Terminal Alkene Formation by the Thioesterase of Curacin A Biosynthesis

16 Pages·2011·7.52 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY VOL.286,NO.16,pp.14445–14454,April22,2011
©2011byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Terminal Alkene Formation by the Thioesterase of Curacin A
Biosynthesis
STRUCTUREOFADECARBOXYLATINGTHIOESTERASE*□S
Receivedforpublication,December21,2010,andinrevisedform,February25,2011 Published,JBCPapersinPress,February27,2011,DOI10.1074/jbc.M110.214635
JenniferJ.Gehret‡§§,LiangcaiGu§§§1,WilliamH.Gerwick¶,PeterWipf(cid:1),DavidH.Sherman§**‡‡§§,
andJanetL.Smithत2
FromtheDepartmentsof‡BiologicalChemistry,§MedicinalChemistry,**Chemistry,and‡‡MicrobiologyandImmunology,and
§§LifeSciencesInstitute,UniversityofMichigan,AnnArbor,Michigan48109,the¶ScrippsInstitutionofOceanography,University
ofCaliforniaatSanDiego,LaJolla,California92093,andthe(cid:1)DepartmentofChemistry,UniversityofPittsburgh,
Pittsburgh,Pennsylvania15260
CuracinAisapolyketidesynthase(PKS)-non-ribosomalpep- Natural products display a remarkable chemical diversity,
tidesynthetase-derivednaturalproductwithpotentanticancer providingadvantagesfortheproducingplantsandmicrobesto
properties generated by the marine cyanobacterium Lyngbya survive and thrive in particular ecological niches. These sec-
majuscula.TypeImodularPKSassemblylinestypicallyemploy ondarymetabolitesandtheirderivativeshaveimportantappli-
athioesterase(TE)domaintooff-loadcarboxylicacidormacro- cationsaspharmaceuticals(1),andsomehavethepotentialto
lactone products from an adjacent acyl carrier protein (ACP) bedevelopedasbiofuels(2).Geneclustersencodingassembly
domain.InastrikingdeparturefromthisschemethecuracinA linebiosyntheticpathwaysforpolyketideandpolypeptidenat-
PKSemploystandemsulfotransferaseandTEdomainstoforma uralproductsareubiquitousinbacterialandfungalgenomes.
terminal alkene moiety. Sulfotransferase sulfonation of (cid:1)-hy- Polyketidesynthase(PKS)3andnon-ribosomalpeptidesynthe-
droxy-acyl-ACPisfollowedbyTEhydrolysis,decarboxylation, tase(NRPS)pathwayshaveacommonmodularorganizationin
andsulfateelimination(Gu,L.,Wang,B.,Kulkarni,A.,Gehret, whichintermediatestetheredtocarrierdomainsbyathioester
J.J.,Lloyd,K.R.,Gerwick,L.,Gerwick,W.H.,Wipf,P.,Håkan- linkagepasssequentiallythroughmodulesoftheassemblyline.
sson,K.,Smith,J.L.,andSherman,D.H.(2009)J.Am.Chem. The final step in the assembly line is typically a thioesterase-
Soc. 131, 16033–16035). With low sequence identity to other catalyzedoff-loadingfromthefinalcarrierdomaintoproducea
PKSTEs(<15%),thecuracinTErepresentsanewthioesterase carboxylate,macrolactone,orcyclicpeptide.
subfamily.The1.7-A˚ curacinTEcrystalstructurerevealshow Inanotableexceptiontothisoff-loadingparadigm,thecura-
thefamiliar(cid:2)/(cid:1)-hydrolasearchitectureisadaptedtospecificity cinAfinalproductcontainsaterminalalkenemoiety.Curacin
for(cid:1)-sulfatedsubstrates.ASer-His-Glucatalytictriadiscen- A, from the marine cyanobacterium Lyngbya majuscula, is a
teredinanopenactivesitecleftbetweenthecoredomainanda mixed polyketide/non-ribosomal peptide with antimitotic
lidsubdomain.UnlikeTEsfromotherPKSs,thelidisfixedinan properties (3). The hybrid PKS/NRPS assembly line pathway
openconformationononesidebydimercontactsofaprotrud- forcuracinA(4)generatesseveralunusualchemicalgroupsin
inghelixandontheothersidebyanarginineanchorfromthelid additiontotheterminalalkene,includingacyclopropylring,a
intothecore.Adjacenttothecatalytictriad,anotherarginine thiazolinering,andacisdoublebond.Wehaveinvestigatedthe
residueispositionedtorecognizethesubstrate(cid:1)-sulfategroup. biosynthetic steps leading to several of these segments (5–9).
The essential features of the curacin TE are conserved in Herein we investigate the structural basis for the unique off-
loadingstrategyleadingtotheterminalalkeneinthecuracinA
sequences of five other putative bacterial ACP-ST-TE trido-
molecule.
mains. Formation of a sulfate leaving group as a biosynthetic
ThecuracinPKShasanunusualterminalmodule,theCurM
strategytofacilitateacylchaindecarboxylationisofpotential
protein,withaC-terminaltridomaincomprisedofanacylcar-
valueasaroutetohydrocarbonbiofuels.
rierprotein(ACP),asulfotransferase(ST),andathioesterase
(TE)(Fig.1A).AnnotationsofbothSTandTEwerebasedon
weak sequence similarity to characterized enzymes. The pre-
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth dictionofanSTwithinaPKSwasunprecedented(4).STsare
GrantsR01DK42303(toJ.L.S.),R01CA108874(toD.H.S.,W.H.G.,and widelydistributedandareknowntohavedetoxification,hor-
P.W.), and U01 TW007404, and a Hans W. Vahlteich Professorship (to mone regulation, or signaling functions (10). They catalyze
D.H.S.).
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains transferofasulfonategroupfromthedonorPAPStoahydroxyl
supplementalFigs.S1–S3andTablesS1. oramineofanacceptorsmallmoleculeorprotein.CurMTE,
Theatomiccoordinatesandstructurefactors(code3QIT)havebeendepositedin
theProteinDataBank,ResearchCollaboratoryforStructuralBioinformatics,
RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/).
1Presentaddress:Dept.ofGenetics,HarvardMedicalSchool,77Ave.Louis 3Theabbreviationsusedare:PKS,polyketidesynthase;NRPS,non-ribosomal
Pasteur,NRB232,Boston,MA02115. peptidesynthetase;TE,thioesterase;ACP,acylcarrierprotein;ST,sulfo-
2Towhomcorrespondenceshouldbeaddressed.Tel.:[phone];Fax: transferase;PAPS,adenosine3(cid:1)-phosphate5(cid:1)-phosphosulfate;PAP,aden-
734-763-6492;E-mail:[email protected]. osine3(cid:1)-phosphate5(cid:1)-phosphate;SeMet,selenomethione.
