X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis

KristinaHaslinger1*,MadeleinePeschke1*,ClaraBrieke1,EgleMaximowitsch1&MaxJ.Cryle1

X-domainofpeptidesynthetasesrecruits

oxygenasescrucialforglycopeptidebiosynthesis

Non-ribosomalpeptidesynthetase(NRPS)mega-enzymecomplexesaremodularassemblylinesthatareinvolvedinthebiosynthesisofnumerouspeptidemetabolitesindependentlyoftheribosome1.ThemultipleinteractionsbetweencatalyticdomainswithintheNRPSmachineryarefurthercomplementedbyadditionalinteractionswithexternalenzymes,particularlyfocusedonthefinalpeptidematura-tionprocess.AnimportantclassofNRPSmetabolitesthatrequireextensiveexternalmodificationoftheNRPS-boundpeptidearetheglycopeptideantibiotics(GPAs),whichincludevancomycinandteicoplanin2,3.TheseclinicallyrelevantpeptideantibioticsundergocytochromeP450-catalysedoxidativecrosslinkingofaromaticsidechainstoachievetheirfinal,activeconformation4–12.However,themechanismunderlyingtherecruitmentofthecytochromeP450oxy-genasestotheNRPS-boundpeptidewaspreviouslyunknown.Hereweshow,throughinvitrostudies,thattheX-domain13,14,aconserveddomainofunknownfunctionpresentinthefinalmoduleofallGPANRPSmachineries,isresponsiblefortherecruitmentofoxygenasestotheNRPS-boundpeptidetoperformtheessentialside-chaincross-linking.X-raycrystallographyshowsthattheX-domainisstructurallyrelatedtocondensationdomains,butthatitsaminoacidsubstitu-tionsrenderitcatalyticallyinactive.WefoundthattheX-domainrecruitscytochromeP450oxygenasestotheNRPSanddeterminedtheinterfacebysolvingthestructureofaP450–X-domaincomplex.Additionally,wedemonstratedthatthemodificationofpeptidepre-cursorsbyoxygenasesinvitro—inparticulartheinstallationofthesecondcrosslinkinGPAbiosynthesis—occursonlyinthepresenceoftheX-domain.Ourresultsindicatethatthepresentationofpep-tidylcarrierprotein(PCP)-boundsubstratesforoxidationinGPAbiosynthesisrequiresthepresenceoftheNRPSX-domaintoensureconversionoftheprecursorpeptideintoamatureaglycone,andthatthecarrierproteindomainaloneisnotalwayssufficienttogenerateacompetentsubstrateforexternalcytochromeP450oxygenases.Fewcompoundclasseshavehadsuchapositiveeffectonhumanhealthasantibiotics15.Concernovertheriseofantibioticresistancemakesitessentialtodevelopnewchemotherapeutics,throughthediscoveryofnovelantibioticsandtherenewedexploitationofexistingones15,16.TheGPAsareagroupofcompoundsincurrentusethatarehighlyef-fectiveagainstGram-positivebacterialinfectionsresistanttootherclassesofantibiotics2:examplesarethenaturalproductsvancomycinandteico-planinandsemi-syntheticderivativesunderdevelopment16.TheGPAsarecomplexmolecules,comprisingheptapeptideaglyconeswithahighpercentageofnon-proteinogenicaminoacids,whicharecrosslinkedthroughmultiplearylandphenoliclinks.Theaglyconesarefurtherdecoratedthroughmanydifferentprocesses,includingglycosylationandsulfonation2.GPAsfunctionthroughtheformationofanon-covalentcomplexofmicromolaraffinitywithapeptidoglycanprecursor(lipidII).Thiscomplexisformedviahydrogenbondsfrombackboneamidegroupsoftheglycopeptideaglycone,andthecrosslinkedaromaticsidechainsarecrucialingivingtheaglyconethethree-dimensionalshaperequiredforbinding2.GPAsarebiosynthesizedwithouttheribosomebyalinearNRPS(Fig.1andExtendedDataFig.1)1,2.Inglycopeptide

