2-Alkylquinolone alkaloid biosynthesis in the medicinal plant Evodia rutaecarpa involves

20 Pages·2017·5.82 MB·English

cros
ARTICLE
2-Alkylquinolone alkaloid biosynthesis in the medicinal plant
Evodia rutaecarpa involves collaboration of two novel type III
polyketide synthases
Receivedforpublication,January29,2017,andinrevisedform,April5,2017Published,PapersinPress,April14,2017,DOI10.1074/jbc.M117.778977
TakashiMatsui‡1,TakeshiKodama‡1,TakahiroMori§,TetsuhiroTadakoshi‡,HiroshiNoguchi¶,IkuroAbe§2,
andHiroyukiMorita‡3
Fromthe‡InstituteofNaturalMedicine,UniversityofToyama,2630-Sugitani,Toyama930-0194,the§GraduateSchoolof
PharmaceuticalSciences,UniversityofTokyo,7-3-1Hongo,Bunkyo-ku,Tokyo113-0033,andthe¶SchoolofPharmaceutical
Sciences,UniversityofShizuoka,52-1Yada,Suruga,Shizuoka422-8526,Japan
EditedbyJosephJez
2-Alkylquinolone(2AQ)alkaloidsarepharmaceuticallyand ADS and AQS, respectively. These results provide additional
biologicallyimportantnaturalproductsproducedbybothbac- insights into the catalytic versatility of the type III PKSs and
teriaandplants,withawiderangeofbiologicaleffects,includ- theirfunctionalandevolutionaryimplicationsfor2AQbiosyn-
ing antibacterial, cytotoxic, anticholinesterase, and quorum- thesisinplantsandbacteria.
sensing signaling activities. These diverse activities and 2AQ
occurrenceinvastlydifferentphylahaveraisedmuchinterestin
thebiosynthesispathwaysleadingtotheirproduction.Previous The2-alkylquinolones(2AQs)4compriseasubclassofalka-
studies in plants have suggested that type III polyketide syn- loidswithawiderangeofbiologicaleffects,suchasantibacte-
thases(PKSs)mightbeinvolvedin2AQbiosynthesis,butthis rial (1), cytotoxic (1), and anticholinesterase (2) activities, as
hypothesisisuntested.Tothisend,weclonedtwonoveltypeIII wellasquorum-sensingsignaling(3).Gram-negativebacteria,
PKSs, alkyldiketide-CoA synthase (ADS) and alkylquinolone Pseudomonasaeruginosa(3),andthemedicinalRutaceaefam-
synthase(AQS),fromthe2AQ-producingmedicinalplant,Evo- ilyplants,Evodiarutaecarpa(1),Zanthoxylumschinifolium(4),
dia rutaecarpa (Rutaceae). Functional analyses revealed that andEsenbeckialeiocarpa(2),produce2AQs.Inthebiosynthe-
collaborationofADSandAQSproduces2AQviacondensations sisof2-heptylquinolinoneproducedbyP.aeruginosa,aprevi-
ofN-methylanthraniloyl-CoA,afattyacyl-CoA,withmalonyl- ousstudysuggestedthata(cid:2)-ketoacylcarrierproteinsynthase
CoA.WeshowthatADSefficientlycatalyzesthedecarboxyla- III (FabH)-like enzyme, PqsD, catalyzes the condensation of
tivecondensationofmalonyl-CoAwithafattyacyl-CoAtopro- anthraniloyl-CoA and malonyl-CoA (1) to produce 2-amino-
duceanalkyldiketide-CoA,whereasAQSspecificallycatalyzes benzoyldiketide-CoA(Fig.1A).Subsequently,thethioesterase
thedecarboxylativecondensationofanalkyldiketideacidwith PqsE converts 2-aminobenzoyldiketide-CoA to the corre-
N-methylanthraniloyl-CoA to generate the 2AQ scaffold via sponding2-aminobenzoyldiketideacid,whichisthenaccepted
C–C/C–N bond formations. Remarkably, the ADS and AQS by the FabH-like enzymes, the PqsB and PqsC heterodimeric
crystal structures at 1.80 and 2.20 Å resolutions, respectively, enzymes, as the extender substrate with octanoyl-CoA (2) as