APRIL22,2011•VOLUME286•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 14445
This is an Open Access article under the CC BY license.
StructureofCurMTE
superfamilywithacatalytictriadactivesitelocatedatthetopof
an(cid:2)/(cid:1)coreandcoveredbyan(cid:2)-helicallidsubdomain.AllPKS
off-loading TEs of known structure are dimers in which two
N-terminal(cid:2)-helicesinthelidformalid-to-liddimerinterface
(12–15). A classic Ser-His-Asp catalytic triad is positioned at
the center of a narrow tunnel formed by the lid. The tunnel
architecturewithopenendsisfixedbythedimerinterface.In
contrasttothesedimericoff-loadingTEs,manyPKSandNRPS
pathwaysalsohaveasecondmonomericthioesterasecalleda
TEII,whichperformsaneditingfunctionwithinthepathway.
TEIIs,aswellasNRPSoff-loadingTEs,aremonomerswitha
flexibleliddomainthatappearstocontrolaccesstotheactive
site (16–20). The curacin TE sequence has low similarity to
sequencesinallpartsoftheTEphylogenictree(11),lacksan
N-terminalextensionfordimerization,andhasalongerinter-
nallidthanotherPKSoff-loadingTEs.
To gain further insights and enable mechanistic studies of
the novel decarboxylation and sulfate elimination, we report
herethecrystalstructureofCurMTE.ThestructureoftheTE
lid and an unusual dimer interface appear to fix the active
siteinaperpetuallyopenstate.Amodelfor(cid:1)-sulfaterecog-
nitionwastestedbysite-directedmutagenesis.Thesimilar-
ity of CurM to other conserved ACP-ST-TE tridomain
FIGURE1.ChainterminationincuracinAbiosynthesis.A,off-loadingreac- sequences strongly suggests that CurM TE is part of a new
tionsinthefinalmodule,CurM.FollowingextensionbytheCurMketosyn-
thase(KS)andacyltransferase(AT)andreductionbytheketoreductase(KR), subfamilyofthioesterases.
STcatalyzestransferofsulfonatetothe(cid:1)-hydroxylfollowedbyTEhydrolysis
ofthethioesterwithconcomitantdecarboxylationandsulfateelimination. EXPERIMENTALPROCEDURES
ThesulfonategroupdonoristhePAPScofactor,whichisconvertedtoPAP.
B,experimentalschemefortheassayofCurMTE.RecombinantCurMACP CloningandSite-directedMutagenesis—Aconstructencod-
loadedwithasubstrateanalogandsulfonatedbySTwasreactedwithexcised ingtheTE(CurMresidues1929–2211)wasamplifiedfromthe
CurMTE.ActivitywasmonitoredbyHPLCofthereactionmixtureanddetec-
cosmid pLM14 (9) and was inserted into pMoCR, a vector
tionoftheholo-ACPproduct.
encodingthefusionproteinHis -Mocrforenhancedsolubility
6
althoughidentifiableasathioesterase,doesnotresembleanyof (21). Site-directed mutagenesis was performed using the
thepreviouslyestablishedPKSorNRPSTEsubfamilies(11). QuikChangeprotocol(Stratagene).Allconstructswereverified
Usingasimplifiedanalogofthepenultimatepathwayinter- by sequencing. The CurM ACP and ST expression plasmids
mediate,werecentlydemonstratedthatoff-loadingandtermi- werepreviouslydescribed(9).
nal alkene formation require ST-mediated sulfonation of the Protein Expression and Purification—Escherichiacoli strain
(cid:1)-hydroxyl group from the PAPS cofactor (9) (Fig. 1A). This BL21(DE3) was transformed with an expression plasmid,
was the first observation of biological substrate activation by grownat37°Cin500mlofTBwith4%glyceroltoanA of1.0,
600
formation of a sulfate leaving group. CurM TE acts upon the cooled to 18°C, induced with isopropyl (cid:1)-D-thiogalactopyra-
(cid:1)-sulfateintermediatetoyieldadecarboxylatedproductwitha noside (final concentration, 0.2 mM), and grown for an addi-
terminal double bond resulting from sulfate elimination (Fig. tional18h.Selenomethionyl(SeMet)proteinwasproducedin
1B). CurM TE catalyzes thioester hydrolysis 800-fold more thesameE.colistraininSelenoMetmedium(AthenaES)con-
slowly on the corresponding substrate bearing a (cid:1)-hydroxyl taining100(cid:3)g/mlofseleno-DL-methionine.
group. The unprecedented requirement of a (cid:1)-sulfate for Allstepswereperformedat4°C.Thecellpelletfrom500ml
thioester hydrolysis as well as the decarboxylation and sul- ofcellculturewasresuspendedin40mlofBufferA(20mMTris,
fate elimination suggests a unique catalytic strategy and pH 7.9, 500 mM NaCl, 20 mM imidazole, 10% glycerol), incu-
activesitestructurefortheTE.Moreover,thiscuracinpath- bated30minwithDNase(2mg),lysozyme(5mg),andMgCl (4
2
way decarboxylation strategy provides an opportunity to mM),lysedbysonication,andthesolublefractionloadedontoa
investigate a new biological route to hydrocarbon produc- 5-mlHisTrapNi-NTAcolumn(GEHealthcare).CurMTEwas
tionfromfattyacids.Thus,inadditiontointerestincuracin eluted with a linear gradient from 20 to 650 mM imidazole
biosynthesis as a route to a potent anti-cancer compound (BufferB).TheHis -Mocrfusionpartnerwasremovedbya2-h
6
(3),thepathwayalsohasrelevancetobiofuelproduction. incubationwith1mMDTTandtobaccoetchvirusprotease(1
Off-loadingTEsfrommanyPKSshavebeenstudiedinclud- mgofprotease/50mgofTE)atroomtemperature.Afterover-
ingtheTEsofthepikromycin(PikTE)(12–14),erythromycin nightdialysisat4°CinBufferC(20mMTris,pH7.9,500mM
(14),andtautomycetin(15)PKS.PKSoff-loadingTEstypically NaCl,10%glycerol)with1mMDTT,theremainingHis-tagged
catalyzeeitherhydrolysistoproducealinearcarboxylicacidor proteinswereremovedbynickel-affinitychromatography,fol-
theattackofanintramolecularhydroxyltoproducealarge-ring lowedbysizeexclusionchromatographywithaHiLoad16/60
macrolactone. PKS TEs are members of the (cid:2)/(cid:1)-hydrolase Superdex 200 column (GE Healthcare) pre-equilibrated with
14446 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER16•APRIL22,2011
StructureofCurMTE
BufferC.CurMTEwasconcentratedto5mg/ml,flashfrozenin and prepare figures (34). CurM TE was aligned with affinity-
liquidN ,andstoredat(cid:2)80°C.Of14TEvariantspurifiedas labeled Pik TE (Protein Data Bank code 2H7X, root mean
2
thewildtype,only6yieldedenoughsolubleproteinforassay. squaredeviation(cid:3)3.309)bysuperpositionofthecoredomains
SeMetTEwaspurifiedasthewildtypewithadditionof2mM (residues55–176and232–292inPikTEtoresidues1–126and
DTTtoallbuffers.Yieldsper500mlofculturewere5mgofTE 217–282inCurMTE).ThePRODRG2server(35)wasusedto
and2mgofSeMetTE. generatecoordinatesandatopologyfileformodelingtheacyl-
Crystallization—Crystals grew at 4°C within 24–48 h by enzymeintermediate,whichwasmodeledmanuallyinCOOT
vapordiffusionfroma1:1mixtureofproteinstock(2mg/mlof usingtheaffinitylabelintheactivesiteofPikTE(12,13)asa
TE,20mMTris,pH7.9,200mMNaCl,2.5%glycerol)andwell guide.