1

biosynthesis,thematurationoftheheptapeptiderequirestheNRPSforinteractionwithexternaloxygenaseenzymes—cytochromesP450(OxyA,B,C,E)—thatcatalysethe(aryl/phenolic)crosslinkingofaromaticaminoacidsidechainsandprovidethefinal,rigidaglyconestructure3.ExtensiveinvivogenedisruptionexperimentshavesuggestedrolesforeachoftheOxyproteinsandalsoaspecificsequenceofoxidation,withOxyBintroducingthefirstcrosslink5–7,11,12.Invitroandinvivoexperi-mentshaveindicatedthatthepeptidesubstratesfortheseP450oxy-genasesremainboundtotheNRPSduringoxidation4,9,10.Ourrecentstudieshaveconcentratedonunderstandingthelater,crucial,stagesofglycopeptidebiosynthesis,inparticularthecyclizationofthelinearpep-tidethroughtheoxidativecrosslinkingofaromaticsidechainsbytheOxyproteins8,17.Wearenowfocusingontheroleofadomainofun-knownfunctionthatispresentinthefinalmoduleofallglycopeptide-typeNRPSmachineries14.Fromphylogeneticanalyses,thisdomainappearstoberelatedtothecondensation/epimerization(C/E)-domainsfoundinNRPSbiosynthesisandmostcloselytoanLCL-typecondensa-tiondomain.However,itseemstobecatalyticallyinactiveduetothemutationofresiduesofthehighlyconservedHHxxxDGmotifessentialforpeptidebondformationandepimerization13,andisreferredtoasthe‘X-domain’.

AsthefinalNRPSmodulesofGPAsallcontainanX-domain,wesoughttoclarifythemolecularfunctionofthisdomain.WedeterminedthecrystalstructureoftheX-domainfromthefinalNRPSmoduleof

?resolution(Fig.2aandExtendedteicoplaninbiosynthesis(Xtei)to2.9A

DataTable1)18–22.TheX-domainadoptsaC/E-typefold,whichischar-acterizedbyaV-shapedarrangementoftwosubdomainsthatbothbe-longtothechloramphenicol-acetyltransferase(CAT)fold.Crossoverelementsbetweenthesubdomainscomprisethe‘floor’oftheV-shapedcleft(I;Fig.2a,magenta)andthecrossover‘latch’(II;Fig.2a,orange).TheX-domainalsocontainsseveralinsertionregionscomparedwithotherstructurallycharacterizedC/E-domains(Fig.2a,red/yellow).Im-portantlyforthefunctionoftheX-domain,theacceptorentrysideoftheX-domainisblockedbytheorientationofseveralloopregions(Fig.2a,cyan)andnotunnelthroughtheX-domaincanbeobserved.Whilethestructuralactive-sitemotifresiduesH140andD145aremaintained,theresiduesmutatedfromthecanonicalC-domainactivesite(HRxxxDD;boldtextindicatesmutatedresidues)impactonthepotentialcatalyticfunctionofthisdomain:thesidechainsofbothresiduesR141andD146projectdirectlyintothepositionexpectedtobeoccupiedbythedonor49-phosphopantetheinegroupduringpeptidebondformationinanactiveC-domain(Fig.2candExtendedDataFig.4a).

SincethestructureoftheX-domainsuggestedthatitsfunctioninglycopeptidebiosynthesiswasunlikelytoberelatedtoamide-bondfor-mation,weconsidereditspossibleroleintheinteractionwithpeptide-modifyingproteinsintrans:theOxyproteinsresponsibleforside-chaincyclizationoftheNRPS-boundpeptide4–8.Invivoexperimentshadpre-viouslyindicatedtheprobableassociationoftheNRPSwiththeoxy-genaseenzymes4,andtheinitialoxidationinvancomycinbiosynthesisbyOxyBvanhasbeenshowntooccurinvitrothroughtheoxidationofsubstratesboundtocarrierproteins9,10.However,wehaverecently

MaxPlanckInstituteforMedicalResearch,Jahnstrasse29,69120Heidelberg,Germany.*Theseauthorscontributedequallytothiswork.