indicatedthattheuniqueactive-sitearchitecturewithTrp-332 thestartersubstratetogenerate2-heptyl-4-quinolone(5–7).In
and Cys-191 and the novel CoA-binding tunnel with Tyr-215 contrast,theheterologouslyexpressedtypeIIIpolyketidesyn-
principally control the substrate and product specificities of thase3(HsPKS3)fromtheLycopodiaceaefamilyplant,Huper-
ziaserrata,yields2AQbycatalyzingfourreactionsteps,con-
This work was supported in part by Grants-in-Aid for Scientific Research sistingofthecondensationreactionof1andfattyacyl-CoAto
fromtheMinistryofEducation,Culture,Sports,ScienceandTechnology,
producealkyldiketide-CoA(AD-CoA),thehydrolysisreaction
Japan (JSPS KAKENHI Grants JP15H01836, JP16H06443, JP25242067,
of AD-CoA to produce the alkyldiketide acid (AD acid), the
JP26282210, and JP26560435); by Grants-in-Aid for the Cooperative
Research Project from the Institute of Natural Medicine, University of coupling reaction of N-methylanthraniloyl-CoA (3) and AD
Toyama,in2013(toH.N.andH.M.)andin2015(toT.M.andH.M.);andby acidtoproducethelinearN-methylanthraniloyldiketideinter-
the Platform Project for Supporting Drug Discovery and Life Science
Research(PlatformforDrugDiscovery,Informatics,andStructuralLifeSci-
ence) from the Japan Agency for Medical Research and Development
(AMED)(toT.M.).Theauthorsdeclarethattheyhavenoconflictsofinterest 4Theabbreviationsusedare:2AQ,2-alkylquinolone;ADacid,alkyldiketide
withthecontentsofthisarticle. acid;AD-CoA,alkyldiketide-CoA;ADS,alkyldiketide-CoAsynthase;AQS,
ThisarticlecontainssupplementalTablesS1–S3. alkylquinolone synthase; 2ATL, 2-alkyltriketide lactone; CsOLS, C.sativa
Thenucleotidesequence(s)reportedinthispaperhasbeensubmittedtotheGen- olivetol synthase; ClDCS, C.longa diketide-CoA synthase; ClCURS1,
BankTM/EBIDataBankwithaccessionnumber(s)LC208543andLC208544. C.longacurcuminsynthase1;QNS,quinolonesynthase;CmQNS,C.micro-
Theatomiccoordinatesandstructurefactors(codes5WX3,5WX4,5WX5,5WX6,and carpaquinolonesynthase;EtOAc,ethylacetate;FabH,(cid:2)-ketoacylcarrier
5WX7)havebeendepositedintheProteinDataBank(http://wwpdb.org/). proteinsynthaseIIIs;HsPKS3,H.serratatypeIIIpolyketidesynthase3;CHS,
1Bothauthorscontributedequallytothiswork. chalconesynthase;MsCHS,M.sativachalconesynthase;OsCUS,O.sativa
2To whom correspondence may be addressed. Tel.: [phone]; Fax: curcuminoidsynthase;PKS,polyketidesynthase;RdORS,R.dauricum
81-3-5841-4744;E-mail:[email protected]. orcinolsynthase;r.m.s.d.,rootmeansquaredeviation;RpBAS,R.palma-
3To whom correspondence may be addressed. Tel.: [phone]; Fax: tumbenzalacetonesynthase;PDB,ProteinDataBank;ESI,electrospray
81-76-434-5059;E-mail:[email protected]. ionization.
This is an Open Access article under the CC BY license.
J.Biol.Chem.(2017)292(22)9117–9135 9117
©2017byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
CharacterizationoftwotypeIIIpolyketidesynthases
Figure1.Enzymaticformationof2AQsandcurcuminoids.A,2-heptyl-4-quinoloneformedbyPqsB,-C,-D,and-E;B,2-heptyl-N-methyl-4-quinolone(4)
formedbyHsPKS3;C,feruloyldiketide-CoAformedbyClDCS;D,curcuminformedbyClCURS1;E,decanoyldiketide-CoA(21)formedbyADS;F,2-nonyl-N-
methyl-4-quinolone(17)formedbyAQS.