solution(27–32%PEG3350,100mMTris,pH8.3–8.5).Micro- EnzymeAssay—CurMTEactivitywasassayedusingamod-
seedingwasrequiredforcrystalgrowthoftheSeMetproteinin ification of our previous protocol (9). Apo-ACP was loaded
similar conditions with 1 mM DTT in the protein solution. withasubstrateanalogbya2-hincubationof50(cid:3)Mapo-ACP,
Crystalswerecryoprotectedinawellsolutionwith15%glyc- 100(cid:3)M(3R)-hydroxy-5-methoxytetradecanoyl-CoA(9),10(cid:3)M
erol,harvestedinloops,andflashcooledinliquidN . S.verticillus Svp(36),10mMMgCl ,100mMTris,pH7.9,at
2 2
Data Collection and Structure Determination—Data were 30°C.CompleteloadingwasconfirmedbyreversephaseHPLC
collected at GM/CA-CAT beamline 23ID-D at the Advanced usingaJupiterC4column(250(cid:4)2.0mm,5(cid:3)m,300Å,Phe-
PhotonSource(APS)attheArgonneNationalLab(Argonne, nomenex)andalinearelutiongradientfrom30to90%CH CN
3
IL).Among25SeMetTEcrystals,onlyonediffractedbeyond4 (0.1% CF CO H)/H O (0.1% CF CO H) over 45 min. After
3 2 2 3 2
Å,butwasvisiblytwocrystalsandhadmultiplelatticesinthe exchange into Buffer C and concentration (Amicon Ultra
diffractionpattern.Aregionvisuallyidentifiedasasinglecrys- 10-kDa concentrators Millipore), substrate-loaded ACP was
talwasprobedinthree10-(cid:3)mstepsusinga20-(cid:3)mmini-beam flashfrozenandstoredat(cid:2)80°C.Togeneratethesulfatedsub-
(22).Thecrystalwascenteredatthepositionwiththestrongest stratefortheTEassay,225(cid:3)MloadedACPwasincubatedwith
diffraction and the least interference from the second lattice, 5(cid:3)MST,1.75mMPAPS(Sigma),100mMTris,pH7.9,atroom
andFriedeldatawerecollectedininverse-beamgeometry((cid:4)(cid:3) temperaturefor10min.Completesulfonationwasconfirmed
0–90oand180–270oaswedgesof45owith1oimages)atthe byHPLC.TheTEreactionwasinitiatedbyadditionofTE(4
wavelengthofpeakabsorptionattheseleniumedge.Thedif- (cid:3)M).After1minthereactionwasquenchedwith10%formic
fractionimagesshowedsignsofdecayattheendofthecollec- acid. Conversion of loaded to holo-ACP was quantified by
tion.Thecrystalwastranslatedtoexposeafreshregiontothe HPLC as described above. Assays with the non-sulfated sub-
beam,androtated90ofromthestartofdatacollection.Eachof stratewereperformedbyincubating1mM(3R)-hydroxy-5-me-
tworegionswasprobedwiththe10-(cid:3)mmini-beamina3(cid:4)3 thoxytetradecanoyl-CoA,50mMTris,pH7.9,with40(cid:3)MTE
rasterof10-(cid:3)msteps.Datawerecollectedatthestrongestdif- for 16 h, including 6% glycerol and 300 mM NaCl for protein
fracting position with a single lattice, again in inverse beam stability.Thereactionwasquenchedwithequalvolumeof1M
geometry((cid:4)(cid:3)90–150oand270–330oas30owedgeswith0.5o CH CO Handneutralizedwith1MNaOH,andcrotonyl-CoA
3 2
images). The two partial datasets were integrated separately was added as an internal standard. Hydrolysis was analyzed
andscaledtogetherusingtheHKL2000suite(23).UsingPhaser usingaLunaC18column(250(cid:4)4.60mm,5(cid:3)m,100Å,Phe-
(24) in the PHENIX (25) software suite, selenium sites were nomenex)withalineargradientfrom10to90%CH OH/H O
3 2
foundforallMetresiduesofthefourpolypeptidesintheasym- (10mMCH CO NH )over20min.
3 2 4
metric unit. Nine Met side chains had two partially occupied
RESULTS
seleniumsitesandanotherMethadthreesitesfor39totalsele-
niumsites(averagefigureofmerit(cid:3)0.401).Afterdensitymod- OverallStructure—ThecuracinTEistheC-terminaldomain
ificationand4-foldnoncrystallographicsymmetryaveragingin ofthe2211-residueCurMpolypeptide.Becauselowsequence
RESOLVE(26)(figureofmerit(cid:3)0.81),an86%completeinitial identity to other TEs prevented accurate definition of the
modelwasbuiltbyAUTOBUILD(27)andcompletedmanually domain N terminus, expression plasmids were constructed
inCOOT(28).A1.7-Ånativedatasetwasusedforrefinement. with three different start sites corresponding to amino acids
REFMAC5(29),fromtheCCP4suite(30),wasusedforrefine- 1917,1929,and1934.Forallthreedomainvariants,anN-ter-
mentwith5translation/libration/screwgroupspermonomer minalfusionoftheproteinMocr(21)wasnecessarytoobtain
(29–31).Non-crystallographicsymmetrywasnotusedduring sufficient soluble protein for purification and crystallization.
any stage of the refinement. Electron density was complete Theconstructencodingaminoacids1929–2211ofCurM(here
throughoutthepolypeptidechainexceptfortwoloopregions, denoted 1–283) yielded crystals after removal of the Mocr
whichhaddifferentdisorderedresiduesinthefourpolypeptide fusion partner. In the CurM TE crystal structure, solved by
chains (monomer A, 132–134; B, 205–206; C, 133–136 and single anomalous diffraction phasing using selenomethionyl
205–216;D,206–207).Althoughnosinglemonomerisacom- CurMTE(Table1),thepolypeptidechainwasorderedtothe
pleteviewoftheseloops,superpositionofthemonomerspro- endsoftheconstruct.