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Tcp9

Module 1

D-Hpg

Tcp10Module 3

D-Dpg

Tcp11

Module 4

Tcp12

Module 6

Module 2

Module 5

D-Hpg

Module 7

L-Dpg

CD

E

B

A

F

G

Teicoplanin-type aglycone

Figure1|Structureoftheteicoplaninaglycone

andaschematicpathwayofteicoplanin

biosynthesisbynon-ribosomalpeptidesynthesis.DomainlabelsforNRPSproteins(Tcp9–12):A,adenylation(selectedaminoacidsindicatedabovethemodule:Hpg,4-hydroxyphenylglycine;Dpg,3,5-dihydroxyphenylglycine);C,condensation;E,epimerization;T,thiolation/peptidylcarrierprotein(PCP);TE,thioesterase;X,domainofunknownfunction.EssentialP450-catalysedaglyconerigidificationtakesplacethroughcrosslinkingofaromaticsidechains(OxyA–C,OxyE).EachcrosslinkingreactionisperformedbyaspecificOxyprotein,withtheproductsofeachOxyproteinindicatedschematically;standardringnomenclatureisindicatedontheteicoplaninaglyconeinredlettering.

Teicoplanin peptide

demonstratedthatOxyBvandisplaysapromiscuityinsubstrateselec-tion17notobservedfortheOxyBproteinfromteicoplanin23,24biosyn-thesis:forOxyBtei,thecarrierproteindomainisinsufficienttomediateefficientcrosslinkingoftheboundpeptide8.WethereforeinvestigatedseveralconstructsfromthelastteicoplaninNRPSmoduletoassesspotentialinteractionsoflargerNRPSconstructswithOxyBtei:toachievethisweusedbothgelfiltrationco-elutionexperimentsandnativepoly-acrylamidegelelectrophoresis(PAGE)mobilityshiftassay.Beginningwiththelargestconstruct,whichcontainsthreeNRPSdomains,PCP7–X–thioesterase(seeFig.1),weobservedco-elutionofOxyBteiwiththe

a

C-terminalsubdomain

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X-domain

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N-terminalsubdomain

OxyBtei

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Crossover II

D146

E391

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H140

I142

α1

R171

2.9

E291αG

E188

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R181

E170

3.1

D162

Figure2|StructuresoftheX-domainandtheX-domain–OxyBteicomplex.a,IsolatedX-domain:thesecondarystructuredisplaysthetopologytypicalofC/E-domains.b,TheX-domain–OxyBteicomplex:selectedOxyBhelices

arelabelled.c,X-domainactivesiteshowingtheeffectsoftheresiduesmutatedfromthecanonicalC-domainactive-siteR141andD146.d–f,Selectedaminoacidsformingimportantinteractionsintheinterfaceareshown(OxyBtei

F-helixD161(d)andD162(e),OxyBteiG-helix(f)).Colourscheme:X-domain

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amino-terminalsubdomain,green;carboxy-terminalsubdomain,blue;

crossoverelementI,magenta,andII,orange;X-domaininsertionsa5/a6,red,andb12/b13,yellow;loopsoccludingtheacceptorsite,cyan;X-domain

proteinsurface,grey;OxyBtei,yellow;watermoleculesmediatinginteractionsbetweentheX-domainandOxyBteiareshownasbluespheres.Residuesdisplayedassticksarelabelled,asareselectedsecondarystructureelements;