mediate,andthesubsequentC–Nbondformationtogenerate loyldiketide acid, the hydrolyzed product of feruloyldiketide-
the2AQscaffoldintheinvitroenzymereaction(8).Forexam- CoA, is accepted by the other type III PKS, named curcumin
ple,HsPKS3reportedlygenerated2-heptyl-N-methyl-4-quin- synthase (ClCURS1), and curcumin is generated from the
olone(4)fromthecondensationof2,3,and1viatheforma- decarboxylative condensation of the diketide acid with feru-
tionsofoctanoyldiketide-CoA(5)andoctanoyldiketideacid(6) loyl-CoA(Fig.1D)(9).Therefore,itismostlikelythattwotype
(Fig.1B).However,becausetherearenoreportsabouttheiso- IIIPKSsareinvolvedinthebiosynthesisofthe2AQalkaloidsin
lationof2AQsfromH.serrata,the2AQsynthesisbyHsPKS3 E.rutaecarpa,inamannersimilartothecurcuminbiosynthesis
ispredictedtobeanon-physiologicalfunctionoftheenzyme inturmeric.
limited to the in vitro enzyme reactions. In contrast, in the Herein,wereporttwouniquetypeIIIPKSsfromthebudsof
biosynthesis of curcumin in the turmeric Curcuma longa, E.rutaecarpa, named alkyldiketide-CoA synthase (ADS) and
feruloyldiketide-CoA is produced by diketide-CoA synthase alkylquinolonesynthase(AQS).ThefunctionalanalysesofADS
(ClDCS),atypeIIIpolyketidesynthase(PKS),fromtheconden- andAQSdemonstratedthatcollaborationofADSandAQSis
sationofferuloyl-CoAwith1(Fig.1C).Subsequently,theferu- required for the generation of the 2AQ scaffold in E.rutae-
9118 J.Biol.Chem.(2017)292(22)9117–9135
CharacterizationoftwotypeIIIpolyketidesynthases
carpa. Furthermore, our functional analyses indicated that Tyr were also found in RdORS (13). Interestingly, AQS com-
ADSproducedAD-CoAfromfattyacyl-CoAand1asthesub- pletelyretainedtheactive-siteresiduesofCmQNSthatspecif-
strates,whereasAQSgenerated2AQfrom3andADacidasthe icallygenerate8(11).Thus,Thr-132,Met-137,Thr-197,Gly-
substrates. Remarkably, unlike the previously reported 2AQ- 256, Phe-265, and Ser-338 in MsCHS were simultaneously
producingHsPKS3,bothenzymeslackedtheabilitytocatalyze substituted with Met, Ile, Tyr, Ala, Leu, and Gly in AQS. A
theone-potformationof2AQbyasingleenzyme.TheX-ray phylogenetic tree analysis suggested that ADS and AQS are
crystal structures of ADS and AQS and the structure-based groupedinthenon-chalcone-producingtypeIIIPKSsandare
site-directed mutagenesis studies revealed that the substrate closesttoCmQNS(Fig.3).
andproductspecificitiesofADSareprincipallyderivedfroma
InvitrofunctionalanalysesofADSandAQSbyco-incubation
uniqueactive-sitegeometry,whereasthoseofAQSareattrib-
utedtoanovelCoA-bindingtunnelarchitecture. Based on the hypothesis that the two type III PKSs from
E.rutaecarpagenerate2AQ,ADSandAQSwereindividually
Results expressed in Escherichia coli M15 as the N-terminally His -
6
taggedproteins,andbothpurifiedenzymesweresubjectedto
SequenceanalysesofADSandAQS
enzymereactions.Thesubstratesandtheproductsobtainedin
Two full-length cDNAs encoding the novel type III PKSs,
thisstudyaresummarizedinFig.4andsupplementalTableS1.