videsacompletemodel. CurMTEpossessesthe(cid:2)/(cid:1)-hydrolasefold,asexpected,with
Sequence Alignment, Structure Alignment, and Substrate residues1–132and215–283comprisingthestructurallycon-
Modeling—The search for ACP-ST-TE homologs was done servedcoredomain(Fig.2,AandB).Residuesofthecatalytic
with BLAST (32), MUSCLE (33) was used for multiple triad (Ser100, Glu124, and His266) are located at the top of the
sequencealignment,andPyMOLwasusedtoalignstructures coredomainasinotherTEs,butwithaGluinplaceofthemore
APRIL22,2011•VOLUME286•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 14447
StructureofCurMTE
TABLE1 threehelices((cid:2)2,(cid:2)3,and(cid:2)4)inthecoreofthepartnersubunit
Crystallographicsummary (Fig. 2C), with additional core-to-core contacts of the (cid:1)4-(cid:2)2
CurMTE(SeMet) CurMTE(native) loops of the two subunits. The subunit interface is predomi-
Diffractiondata nantlyhydrophobicandlarge(buriedsurfaceareaof1220Å2
Spacegroup P2 P2
1 1 permonomer).TheNterminiaredistantfromthedimerinter-
X-raysource APS23-ID-D APS23-ID-D
a,b,c(Å) 74.1,86.9,87.1 74.5,86.9,87.6 face(Fig.2C),allowingforfusiontothemonomericSTdomain
(cid:2),(cid:1),(cid:5)(o) 90,90.4,90 90,90.8,90 as well as dimerization of the TE domain within the dimeric
Wavelength(Å) 0.97948 1.0332
d (Å) 2.14(2.22-2.14)a 1.68(1.74-1.68) CurMmodule.Thisisconsistentwiththeobservationthatthe
AmvignerageI/(cid:6)I 16.3(5.5) 18.1(2.1) naturalCurMST-TEdi-domainisdimericinsolution(datanot
R b 0.113(0.362) 0.059(0.384)
Csoymmpleteness 99.5(99.9) 90.2(49.8) shown).
Averageredundancy 6.4(6.3) 3.4(2.0) Thecrystalasymmetricunitcontainstwodimersthatdiffer
Uniquereflections 60,147 114,584
byaslightflexure(3°)atthedimerinterface,accountingforthe
Refinement
Datarange(Å) 39.10–1.68 pooreroverallfitofdimers(rootmeansquaredeviation(cid:3)1.05
No.reflections 108,718 Åfor486C(cid:2)atoms)comparedwithmonomers.Theprotrud-
Rwork/Rfreec 0.164/0.209 inglidhelix,(cid:2)L1,moveswiththepartnersubunitasthesub-
Rootmeansquaredeviations
Bonds(Å) 0.011 unitsflex(supplementalFig.S1B).Thus,thelid-to-coredimer
Angles(o) 1.268
contacthelpsfixthelidinanopenorientation(Fig.3B).Thisis
AvgB-factors(Å2)
Protein 29.5 surprisinganduniquecomparedwithoff-loadingTEsofother
Water 39.9
PKSsinwhichlid-to-liddimercontactsfixthelidinaclosed
Ramachandran
Allowed 99.9% orientation. In these other TEs, the lid-to-lid dimer contact
Outliers 0.1%
stabilizesanopen-endedtunnelwiththeactivesiteatitscenter
Proteinatoms 8817
Watermolecules 1203 (Fig.3C).Incontrast,thedimer-enforcedopenlidofCurMTE
aOutermostshellinparentheses. resultsinahighlyexposed,opencleftactivesite(Fig.3B).Thus,
bIncludinganomalousdifferences.
thedimerinterfacecreatesaverydifferentactivesiteenviron-
cTheR datasetincludedarandom5%ofreflections.
free
mentinCurMTEandtheotheroff-loadingPKSTEs.
common Asp (Fig. 2B). The catalytic triad faces into a cleft CurM TE Is Not Alone—We identified five open reading
between the core and lid. The lid subdomain (residues 133– framesinthesequencedatabasewithsubstantialidentity(33to
214)is(cid:5)20residueslongerthantheanalogousregioninother 51%)totheCurMACP-ST-TEtridomain.Thesequencesare
TEs,andincludesthreehelices((cid:2)L1,(cid:2)L2,and(cid:2)L3)andasmall uncharacterizedproteinproductsfromgenomesofcyanobac-
(cid:1)-hairpin ((cid:1)L1 and (cid:1)L2) (Fig. 2, A and B). The first lid helix teria (Synechococcus and Cyanothece) and proteobacteria
((cid:2)L1) is designated the “protruding helix” because it has few (PseudomonasandHaliangium).Basedontheconservationof
contactswiththelidandnonewiththecoreofthepolypeptide. allthreedomains,including,forexample,theTEcatalytictriad,
Theorientationofthelidwithrespecttothecoreisidentical weassumethattheothergeneproductsalsocatalyzesequential
inthefourindependentcopiesoftheCurMTEpolypeptidein sulfonation, hydrolysis, decarboxylation, and sulfate-elimina-
theasymmetricunitofcrystals(rootmeansquaredeviation(cid:3) tion reactions, and that conserved residues within these
0.29Åfor216C(cid:2)atoms,supplementalFig.S1A),demonstrat- sequencesmayilluminateareasofthestructurethatareimpor-
ingthattheactivesitecleftisidenticallyopeninallfourpoly- tantforfunction.ConservedresiduesinCurMTEandthefive
peptides.Thepositionofthelidismaintainedbycomplemen- other putative TE sequences (Fig. 4) were mapped onto the
tarysurfacecontactsoflidhelix(cid:2)L3(residues176–186)with CurMTEstructure,revealingadenseareaofconservationin
severalloopsinthecore.Mostofthecontactsarehydrophobic. theactivesitecleft(Fig.5A).Sequenceconservationindicates
Specificityforthefixedlid-coreorientationisprovidedbyan thatCurMTEandtheothergeneproductshavethesamelid
“arginineanchor”inwhichthesidechainofArg185inlidhelix structure and orientation and the same dimer interface. The
(cid:2)L3extendsintothecoredomainwhereitformsafullsetoffive essential features of the lid-to-core arginine anchor (Arg185,
hydrogen bonds with core residues, including a buried salt Asp57, and Gln35) are conserved. The protruding helix ((cid:2)L1)
bridge with Asp57 and hydrogen bonds with Gln35 and Glu3 hasaconservedhydrophobiccharacter,asdothesurfacesofthe
(supplementalFig.S2).Additionally,theGln35sidechainamide core that it contacts in the dimer. The lid-to-core TE dimer
ishydrogenbondedwiththebackbonecarbonylofLeu182inlid interface,theopenactivesitecleft,andthesurfaceofthecleft
helix(cid:2)L3.Incontrasttotheremarkablelid-coresurfacecom- arelikelyconservedamongtheseTEs.