?).hydrogenbondsareindicatedbydashedlineswithdistances(A

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NRPStri-domainbygelfiltration(ExtendedDataFig.2a).Thisasso-ciationwasconfirmedbytheappearanceofabandinthenative-PAGEassayoflowerelectrophoreticmobilitythatcontainedbothOxyBteiandtheNRPStri-domain(ExtendedDataFig.3a),asconfirmedbypeptidemassfingerprinting.RepetitionoftheseexperimentswithsmallerNRPSconstructsshowedthattheminimalconstructrequiredforOxyBteiin-teractionfromthefinalNRPSmoduleofteicoplaninwastheX-domainitself(Fig.3andExtendedDataFigs2a,3a).Native-PAGEanalysisofOxyBteiinthepresenceofincreasingconcentrationsofX-domainshowedthatatequimolarconcentrationsOxyBisfullyboundandthattheappar-entdissociationconstantisinthelowmicromolarrange(ExtendedDataFig.3c).ThisiswithintherangeobservedforotherP450–NRPSinter-actions—albeithereintheabsenceoftheactualP450substrate9,25–27.WiththeinteractionofOxyBteiandtheX-domainofteicoplanindem-onstrated,werepeatedtheseexperimentsforvancomycin-typesystemsandfoundcomparableresults(Fig.3andExtendedDataFig.3a).ThecrossoverexperimentwithalternateP450–NRPSpairsalsoindicatedthatOxyBvancaninteractwiththeteicoplanin-producingNRPSdomain(ExtendedDataFig.3a)—whichagreeswiththeinvivoactivityobservedforoxyBcross-complementationstudies11,12.Theseexperimentsindi-catethattheinteractionoftheX-domainwithOxyBisaconservedfeatureinglycopeptidebiosynthesis.

Wethendeterminedacrystalstructureoftheproteincomplexbe-?resolution(Fig.2b,ExtendedtweentheX-domainandOxyBteito2.5A

DataFigs4b,5aandExtendedDataTable1)Theorientationofthecom-plexplacestheuppersurfaceoftheP450(withthehaem-centredactivesite)facingtowardsthedonorsiteoftheX-domaincleft,http://wendang.chazidian.comparisonofthecomplexwiththeisolatedP450(ref.8)andX-domainstructuresindicatesthatthereislittlerearrangementofeither

?,proteinoninteraction(rootmeansquareddeviation1.1and0.9A

respectively);theinteractionismoreofarigidbodydockingandismediatedthroughapproximately20residuesoneachprotein.Thein-teractionsbetweenthetwoproteinsaremainlyviahydrogenbondsandsaltbridges;hydrophobicinteractions,asareoftenseeninintramole-cularNRPSdomaininteractions,arelimitedtoisolatedresiduesintheX-domain–OxyBinterface(Fig.2d–f).ThestartoftheF-helix(PRDD)isconservedintheOxyproteins(ExtendedDataFig.5b)andiscritical

a

Molecular mass (×105)

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tei

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cepNative PAGE1: GB1–X2: OxyA3: OxyB4: OxyC

5: OxyA+GB1–X6: OxyB+GB1–X7: OxyC+GB1–X8: OxyE+GB1–X

Elution profiles:

P450, 415 nm

P450–NRPS mix (1:3), 415 nmP450–NRPS mix (1:3), 280 nmNRPS, 280 nm

Molecular massdistributions:P450

P450–NRPS mix (1:3)NRPS

Figure3|InteractionoftheX-domainwithOxyproteins.a,Elutionprofilesofanalyticalsize-exclusionchromatography(solidlines415nm,dashedlines280nmdetection)andmolecularmassesoftheindividualspecies

observedbymulti-anglelightscatteringofindividuallysampledandco-elutedOxyBteiandXteifusedtoGB1(proteinG,B1domain;1:3mixture).AU,

arbitraryunits.b,NativePAGEofteicoplaninXtei-domainandOxyteiproteins(1–4)and3:1mixturesthereof(5–8;asterisksindicatethenewbandsoflowelectrophoreticmobility,triangleindicatesthebandofOxyEteithatdoesnotco-migratewithGB1–Xtei).c,NativePAGEofvancomycin-producingXcep-domainandOxyvanproteins(sampleorderasinbwiththesquareindicatingthebandofOxyCvanthatdoesnotco-migratewithGB1–Xcep).