ADSandAQS,wereclonedfromthebudsofE.rutaecarpaby
Because 2AQs were extracted with organic solvents from the
the RT-PCR method. The full-length ADS and AQS cDNAs
enzymereactionmixture,theinitialfunctionalanalysesofADS
contained1,155-and1,200-bpopenreadingframesencoding
andAQSwereperformedfortheethylacetate(EtOAc)-soluble
M 42,705and44,065proteinswith384and399aminoacids,
r portions of the enzyme reaction mixtures, using 3, 1, and
respectively. ADS and AQS shared 76–46% amino acid
C –C fattyacyl-CoAs(hexanoyl-CoA(10),2,decanoyl-CoA
6 16
sequenceidentitieswithotherfunctionallycharacterizedplant
(11), lauroyl-CoA (12), myristoyl-CoA (13), and palmitoyl-
type III PKSs (Fig. 2). ADS shared 62% amino acid sequence
CoA (14)) as the substrates (Fig. 5A). The enzyme reaction
identity with Medicago sativa chalcone synthase (MsCHS),
products were determined by comparing the retention times
whichproducesnaringeninchalconefromthecondensationof
and the UV and MS spectra with those of authentic com-
threemoleculesof1withp-coumaroyl-CoA(7)(10);61%iden- pounds.Theco-incubationofADSandAQSandthesoleincu-
titywithAQS;59%identitywithCitrusmicrocarpaquinolone bation of ADS produced 2-methyltriketide lactone (15) and
synthase (CmQNS), which produces 4-hydroxy-N-methyl-2- 2-acetylmethyltetraketidelactone(16),derivedfromthreeand
quinolone(8)fromthedecarboxylativecondensationof1with fourmoleculesof1,respectively(Fig.5,AandB).However,the
N-methylanthraniloyl-CoA(3)(11);59%identitywithRheum co-incubation of ADS and AQS with 3, 1, and the C fatty
10
palmatum benzalacetone synthase (RpBAS), which produces acyl-CoA 11 generated 2-nonyl-N-methyl-4-quinolone (17),
benzalacetonefromthedecarboxylativecondensationof1with derivedfromthesethreesubstratesasthemajorproduct,along
7(12);54%identitywithClDCS(9);51%identitywithRhodo- witha2-alkyltriketidelactone(2ATL),2-nonyltriketidelactone
dendrondauricumorcinolsynthase(RdORS),whichgenerates (18), derived from 11 and two molecules of 1 as the minor
orcinol from the condensation of three molecules of 1 with product.Theco-incubationofADSandAQSwith3,1,andthe
acetyl-CoA(9)(13);50%identitywithHsPKS3(8);49%identity C fattyacyl-CoA2alsoyieldedthecorresponding2AQ4asthe
8
withClCURS1(9);and46%identitywithOrizasativacurcumi- major product, along with 2-heptyltriketide lactone (19) as
noidsynthase(OsCUS),whichcatalyzestheone-potformation theminorproduct.Incontrast,theco-incubationwith3,1,and
ofbisdemethoxycurcuminfromthecondensationoftwomol- theC fattyacyl-CoA12onlygenerated2-undecyl-N-methyl-
12
eculesof7andonemoleculeof1(14).AQSshared76%amino 4-quinolone(20).Amongtheproduced2AQs,17waspredom-
acidsequenceidentitywithCmQNS,61%identitywithRpBAS, inantlyyieldedby21.3%(Fig.5A).However,noproductswere
60%identitywithMsCHS,56%identitywithClDCS,53%iden- producedbytheADSandAQSco-incubationreactionsusing3
titywithClCURS1,50%identitywithOsCUS,50%identitywith and1witheither7ortheC ,C andC fattyacyl-CoAs10,
6 14 16
HsPKS3,and50%identitywithRdORS.NoadditionalcDNAs 13,and14asthesubstrates(Fig.5A).Furthermore,theco-in-
encoding other type III PKS isomers were obtained in this cubationsofCmQNSwithADSorADS,andthesoleincuba-
study. tionofCmQNSinthepresenceof3,1,andeithertheC –C
6 16
ADSandAQSconservedthecatalytictriadoftypeIIIPKS fatty acyl-CoAs 10, 2, and 11–13 or 7 as the substrates only
(Cys-164,His-303,andAsn-336)andtheactive-siteresiduesof afforded 8 (Fig. 6). Interestingly, the sole incubations of ADS
CHS(Ser-133,Pro-375,andthe“gatekeeper”Phe-215(MsCHS using 3, 1, and either the C –C fatty acyl-CoAs or 7 as the
6 16
numbering))(Fig.2).Incontrast,Thr-132,Thr-194,Thr-197, substrates revealed that ADS lacks the 2AQ-forming activity;
Ser-338,andthegatekeeperPhe-265,liningtheactive-sitecav- ADSonlyaffordedthe2ATLs19and18,derivedfromC and
8
ityofCHS,wereuniquelyalteredwithSer,Met,Cys,Trp,and C fattyacyl-CoAs2and11andtwomoleculesof1,respec-
10
Tyr, respectively, in ADS. These residues differ in numerous tively (Fig. 5B). Furthermore, AQS no longer yielded any
functionallydistincttypeIIIPKSsandarethoughttocontrol productsfromthesetsofthethreesubstrates(Fig.5C).These
thesubstrateandproductspecificitiesoftheenzymes(15,16). observationsclearlysuggestedthatbothADSandAQSlackthe
ThesubstitutionoftheconservedThr-197ofCHSwithCyswas one-pot2AQ-formingactivity,incontrasttoHsPKS3,andthe
alsoobservedinRpBAS(12).Simultaneoussmall-to-largesub- collaborationofADSandAQSisrequiredfortheformationof
stitutionsofThr-194,Ser-338,andPhe-265withMet,Trp,and 2AQ.