plementarityononesideoftheactivesitecleft,thelinkerpep- Active Site—The open cleft active site and intact catalytic
tides(residues129–136and205–215)ontheoppositesideof triad(Fig.3)appearpoisedtohydrolyzeabroadarrayofthio-
the cleft are dynamic with some residues disordered in some estersubstrates.TounderstandhowCurMTEselectsasulfated
subunits(supplementalFig.S1A). substrate, we modeled the acyl-enzyme intermediate of the
NovelDimerInterface—CurMTEisdimericinsolution(sup- hydrolyticreaction(Scheme1)usingourknowledgeofcatalytic
plementalFig.S3)andalsointhecrystalstructure(Fig.2C).The triad catalysis and geometry and the structure of affinity-la-
modeofdimerformationisradicallydifferentfromotherPKS beledPikTE(12)(Fig.5B).Constraintsonthemodelincluded
off-loadingTEseventhoughthecorestructuresandcatalytic acylationoftheSer100nucleophile,carbonyl-oxygenbindingin
triads are similar (Fig. 3A). The primary dimer contact is theoxyanionhole(NHsofIle32andMet101),the(R)-(cid:1)-sulfate
betweentheprotrudinghelix((cid:2)L1)inthelidofonesubunitand isomer,contactofsulfatewiththeenzyme,andplacementof
14448 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER16•APRIL22,2011
StructureofCurMTE
FIGURE2.StructureofcuracinAthioesterase.A,CurMTEpolypeptide.ThestereoribbondiagramiscoloredasarainbowfromblueattheNterminustored
attheCterminuswiththecatalytictriadresiduesinstickformwithamagentaC.B,topologydiagram.CurMTEhasan(cid:2)/(cid:1)-hydrolasefoldinthecoredomain
andanovellidtopology.Residuesofthecatalytictriad(Ser100,Glu124,andHis266)arelabeled.C,backbonetraceoftheCurMTEdimerviewedalongthe
moleculardyad.Monomersarecoloredasarainbow(right)andinyellow(left),withthecatalytictriadasinA,andNandCterminishownasspheresofthesame
colorastheterminalresidue.
thelongacylchainawayfromthecatalyticHis266.Giventhese well as Ala substitutions at Asn267, which is adjacent to the
constraints,theacyl-enzymewaseasilymodeledwithfavorable catalytic His, and at Asn211, which is hydrogen bonded with
bond rotations and without change to the protein structure. Arg205(Fig.5B).
Theacylchainliesinanarrowcleftbetweenthelidandcore TheTEwildtypeandvariantsweretestedusingaone-pot,
domains, and the (R)-(cid:1)-sulfate occupies a niche adjacent to multistep assay (9) in which recombinant CurM ACP was
Arg205(Fig.5A).Themodelisconsistentwiththestrongstere- loadedwithasyntheticsubstratemimic(Fig.1B)andreacted
oselectivity of CurM TE for the (R)-(cid:1)-sulfate over the corre- sequentiallywithrecombinantCurMSTandCurMTE.ACP
sponding(S)isomer(9),asmodelingofthe(S)-(cid:1)-sulfateformof substrates and ACP hydrolysis products were analyzed by
thesubstrateresultedinsulfatecontactswiththehydrophobic HPLC(supplementalFig.S3).ActivitiesoftheCurMTEvari-
surfaceoftheprotein.Themodelalsosuggeststhatthehydro- antswerenormalizedtotheactivityofthewildtype(Table2).
gen-bondedsidechainsofArg205andAsn211mayrecognizethe The three substitutions at Arg205 (R205Q, R205E, and
(cid:1)-sulfatemoiety.Toexploretheseissuesfurther,thefunctions R205A) resulted in significantly reduced activity, compara-
ofthepresumedsulfate-recognitionresiduesandothersinthe blewithanegative-controlsubstitutioninthecatalytictriad
active site cleft were probed by site-directed mutagenesis. (H266R). Substitutions at two Asn residues (N211A and
ThreesubstitutionsweremadeatArg205(Gln,Glu,andAla),as N267A) that are conserved in the ACP-ST-TE group of
APRIL22,2011•VOLUME286•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 14449
StructureofCurMTE
substrate (supplemental Table S1). This result supports a
vitalroleforArg205insulfaterecognition.
DISCUSSION
A New Thioesterase Subfamily—The CurM TE structure
definesanewthioesterasesubfamilywiththeremarkablecata-
lyticactivityofhydrolysis,decarboxylation,andsulfateelimi-
nationtoformalkeneproductsfrom(cid:1)-sulfatedacyl-ACPsub-
strates.ThestructuralfeaturesthatdistinguishCurMTEfrom
other thioesterase subfamilies are conserved in sequences of
fiveotherputativeACP-ST-TEtridomains,indicatingthatall
ofthemcatalyzeterminalalkeneformation.TheCurMTElid
subdomain,whichsitsabovetheactivesite,issubstantiallylon-
gerthantheanalogousregioninotherTEsubfamilies.Thelid
createsanopenactivesitecleft,andisfixedinanopenorien-
tationbykeyinteractionsuniquetothisTEsubfamily(Figs.2C
and3B).Onesideofthelidissecuredbythesurfacecomple-
mentarityoflidhelix(cid:2)L3withthecoredomainandbyanargi-
nine anchor in which Arg185 in the lid extends deep into the
coredomaintoformasaltbridgewithAsp57.Theothersideof
the lid is secured by dimer contacts of lid helix (cid:2)L1 with the
coredomainofthepartnersubunit.
The characteristics of the CurM TE lid provide a striking
contrasttootherPKSandNRPSTEsubfamilies.DimericPKS
off-loadingTEshaveaclosedlid,formedbyalid-to-liddimer
contact, which creates an open-ended tunnel with the active
site at its center (Fig. 3C). Monomeric editing TE II enzymes
andNRPSoff-loadingTEshaveaflexiblelidthatmaycontrol
accessofthephosphopantetheinearmtotheactivesitebyits
abilitytoopenandclose.Theregionofsubstrateentrancein
theseotherTEs(theactivesitetunnelinotherdimericTEsand
theflexiblepartofthemovablelidofmonomericTEs)isanal-
ogoustotheregioninCurMTEsecuredbythearginineanchor.