G2015

forinteractionwiththeX-domain.TheB–B2loopregion(66–77),typ-icallydisorderedintheabsenceofaboundsubstrateinP450oxyge-nases,remainsunresolved,indicatingthatthebindingoftheX-domaindoesnotstimulateclosureoftheP450activesitebutratheractstodockontotheP450topresenttheneighbouringPCP7-peptidesubstrate.WemodelledthepositionofthePCP7-domainbasedonthecomplexstruc-tureofthetwoknownP450–carrierproteincomplexes;26,28thisindicatesthatthePCPorientationfromtheskyllamycinP450–PCPcomplexiscompatiblewiththeX–P450complexstructure(ExtendedDataFig.6).MutationofspecificresiduesineachofthethreeX-domaininteractionregionsrevealedthattheloopregionafterhelixa4thatcontainsresi-duesR167andR171(whichcontacttheconservedPRDDOxymotif)isessentialfortheinteraction(ExtendedDataFig.2b).

SincenoinvitroactivityforanyOxyproteinbeyondtheinitialcy-clizationstepcatalysedbyOxyBvanhasbeendescribedsofar9,10,17,weexaminedwhethertheX-domaininteractedwiththeremainingoxy-genases(OxyAtei,OxyCteiandOxyEtei)usingtheinteractionassaysde-scribedearlierforOxyB(Fig.3andExtendedDataFigs2c,3b).TheseexperimentsshowedthatbothOxyAteiandOxyCteiinteractwithamin-imalX-domainconstructfromteicoplaninbiosynthesis;thesameholdstrueforvancomycin-typesystems.TheaffinitiesoftheteicoplaninOxyenzymesfortheX-domain,asdeterminedbygelfiltration,appearedtobelowerthanthatofOxyB,withOxyAbindingmoretightlythanOxyC.DifferentialaffinityfortheX-domainimpliesthatadditionalselectivityforOxybindingisbasedonthestateofthecrosslinkingofthepeptide.Thus,theroleoftheX-domainintheglycopeptideNRPSistoactasageneralrecruitmentplatformforoxygenaseenzymesingly-copeptidetailoring:thisdiffersfromothercarrierprotein–P450sys-temssuchasthosefoundinaminoacyl-PCPoxidation25–27orbiotinbiosynthesis28—theserelyonthecarrierprotein–substratepairforsub-straterecognition.

Curiously,OxyE—theoxygenaseresponsiblefortheF-O-Gringin-stallationinteicoplaninbiosynthesis—didnotdisplayaninteractionwiththeteicoplaninX-domain(Fig.3andExtendedDataFig.2c).How-ever,asOxyEteicatalysesaphenoliccouplingstepwithminimalpeptidepenetrationintotheP450activesite(residues1and3),itisplausiblethatthisP450requiresthepresenceofthePCP7-boundpeptidetomain-tainatightinteractiontotheNRPS.AstheF-O-Gringisnotrequiredforantibioticactivitybutrathermoderatesselectivityagainstdifferentbacterialtargets2,16,alloxygenaseenzymescrucialforpeptidetailoringinGPAbiosynthesisarerecruitedtotheNRPS-boundheptapeptidebytheX-domain.