J.Biol.Chem.(2017)292(22)9117–9135 9119
CharacterizationoftwotypeIIIpolyketidesynthases
9120 J.Biol.Chem.(2017)292(22)9117–9135
CharacterizationoftwotypeIIIpolyketidesynthases
Figure3.PhylogenetictreeanalysisofplanttypeIIIPKSs.Thebacterial(cid:2)-ketoacylcarrierproteinsynthaseIIIs(KASIIIandFABH)fromE.coliwereemployed
asoutgroups.Thescalerepresents0.1aminoacidsubstitutionspersite.ADSandAQSarehighlightedwitharrows.ACS,acridonesynthase;ALS,aloesone
synthase;BAS,benzalacetonesynthase;CURS1,curcuminsynthase1;CUS,curcuminoidsynthase;DCS,diketide-CoAsynthase;OKS,octaketidesynthase;OLS,
olivetolsynthase;ORS,orcinolsynthase;PCS,pentaketidechromonesynthase;2PS,2-pyronesynthase;STS,stilbenesynthase;THNS,tetrahydroxynaphthalene
synthase;VAS,phloroisovalerophenonesynthase;VPS,valerophenonesynthase.GenBankTMregistrationnumbersareshowninparentheses.
InvitrofunctionalanalysisofADS pH 7.5, and instead, that of 22 was remarkably increased, as
TofurtherclarifythecatalyticroleofADSintheformationof comparedwiththoseofthereactionatpH7.0(Fig.7A).These
2AQ,thesubstrateandproductspecificitiesofADSwereinves- observations suggested that either ADS lacks thioesterase
tigatedbydirectlysubjectingtheenzymereactionmixturesto activitytoward21orthisactivitytoward21wasdisruptedby
HPLC,withoutextractingtheproductswithEtOAc.TheHPLC theacidicconditions.TotestthethioesteraseactivityofADS,
analysis revealed the presence of decanoyldiketide-CoA (21) ADSwasreactedwiththeC AD-CoA21asthesubstrateat
10
anddecanoyldiketideacid(22)intheenzymereactionmixture, pH6.5,7.0,and7.5,asthepossibleassaysystem.However,the
whenADSwasincubatedwith1andtheC fattyacyl-CoA11 HPLCanalysesdidnotrevealanydifferencesintheproduction
10
at pH 7.0 (Fig. 7A). The productions of 21 and 22 were not of22atthesedifferentpHvalues,ascomparedwiththoseinthe
observedinacontrolexperimentusingboiledADS(Fig.7A). control experiment using the boiled ADS (Fig. 7B). These
However,ADSpredominantlyafforded21atpH6.5,whereas resultsstronglysuggestedthatADScatalyzesthedecarboxyla-
theproductionof22wasdrasticallydecreasedatthispH.Fur- tivecondensationof1withtheC fattyacyl-CoA11topro-
10
thermore,theproductionof21wasdramaticallydecreasedat ducethecorrespondingAD-CoA21,whereasthecorrespond-
Figure2.ComparisonoftheprimarysequencesofADS,AQS,andothertypeIIIPKSs.ThesecondarystructuresofADSaredelineatedasfollows:(cid:3)-helices
(rectangles)and(cid:2)-strands(arrows).TheCys,His,andAsncatalytictriadandtheresiduesliningtheactive-sitecavityofCHSarecoloredredandblue,respectively.
Tyr-215inAQSandthecorrespondingresiduesinothertypeIIIPKSsarehighlightedingreen.GenBankTMaccessionnumbersareasfollows:MsCHS,L02902;
CmQNS,AB823730;ClDCS,AB535216;ClCURS1,AB495007;OsCUS,BAC79571;RpBAS,AF326911;RdORS,LC133082.ThesequenceofHsPKS3hasnotyetbeen
releasedfromGenBankTM.
J.Biol.Chem.(2017)292(22)9117–9135 9121
CharacterizationoftwotypeIIIpolyketidesynthases
Figure4.Substratesusedandproductsobtainedinthisstudy.