Hence, the usual entrance route for acyl-ACP substrates is
blockedinCurMTEbytheanchoringoflidhelix(cid:2)L3tothe
coredomain.Instead,wepresumethatthesubstrateentersthe
FIGURE3.ComparisonofcuracinandpikromycinTEs.A,structurealign- CurMTEactivesiteviatheconservedcleftthatextendsaway
mentofthecoreofCurMTE(green)andPikTE(cyan,ProteinDataBankcode
2H7X,(12))(rootmeansquaredeviation(cid:3)1.5Åfor95C(cid:2)atoms).Bothstruc- fromthecatalytictriadtowardthetopofthelid(Fig.5A).In
tureshavetheconserved(cid:2)/(cid:1)-hydrolasecore,butthelidsdiffer.Thezoom contrasttothefirmanchoringontheentrancesideoftheCurM
viewshowstheactivesiteconservationinthecatalytictriadofCurMTE TE active site cleft, the opposite side of the cleft has flexible
(magenta)andPikTE(cyan)withatriketideaffinitylabel(gray).Theviewis
lid-to-corelinkersandlessconservation.
similar to Fig. 2A. B, surface representation of the CurM TE dimer. The
primarydimercontactisbetweenthelid(subunitsintwoshadesofgreen) SubstrateSpecificity—Despitethedeparturefromprecedent
andcore(subunitsinyellowandorange).Theactivesite(magenta)isinan
inthelidarchitectureandsubstrateentrance,thecatalytictriad
opencleftbetweenthecoreandlid.C,surfacerepresentationofthePikTE
dimer.Thedimercontactisexclusivelybetweenthelidsubdomains(sub- ofCurMTEisfullyformedandappearstobepoisedforhydrol-
unitsintwoshadesofblue)withnocontactsofcoredomains(cyan).The ysis (Fig. 3A). The question remains why CurM TE does not
activesite(magenta)withanaffinitylabel(graysticks)isatthecenterofan
hydrolyze any thioester substrate given the positions of the
open-endedtunnel(12).TheviewsinBandChighlightthedifferencesin
activesiteaccess,whicharedistinctfortheCurMTEandPikTEenzymes. Ser100nucleophile,His266base,andIle32/Met101oxyanionhole.
Ananswerisfoundinthewideopenarchitectureoftheactive
sequences but not in the wider TE family resulted in only site, which provides few obvious substrate recognition ele-
2–3-foldreducedcatalyticactivity.TotesttheroleofArg205 ments.CurMTEdoesnotneedexquisiterecognitionofasub-
insulfaterecognition,theArg205variantswereassayedwith stratethatispresentatahighlyeffectiveconcentrationviateth-
anon-sulfatedsubstrate.Duetothe(cid:5)800-foldloweractivity eringtoCurMACP,whichitselfisfusedtotheTE.However,
ofwildtypeTEonnon-sulfatedsubstratesthanonsulfated CurMTEmustselectthe(R)-(cid:1)-sulfatedsubstrateandnotother
substrates (9), these assays were performed with a CoA- acylatedformsofCurMACP(malonyl,(cid:1)-keto,and(R)-(cid:1)-hy-
linked (cid:1)-OH substrate available at a higher concentration droxy),whicharealsopresentatahighlyeffectiveconcentra-
than the ACP-linked substrate. In striking contrast to their tionbeforetheyaretransformedbytheothercatalyticdomains
lack of activity with sulfated substrates, the Arg205 variants of CurM (ketosynthase, ketoreductase, and sulfotransferase,
hadactivitycomparablewiththewildtypeandnon-sulfated respectively).OurmodeloftheTEacyl-enzymeintermediate
14450 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER16•APRIL22,2011
StructureofCurMTE
FIGURE4.SequencealignmentofCurMTEwithputativeTEsfromopenreadingframesencodingtandemACP-ST-TEtridomains.Invariantresiduesare
highlightedinred,conservedresiduesareprintedinred.Theconservedliddomainishighlightedinyellow.Greenstarsindicateactivesiteresiduesandbluecircles
indicateresiduesprobedbysite-directedmutagenesis.SequencealignmentwasperformedbyMUSCLE(33),andthefigurewaspreparedusingESPript(42).
GenBank entries are: Pseudomonas entomophila L48 (GenBank YP_610919), H. ochraceum DSM 14365 (YP_003265308), Synechococcus PCC 7002
(YP_001734428),CyanothecePCC7424(YP_002377174),andCyanothecePCC7822(ZP_03153601).
suggestedthatArg205recognizesthe(R)-(cid:1)-sulfatemoiety(Fig. occur for the corresponding (S)-(cid:1)-sulfated isomer. Intrigu-
5),andthiswasconsistentwiththesite-directedmutagenesis ingly,Arg205isamongtheflexibleresiduesinthelinkercon-
data (Table 2, supplemental Table S1). The Arg205 variants nectingtheCurMTEcoreandlid,andthusmayprovidean
lackedactivitywithasulfatedsubstratebuthadactivitycom- early interaction with incoming sulfated substrates. The
parablewiththewildtypeinthemuchslowerreactionwitha flexibilityofthisregionisconsistentwiththelackofconser-
non-sulfated substrate. Thus Arg205 may be the only positive vation of Arg205 in the homolog from Haliangium ochra-
substrate-recognitionelement,theactivesitemayhavelittleor ceum, in which nearby arginine side chains may serve the
noaffinityfortherestoftheacylchain,andnon-sulfatedsub- samefunction.
stratesmaybeonlyrarelypositionedproperlyforcatalysis.In CatalyticMechanism—TheCurMTEstructurerevealsan
thismanner,the(cid:1)-sulfateactsasa“handle”forArg205toassist intacthydrolyticactivesiteintowhichanacyl-enzymeinter-
ineffectivethioesterbindingtothecatalyticmachineryinthe mediatewasmodeledeasilywithouttheneedtomoveinter-
large open active site cleft, whereas non-sulfated substrates acting groups (Fig. 5). This strongly suggests that acyl-en-
havenosuchguidance.ThegeometryofArg205withrelationto zyme formation precedes other catalytic events. Therefore,
theactivesitealsoensuresthat(R)-(cid:1)-sulfatedsubstratescanbe we propose that hydrolysis of the acyl-enzyme precedes
properlypositionedforcatalysis,whereasstericclasheswould decarboxylationandsulfategroupelimination,basedonour
APRIL22,2011•VOLUME286•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 14451
StructureofCurMTE
FIGURE5.CurMTEactivesite.A,surfaceoftheactivesitecleft.Inthemolecularsurfaceview,theactivesite(magentacatalytictriad)isinacleftinwhichthe
presumedphosphopantetheineentranceislinedwithconservedresidues(cyan),withotherpartsoftheproteinsurfaceingreenandthemodeledacyl-enzyme
intermediateingraysticks.B,modeledacyl-enzymeintermediate(grayC).Inthisstereoview,theintermediateissurroundedbyconservedaminoacidresidues
(cyanC)andthecatalytictriad(Ser100,Glu124,andHis266)(magentaC).Thecarbonyloxygenisboundintheoxyanionhole(hydrogenbondstotheNHsofthe
Ile32andMet101).