Thewell-studiedOxyBenzymefromthevancomycinsystem(OxyBvan)hasbeenshowntocatalysetheinvitrooxidationofheptapeptideswithdecreasedefficiencycomparedwithitsefficientturnoverofhexapep-tidespresentedbythePCP-domainfrommodulesevenoftheNRPS(PCP7),leavingthequestionofitsnaturalsubstrateunanswered9.WesoughttoreconstitutetheenzymaticactivityofOxyBvaninamorenat-uralcontext,comprisingasimplifiedheptapeptide(SupplementaryFig.1)presentedbyaPCP7-domainintheabsenceandpresenceoftheX-domain.WiththisapproachweobservedamoderateincreaseinturnoveryieldbyusingthePCP7–Xconstruct(Fig.4andExtendedDataFig.7c).TheeffectsoftheX-domainweremorepronouncedforOxyBteiduetothecomparativelylowactivityofOxyBteiintheinvitroturnoverofPCP7-boundmodelhexapeptides:8indeed,OxyBteiishighlyactiveincyclizingheptapeptidesboundtothePCP7–Xconstructs,whereasitisonlyabletocatalyse,25%C-O-DringformationinPCP7-boundteicoplanin-likeheptapeptidesunderthesameconditions(Fig.4andExtendedDataFig.7a).ActivityassaysperformedwiththePCP7–X-domaininterfacemutantsconfirmtheresultsfromtheinter-actionstudies(ExtendedDataFig.2b),withnoactivityobservedfortheX1variant(Fig.4andExtendedDataFig.8).MutantvariantsthatretaintheabilitytobindOxyBteiretainP450activitycomparablewiththewild-typePCP7–Xprotein,underpinningtheimportanceoftheX-domainfortheOxyBteiturnoverreaction.WeexaminedwhetherthedifferencesobservedinthepresenceoftheX-domaininthesesystemsweredueto

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a

Tei7(Hpg3,7)a

MonocyclicOxyBtei

Bicyclic

OxyBtei + OxyAtei

b

Yield (%)

Yield (%)

Relative intensity

PCtei

P–XPCteP–iXPC1teiP–X2PCteiP–PCXP–3teiX

PCpPPCcepP–X

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PCPPCteiP–X

1–3tei

PCP

cep

tei

ce

X1: R167A, R171A X2: E290A, D291A X3: E377A, R382AX1–3: R167A, R171A, E290A, D291A, E377A, R382A

teitei

b

Figure5|CoupledinvitroactivityofOxyAteiandOxyBtei.a,TurnoverofheptapeptidesubstratesboundtoPCP7andPCP7–X(GB1fusionproteins)expressedasapercentageofthetotalpeptidedetectedandshownasstackedbars(averageoftriplicateexperiments,errorbarsindicates.d.).b,ESI-MS/MSanalysisofHPLC-purifiedbicyclicteicoplanin-likeOxyB/OxyAturnoverproduct,demonstratingtheanticipatedfragmentationforaC-O-D/D-O-Ebicyclicpeptidestructure(SupplementaryFig.6).

Relative intensity

Figure4|InvitroactivityofOxyBteianditshomologues.a,TurnoverofheptapeptidesubstratesboundtoPCP7andPCP7–X(GB1fusionproteins)fromtheteicoplaninandchloroerymomycinNRPSwithOxyBenzymesfromteicoplanin-likesystems(OxyBtei,StaH)andvancomycin-likesystems(OxyBvan,CepF)expressedasapercentageofthetotalpeptidedetected.HeptapeptidesubstratesloadedontothePCP7/PCP7–Xconstructsare

indicatedabovetherespectiveOxyBenzymes;X-domainvariantsX1–X3andX1–3oftheteicoplaninNRPS(describedbelowthegraph)showstronglyreducedactivityforconstructsX1andX1–3thatpossessR167AandR171AmutationscontactingtheconservedPRDDOxymotifinthewild-typeprotein(averageoftriplicateexperiments;errorbarsindicatestandarddeviation(s.d.)).b,Electrosprayionizationtandemmassspectrometry(ESI-MS/MS)analysisofhigh-performanceliquidchromatography(HPLC)-purifiedmonocyclicteicoplanin-likeOxyBturnoverproduct,demonstratingtheanticipated

fragmentationforaC-O-Dmonocyclicpeptidestructure(SupplementaryFig.5).