9122 J.Biol.Chem.(2017)292(22)9117–9135
CharacterizationoftwotypeIIIpolyketidesynthases
thecondensationof1withtheC fattyacyl-CoA12(Figs.8D
12
and9C).Incontrast,ADSlackedtheabilitiestoaccepttheC ,
6
C ,andC fattyacyl-CoAs10,13,and14asthestartersub-
14 16
strates as well as 3 and 7 as the starter substrates to produce
startersubstrate-derivedproducts(Fig.8,AandE–H).TheAD-
CoA-and2ATL-formingactivitiesobservedweremaximalat
pH7.5andpH6.0,respectively,withintherangeofpH5.5–8.0
(Fig.9).
In the production of AD-CoAs and 2ATLs, 21 (7.0% yield,
accordingtotheyieldof22atpH7.5)and19(4.6%yieldatpH
6.0) were the best products, respectively (Fig. 9). The higher
yieldoftheAD-CoAover2ATLandthelackofreportsforthe
isolation of the 2ATL derivatives from E.rutaecarpa might
indicatethatthe2ATLsareby-productslimitedtotheinvitro
enzymereaction.Interestingly,despitethefactthatADScon-
servestheCHSThr-194,Ser-338,andPhe-265withMet,Trp,
andTyr,asinthecaseofRdORS,ourfunctionalanalysisofADS
using9and1asthesubstratesdidnotaffordtheRdORS-pro-
ducingorcinol;ADSonlyproduced15and16(Fig.8I).Thus,
ADSisanoveltypeIIIPKSthatgeneratesAD-CoAinamanner
similartothatofClDCS(Fig.1,CandE).
ClDCSwasreportedasanallostericenzyme,duetothesig-
moidal curve in the rate-feruloyl-CoA concentration profile
(9).Incontrast,therate-fattyacyl-CoAconcentrationprofiles
withADSgeneratedahyperboliccurve,whichisafeatureofthe
Michaelis-Mentenmodel.ADSexhibitedaK valueof12.5(cid:4)M
m
andak /K valueof3.84min(cid:2)1mM(cid:2)1fortheC fattyacyl-
cat m 10
CoA11,withrespecttothe21-formingactivityatthepHopti-
mumof7.5(Table1),whichare5.9and1.2timeshigherthan
those of 74.4(cid:4)M and 3.23 min(cid:2)1 mM(cid:2)1 for the C fatty acyl-
8
CoA 2, respectively. Thus, ADS preferred 11 over 2 as the
startersubstrateandefficientlyproduced21,asobservedinthe
functional analysis of ADS described above. The steady-state
kineticanalysisrevealedthatADSexhibitedak /K valueof
cat m
1.65min(cid:2)1mM(cid:2)1for1,withrespecttothe21-formingactivity
atthepHoptimumof7.5(Table1).
Figure5.Enzymereactionproductsbyco-incubationandsoleincuba-
tions of ADS and AQS. Shown are the enzyme reaction products from InvitrofunctionalanalysisofAQS
N-methylanthraniloyl-CoA(3),malonyl-CoA(1),andfattyacyl-CoAs(hexa-
noyl(10)-,octanoyl(2)-,decanoyl(11)-,lauroyl(12)-,myristoyl(13)-,orpalmi- Next,weinvestigatedthesubstrateandproductspecificities
toyl(14)-CoAs)orp-coumaroyl-CoA(7)bytheco-incubationofADSandAQS
ofAQS,bydirectlysubjectingtheenzymereactionmixturesto
(A),thesoleincubationofADS(B),andthesoleincubationofAQS(C).
HPLC. In contrast to the other type III PKSs, AQS did not
ingADacid22isaby-productgeneratedbythenon-enzymatic accept3,7,andtheC –C fattyacyl-CoAs10,2,and11-14as
6 16
hydrolysis of 21, in a manner similar to the productions of the starter substrates with 1 to generate products (Fig. 6D).