SCHEME1.PredictedreactionsequenceforCurMTEhydrolysis,sulfategroupelimination,anddecarboxylation.Arg205recognizesthe(R)-(cid:1)-sulfate,
facilitatinghydrolysisbythecatalytictriad.ThecatalyticHis266ispositionedtoassistindecarboxylationfollowedbysulfateelimination.
detection of both the terminal alkene product and the sub- step.Inthereactionscheme,CurMTEassistsdecarboxyla-
strate analog bearing intact (cid:1)-sulfate and carboxylic acid tionandsulfateeliminationinatleastthreeways.First,the
groups(i.e.theinitialTEserineesterhydrolysisproduct)(9). enzyme binds the carboxylic acid hydrolysis product in a
The CurM TE assures decarboxylation and sulfate group conformation that prevents resonance of COOH with the
elimination in vivo, as the corresponding carboxylated and C –C bond to be broken upon decarboxylation. Arg205,
1 2
sulfatedcuracinAintermediatewasnotdetectedincultures through its interaction with the sulfate at C , may assist in
3
ofL.majuscula(Scheme1)(37).Throughinteractionswith achieving the optimal conformation. Second, the catalytic
theoxyanionholeandArg205,thecarboxylicacidhydrolysis His266likelypromotesdeprotonationofthecarboxylicacid
product remains bound to CurM TE long enough for the hydrolysis product to facilitate loss of CO . Stabilization of
2
enzyme to promote loss of CO and SO2- in one concerted theadditionalnegativechargeonthedepartingSO2-offered
2 4 4
14452 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER16•APRIL22,2011
StructureofCurMTE
TABLE2
Acknowledgments—We thank Drs. Amol Kulkarni and Sarang
ActivityofCurMTEvariants
Kulkarniforthesynthesisofsubstrateanalogs.Beamline23ID-Dissup-
Activitya
portedbytheNationalInstitutesofHealth,NationalInstituteofGeneral
% Medical Sciences Grant Y1-GM-1104 and National Cancer Institute
WT 100
R205Q 1.8(cid:6)0.3 GrantY1-CO-1020throughtheGM/CACollaborativeAccessTeamat
R205E 2(cid:6)2 theAPS,whichissupportedbytheUnitedStatesDepartmentofEnergy.
R205A 6(cid:6)3
N267A 34(cid:6)3
N211A 55(cid:6)2 REFERENCES
H266R 1.4(cid:6)0.2
aRawHPLCchromatogrampeakareafortheholo-ACPproductwithbackground 1. Newman,D.J.,andCragg,G.M.(2007)J.Nat.Prod.70,461–477
subtractedandnormalizedwithaninternalstandardtocorrectfordifferences 2. Fortman,J.L.,Chhabra,S.,Mukhopadhyay,A.,Chou,H.,Lee,T.S.,Steen,
insampleinjection.Theareaforeachmutantwasnormalizedtothewildtype E.,andKeasling,J.D.(2008)TrendsBiotechnol.26,375–381
withineachreplicate.Mean(cid:6)S.D.fromduplicateexperimentsareshown. 3. Verdier-Pinard,P.,Lai,J.Y.,Yoo,H.D.,Yu,J.,Marquez,B.,Nagle,D.G.,
Nambu,M.,White,J.D.,Falck,J.R.,Gerwick,W.H.,Day,B.W.,and
by Arg205 may further reduce energy barriers for initiating Hamel,E.(1998)Mol.Pharmacol.53,62–76
4. Chang,Z.,Sitachitta,N.,Rossi,J.V.,Roberts,M.A.,Flatt,P.M.,Jia,J.,
thisremarkabledecarboxylativeeliminationprocess.
Sherman,D.H.,andGerwick,W.H.(2004)J.Nat.Prod.67,1356–1367
Potential for Hydrocarbon Production—CurM TE is the
5. Gu,L.,Geders,T.W.,Wang,B.,Gerwick,W.H.,Håkansson,K.,Smith,
firstmemberofabranchoftheTEfamilyoptimizedtowork
J.L.,andSherman,D.H.(2007)Science318,970–974
in concert with a sulfotransferase to eliminate a carboxyl 6. Geders,T.W.,Gu,L.,Mowers,J.C.,Liu,H.,Gerwick,W.H.,Håkansson,
functional group and create a terminal double bond. The K., Sherman, D. H., and Smith, J. L. (2007) J. Biol. Chem. 282,
ST-TEsystemisthefirstexampleofabiologicalsulfonation 35954–35963
7. Akey,D.L.,Razelun,J.R.,Tehranisa,J.,Sherman,D.H.,Gerwick,W.H.,
employedforchemicalactivation(9).TheACP-ST-TEsys-
andSmith,J.L.(2010)Structure18,94–105
tem exists as an uncharacterized open reading frame in
8. Gu,L.,Wang,B.,Kulkarni,A.,Geders,T.W.,Grindberg,R.V.,Gerwick,
genomesequencesoffiveotherbacteria,withahighlevelof L.,Håkansson,K.,Wipf,P.,Smith,J.L.,Gerwick,W.H.,andSherman,
conservation of residues within and surrounding the TE D.H.(2009)Nature459,731–735
active site and in key contacts between domains and sub- 9. Gu, L., Wang, B., Kulkarni, A., Gehret, J. J., Lloyd, K. R., Gerwick, L.,
units. In CurM and in some ST-TE homologs, the sulfon- Gerwick,W.H.,Wipf,P.,Håkansson,K.,Smith,J.L.,andSherman,D.H.
(2009)J.Am.Chem.Soc.131,16033–16035
ation-decarboxylation system seems designed to eliminate
10. Chapman,E.,Best,M.D.,Hanson,S.R.,andWong,C.H.(2004)Angew.
the terminal carboxylate that would result from canonical
Chem.Int.Ed.Engl.43,3526–3548
TE hydrolysis of an acyl-ACP. Elimination of the terminal 11. Roongsawang,N.,Washio,K.,andMorikawa,M.(2007)Chembiochem8,
carboxyl group is a challenge for efforts to develop liquid 501–512
biofuels.ThustheST-TEsystemisofpotentialbenefitinthe 12. Giraldes,J.W.,Akey,D.L.,Kittendorf,J.D.,Sherman,D.H.,Smith,J.L.,
andFecik,R.A.(2006)Nat.Chem.Biol.2,531–536
development of a direct biosynthetic route to medium and
13. Akey,D.L.,Kittendorf,J.D.,Giraldes,J.W.,Fecik,R.A.,Sherman,D.H.,
long chain hydrocarbons from fatty acids. This could be
andSmith,J.L.(2006)Nat.Chem.Biol.2,537–542
achievedthroughengineeringa(cid:1)-hydroxyfattyacidbiosyn-
14. Tsai,S.C.,Miercke,L.J.,Krucinski,J.,Gokhale,R.,Chen,J.C.,Foster,
theticpathwayforoff-loadingusingtheST-TEmechanism. P.G.,Cane,D.E.,Khosla,C.,andStroud,R.M.(2001)Proc.Natl.Acad.Sci.