OnemajorhurdleinGPAbiosynthesishasbeenthelackofinvitroactivityofanyOxyproteinbeyondtheC-O-DringformationcatalysedbyOxyB.WithresultsindicatingthattheX-domainwasabletorecruitallessentialOxyenzymes(OxyA–C),weinvestigatedwhetherthepres-enceoftheX-domainwouldsupporttheactivityofadditionalOxyen-zymes.Incoupledassays,thecombinationofOxyBteiandOxyAteiwasnowabletoinstallbothC-O-DandD-O-EcrosslinksinaPCP7–X-boundheptapeptidesubstrate(Fig.5andExtendedDataFig.9a).Inagree-mentwiththereportedinvivooxidationorder11,12,thecombinationofOxyBteiandOxyCteididnotleadtothegenerationofabicyclicpeptideproduct(ExtendedDataFig.9b).However,thelackofactivityinthisassaycouldhavebeenduetothelimitedcatalyticcompetenceoftheOxyCteienzyme(P420signalfortheCO-complex);thisalsopreventedtherecreationoftheOxyA–Ccascade(ExtendedDataFig.9c).Inspiteofthis,ourdemonstrationofinvitroOxyAactivityclearlyindicatestheimportanceoftheX-domainasarecruitmentplatformintheOxy-catalysedinstallationofcrosslinksinGPAbiosynthesisandopensthedoortothereconstitutionofGPAaglyconebiosynthesisinvitro.Morebroadly,ourresultsshowthattherecruitmentofenzymestoNRPSsystemscanbemediatedbydomainsbeyondcarrierproteinsandthatsuchmechanismsmayhaveawiderroleinbiosynthesisbymegaen-zymesynthetasemachineriesthanhasbeenacknowledged.

OnlineContentMethods,alongwithanyadditionalExtendedDatadisplayitemsandSourceData,areavailableintheonlineversionofthepaper;referencesuniquetothesesectionsappearonlyintheonlinepaper.Received10July;accepted5December2014.Publishedonline9February2015.1.2.3.4.5.6.7.

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ToclarifyfurthertheroleoftheX-domaininOxyrecruitment,wetestedtheactivityofOxyBteionahexapeptideboundtothePCP-domainfrommodulesixoftheNRPS(PCP6):theseturnoversshowednoactiv-ityofOxyBtei(ExtendedDataFig.9d).Thisagreeswithpreviousinvivoexperimentsthatreportverylittleisolationofcrosslinkedhexapeptidescomparedwiththefinalaglycone11.Takentogether,theseresultsstronglysuggestthatcrosslinkedhexapeptideproductsonlyappearfromminorshuntpathwaysanddonotrepresentthenormalbiosynthesisrouteinvivo,whichinsteadreliesonX-domain-mediatedoxygenaserecruit-menttothemodule-seven-boundheptapeptide.

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AcknowledgementsTheauthorsthankA.Kochforassistancewithproteinexpression;S.Bellforredoxproteins;M.Gradlforassistancewithmassspectrometry;M.TarnawskiandA.Meinhartforassistancewithcrystalharvestinganddataprocessing;

¨ssmuthandI.SchlichtingandJ.Wrayfordiscussions;C.RoomeforITsupport;R.Su

A.Trumanforsharingunpublisheddata.DiffractiondatawerecollectedattheSwissLightSource,X10SAbeamline,PaulScherrerInstitute,Villigen,Switzerland.WethanktheHeidelbergteamfordatacollectionandthePXIIstafffortheirsupportinsettingupthebeamline.M.J.C.isgratefultoI.SchlichtingforconstantencouragementandtotheDeutscheForschungsgemeinschaft(Emmy2NoetherProgram,CR392/1-1)forfinancialsupport.

AuthorContributionsM.J.C.designedthestudy.K.H.,M.P.andE.M.performedthebiochemicalexperiments.C.B.performedthechemicalsynthesisandcompoundcharacterization.M.P.,K.H.andM.J.C.solvedthestructuresandperformedtheanalysis.M.J.C.wrotethemanuscripttogetherwithcontributionsfromK.H.,http://wendang.chazidian.com/reprints.Theauthorsdeclarenocompetingfinancialinterests.Readersarewelcometocommentontheonlineversionofthepaper.CorrespondenceandrequestsformaterialsshouldbeaddressedtoM.J.C.(Max.Cryle@mpimf-heidelberg.mpg.de).

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