feruloyldiketide-CoA and the corresponding diketide acid, as However,theformationsoftheC –C AD-CoAs5,21,and23
8 12
observedintheClDCSenzymereaction(9). byADS(Fig.8)andtheC –C 2AQs4,17,and20byADSand
7 11
ToevaluatethesubstrateandproductspecificitiesofADS,all AQSfrom3,1,andtheC –C fattyacyl-CoAs2,11,and12
8 12
oftheADS-producingAD-CoAswereconvertedtothecorre- (Fig.5A)encouragedustoinvestigatetheAQSreactionprod-
spondingADacidsbythealkalinehydrolysisreaction,andthen uctsderivedfrom3asastartersubstrateandtheC AD-CoA
10
the AD-CoA-forming activity of ADS was evaluated by TLC- 21 as an extender substrate. As expected, the HPLC analysis
basedassays,utilizing2-14C-labeled1astheextendersubstrate, indicatedthatAQSaccepts21astheextendersubstratewith3,
becausetheUVabsorptionoftheADacidsisquitelow(Fig.8). toproducethecorresponding2AQ17(Fig.10A)viaC–C/C–N
The TLC-based assays revealed that ADS accepts the C and bond formations. However, 21 was retained in the AQS and
8
C fattyacyl-CoAs2and11asstartersubstrates,toproduce boiledAQSreactionmixturesatcomparablelevels.Incontrast,
10
theAD-CoAs5and21(6and22intheTLC-basedassay)asthe thehydrolyzedproductof21,22,wasobviouslyconsumedin
majorproducts,alongwiththe2ATLs19and18,respectively the AQS reaction (Fig. 10A). This result suggested that AQS
(Figs.8(BandC)and9(AandB)).Furthermore,ADSproduced utilizes22astheextendersubstratebutlacksthethioesterase
a trace amount of lauroyldiketide-CoA (23) (lauroyldiketide activityfortheformationof22from21.Indeed,AQSaccepted
acid(24)intheTLC-basedassay)asthesingleproduct,from 22astheextendersubstratewith3andprovidedtheC 2AQ
10
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CharacterizationoftwotypeIIIpolyketidesynthases
Figure6.HPLCelutionprofilesofEtOAc-solubleenzymereactionproductsbytheco-incubationofADS,AQS,andCmQNSandbythesoleincubation
ofCmQNSandAQS.ShownareenzymereactionproductsfromN-methylanthraniloyl-CoA(3),malonyl-CoA(1),andfattyacyl-CoAs(hexanoyl-CoA(10),
octanoyl-CoA(2),decanoyl-CoA(11),lauroyl-CoA(12),ormyristoyl-CoA(13))orp-coumaroyl-CoA(7)bytheco-incubationofADSandCmQNS(A),the
co-incubationofAQSandCmQNS(B),andthesoleincubationofCmQNS(C).D,enzymereactionproductsfrom1and10,2,11-14,3,or7bythesoleincubation
ofAQS.
17 with 7.2 times higher efficiency than that from 21 at the
optimumpH7.5(Fig.10A).Quiteinterestingly,AQSwascapa-
ble of accepting the C –C AD acids (hexanoyldiketide acid
6 16
(25),6,22,24,andmyristoyl(26)-andpalmitoyl(27)-diketide
acids)with3toproducethecorresponding2AQs,2-pentyl-N-
methyl-4-quinolone (28), 4, 17, 20, and 2-tridecyl (29)-, and
2-pentadecyl (30)-N-methyl-4-quinolones, respectively (Fig.
10B).However,AQSlackedtheabilitytoaccept7asthestarter
substrate and did not yield any products (Fig. 10B). These
observations suggested that AQS is a novel type III PKS that
specifically catalyzes the decarboxylative condensation of AD
acidwith3togenerate2AQviatheC–Nbondformation,ina
mannersimilartothatofClCURS1(Fig.1,DandF).
Thesteady-statekineticparametersrevealedthattheC AD
10
acid 22 is the best extender substrate (Table 1) of AQS and
efficientlygeneratestheC 2AQ17,ingoodagreementwith
10
thebestproductionof17intheADSandAQSco-incubation
reactions(Fig.5A).AQSaccepted3asthestartersubstratewith
a K value of 22.9 (cid:4)M, which is 1.6 times higher than that of
m
CmQNS(K (cid:3)37.8(cid:4)M(11))(Table1).