The ST-TE route to hydrocarbons is biochemically unique U.S.A.98,14808–14813
comparedwithotherrecentapproaches,includingacyano- 15. Scaglione,J.B.,Akey,D.L.,Sullivan,R.,Kittendorf,J.D.,Rath,C.M.,Kim,
E.S.,Smith,J.L.,andSherman,D.H.(2010)Angew.Chem.Int.Ed.Engl.
bacterial system using acyl-ACP reductase and aldehyde
49,5726–5730
decarbonylase with fatty-acyl-ACP substrates (38) and an
16. Bruner,S.D.,Weber,T.,Kohli,R.M.,Schwarzer,D.,Marahiel,M.A.,
E.coli system with fatty acid biosynthesis engineered for Walsh,C.T.,andStubbs,M.T.(2002)Structure10,301–310
hydrocarbon production (39). Other organisms found to 17. Samel,S.A.,Wagner,B.,Marahiel,M.A.,andEssen,L.O.(2006)J.Mol.
produce hydrocarbons with terminal alkenes, such as Bot- Biol.359,876–889
ryococcusbraunii(40),probablyemployasimilarST-TEoff- 18. Koglin,A.,Lo¨hr,F.,Bernhard,F.,Rogov,V.V.,Frueh,D.P.,Strieter,E.R.,
Mofid,M.R.,Gu¨ntert,P.,Wagner,G.,Walsh,C.T.,Marahiel,M.A.,and
loadingstrategy.Indeed,B.brauniiisknowntouseverylong
Do¨tsch,V.(2008)Nature454,907–911
chainfattyacidsactivatedatthe (cid:1)positiontoproduceter-
19. Frueh,D.P.,Arthanari,H.,Koglin,A.,Vosburg,D.A.,Bennett,A.E.,
minal olefin dienes and trienes through a decarboxylative Walsh,C.T.,andWagner,G.(2008)Nature454,903–906
process(41). 20. Claxton,H.B.,Akey,D.L.,Silver,M.K.,Admiraal,S.J.,andSmith,J.L.
Conclusion—The curacin TE illustrates the remarkable (2009)J.Biol.Chem.284,5021–5029
adaptability of the thioesterase structural framework, here 21. DelProposto,J.,Majmudar,C.Y.,Smith,J.L.,andBrown,W.C.(2009)
ProteinExpr.Purif.63,40–49
adaptedforonlythoseacyl-ACPsubstratesbearingasulfate
22. Fischetti,R.F.,Xu,S.,Yoder,D.W.,Becker,M.,Nagarajan,V.,Sanishvili,
substituentatthe(cid:1)position.TheCurMTEisakeycompo-
R.,Hilgart,M.C.,Stepanov,S.,Makarov,O.,andSmith,J.L.(2009)J.
nent of an unprecedented system for thioester hydrolysis SynchrotronRadiat.16,217–225
anddecarboxylation,workinginconcertwithafusedsulfo- 23. Otwinowski,Z.,andMinor,W.(1997)MethodsEnzymol.276,307–326
transferasetogenerateahighlyfavorableleavinggroup.The 24. Zwart,P.H.,Afonine,P.V.,Grosse-Kunstleve,R.W.,Hung,L.W.,Ioerger,
T.R.,McCoy,A.J.,McKee,E.,Moriarty,N.W.,Read,R.J.,Sacchettini,
CurM TE crystal structure reveals a fully developed hydro-
lyticactivesite,anarginineresiduefor(R)-(cid:1)-sulfaterecog- J.C.,Sauter,N.K.,Storoni,L.C.,Terwilliger,T.C.,andAdams,P.D.
(2008)MethodsMol.Biol.426,419–435
nition,andanactivesitecleftthatmayaccommodateawide
25. Adams,P.D.,Grosse-Kunstleve,R.W.,Hung,L.W.,Ioerger,T.R.,Mc-
varietyofacylsubstrates. Coy,A.J.,Moriarty,N.W.,Read,R.J.,Sacchettini,J.C.,Sauter,N.K.,and
APRIL22,2011•VOLUME286•NUMBER16 JOURNALOFBIOLOGICALCHEMISTRY 14453
StructureofCurMTE
Terwilliger, T. C. (2002) Acta Crystallogr. D Biol. Crystallogr 58, Scientific,PaloAlto,CA
1948–1954 35. Schu¨ttelkopf,A.W.,andvanAalten,D.M.(2004)ActaCrystallogr.DBiol.
26. Terwilliger, T. C. (2000) Acta Crystallogr. D Biol. Crystallogr. 56, Crystallogr.60,1355–1363
965–972 36. Sa´nchez,C.,Du,L.,Edwards,D.J.,Toney,M.D.,andShen,B.(2001)
27. Terwilliger,T.C.(2003)ActaCrystallogr.DBiol.Crystallogr.59,38–44 Chem.Biol.8,725–738
28. Emsley,P.,andCowtan,K.(2004)ActaCrystallogr.DBiol.Crystallogr.60, 37. Gerwick,W.H.,Proteau,P.J.,Nagle,D.G.,Hamel,E.,Blokhin,A.,and
2126–2132 Slate,D.L.(1994)J.Org.Chem.59,1243–1245
29. Murshudov,G.N.,Vagin,A.A.,andDodson,E.J.(1997)ActaCrystallogr. 38. Schirmer,A.,Rude,M.A.,Li,X.,Popova,E.,anddelCardayre,S.B.(2010)
DBiol.Crystallogr.53,240–255 Science329,559–562
30. CollaborativeComputationalProjectNumber4(1994)ActaCrystallogr. 39. Steen,E.J.,Kang,Y.,Bokinsky,G.,Hu,Z.,Schirmer,A.,McClure,A.,Del
DBiol.Crystallogr.50,760–763 Cardayre,S.B.,andKeasling,J.D.(2010)Nature463,559–562
31. Painter,J.,andMerritt,E.A.(2006)ActaCrystallogr.DBiol.Crystallogr. 40. Metzger, P., and Largeau, C. (2005) Appl. Microbiol. Biotechnol. 66,
62,439–450 486–496
32. Altschul,S.F.,Gish,W.,Miller,W.,Myers,E.W.,andLipman,D.J.(1990) 41. ChanYoung,T.,Largeau,C.,andCasadevall,E.(1986)NewJ.Chem.10,
J.Mol.Biol.215,403–410 701–707
33. Edgar,R.C.(2004)BMCBioinformatics5,113 42. Gouet,P.,Courcelle,E.,Stuart,D.I.,andMe´toz,F.(1999)Bioinformatics
34. DeLano,W.L.(2002)ThePyMOLMolecularGraphicsSystem,DeLano 15,305–308
14454 JOURNALOFBIOLOGICALCHEMISTRY VOLUME286•NUMBER16•APRIL22,2011