurfacepotentialcomparedwiththenegativepatch
transientinvivo.Inregion1,H2andtheCterminusofS1are seeninPseudomonas.Overall,theArabidopsismodelexhibits
wellconservedintheprokaryoticKATs;however,H1andthe moreelectrostaticpotential(10–20%morechargedresiduesat
restofthe(cid:1)-hairpin1seemtobefairlyvariable(Fig.5).Elec- interface)ateachoftheinterfacesthanthePseudomonasstruc-
trostaticinteractionsareevidentbetweenLys146ofH2andthe ture, which could play a role in interaction specificity of the
conserved negative surface of thePfMFP (Asp162 and Glu166) isoformsinArabidopsisandmaymakethecomplexlesstightly
(Fig.5,AandC).Asp200inthe(cid:1)-hairpinisinthevicinityofa boundinconventionalproteinextractionprocedures.Inaddi-
positivelychargedpatchonthesurfaceoftheMFPinthecaseof tion,inplantsthereisthepotentialtoformcation-piinterac-
24086 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER31•JULY30,2010
RedoxControlofPeroxisomalThiolase
tionsbetweenahighlyconservedLys281inAtKAT2withPhe233 Carde,J.P.,Bryce,J.H.,Graham,I.A.,andSmith,S.M.(2001)PlantJ.28,
inAtMFP2(conservedinplantMFP2-likebutnotAIM1-like 1–12
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isoforms).Thus,weproposethatasimilarinteractiontothat
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foundinPseudomonascouldexistbetweenKAT2andMFP2in
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theperoxisomalplantsystem;however,wewouldsuggestthat
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Acknowledgments—We thank beamline scientists at stations X12,
34. Nicholls,P.,andSchonbaum,G.R.(1963)inTheEnzymes,2ndEd.,pp.
DESY, Hamburg, and ID23, ESRF, France for technical assistance
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duringdatacollection.WethankAnnetteKureAndreassenforgen-
35. Sinclair, A. M., Trobacher, C. P., Mathur, N., Greenwood, J. S., and
erallaboratoryassistance,andwearealsoverygratefultoDr.Anders
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