m
OverallstructuresofADSandAQS
To clarify the structural details of the AD-CoA- and 2AQ-
Figure7.AnalysisofallADSenzymereactionproducts.A,theADS
reactionproductsfromdecanoyl-CoA(11)andmalonyl-CoA(1)atpH6.5, forming catalytic mechanisms of ADS and AQS, the crystal
7.0, and 7.5. B, analysis of the thioesterase activity of ADS with structureofADScomplexedwithCoASHandtheapo-crystal
decanoyldiketide-CoA(21)atpH6.5,7.0,and7.5.TheHPLCelutionpro-
structure of AQS were solved at 1.80 and 2.20 Å resolutions,
filesobtainedbyADSandboiledADSareindicatedasblackandredlines,
respectively. respectively(Fig.11).Thecrystallographicdataandrefinement
9124 J.Biol.Chem.(2017)292(22)9117–9135
CharacterizationoftwotypeIIIpolyketidesynthases
Figure8.EnzymereactionproductsfromADSandtheADSmutantenzymeswithstructurallydistinctstartersubstrates.A–H,theTLC-basedanalysisof
radiolabeledproductsfrommalonyl-CoA(1)andhexanoyl-CoA(10)(A),octanoyl-CoA(2)(B),decanoyl-CoA(11)(C),lauroyl-CoA(12)(D),myristyl-CoA(13)(E),
palmitoyl-CoA(14)(F),N-methylanthraniloyl-CoA(3)(G),andp-coumaroyl-CoA(7)(H)bytheADSandADSmutantenzymes.I,HPLCelutionprofilesofenzyme
reactionproductsobtainedbyADSandtheboiledADSreactionswithacetyl-CoA(9)and1asthesubstrates.
Figure9.pH-dependentADacid-and2ATL-formingactivitiesofADS.Shownaretherelativeyieldsofoctanoyldiketideacid(6)and2-heptyltrikeide
lactone(19)frommalonyl-CoA(1)andoctanoyl-CoA(2)(A),decanoyldiketideacid(22)and2-nonyltriketidelactone(18)from1anddecanoyl-CoA(11)(B),and
lauroyldiketideacid(24)from1andlauroyl-CoA(12)(C).TheyieldsofADacidsatpH6.0–8.0,2ATLatpH5.5andpH6.0,and2ATLatpH6.0–8.0areindicated
asclosedsquaresandclosedandopencircles,respectively.
statisticsaresummarizedinsupplementalTableS2.Theasym- revealedthetypicaltypeIIIPKS-folds.Thecatalytictriad(Cys-
metricunitsofADSandAQScontainedfourandthreemono- 157/His-297/Asn-330 in ADS and Cys-170/His-309/Asn-342
mers, respectively. Significant backbone changes were not inAQS)andthe16-Å-longCoA-bindingtunnelarealsocon-
observedbetweenthemonomersinthestructuresofADSand servedinthestructures,inlocationsandorientationsverysim-
AQS,respectively.ThestructuresofADSandAQSarenearly ilartothoseinotherplanttypeIIIPKSs.Theoverallstructures
identical,witharoot-mean-square-deviation(r.m.s.d.)of1.0Å ofADSandAQSarehighlyhomologoustothoseofthestruc-
for the C(cid:3)-atoms. The overall structures of ADS and AQS turallycharacterizedplanttypeIIIPKSs(r.m.s.d.valuesof0.8–
J.Biol.Chem.(2017)292(22)9117–9135 9125
CharacterizationoftwotypeIIIpolyketidesynthases
Table1 acceptance of the C –C fatty acyl-CoAs by ADS, the side
8 12
Steady-statekineticparametersofADSandAQS chain(s) of one or more residues lining the active-site cavity
Startersubstrate K k k /K might move flexibly and open the gate to a hidden space, to
m cat cat m
(cid:4)M min(cid:2)1 min(cid:2)1mM(cid:2)1 accepttheC12fattyacylmoietyofthesubstrate.
ADS
Onthebasisoftheseobservations,weconstructedasetof
2 74.4 0.24 3.23
11 12.5 0.05 3.84 ADSsite-directedmutants,inwhichTrp-332wassubstituted
12a NDb ND ND withGln,Ile,Val,Ala,andGly.WealsoconstructedtheADS
1 35.8 0.06 1.65
C191G/Amutantenzymes,becausethecorrespondingresidue
AQS
3 22.9 1.20 51.6 atthispositionoftencontrolsthesubstratespecificityandthe
25 9.0 0.01 1.10 polyketide-chain elongation reaction in numerous type III
6 11.8 0.33 28.0
22 5.0 0.30 60.0 PKSs.Amongthem,theADSW332Smutantenzymeformed
24 9.0 0.49 54.4 inclusionbodiesinE.coli,andthusthemutantenzymewasnot
26 9.0 0.01 1.44
27 7.1 0.01 1.43 analyzedinourstudy.TheTLC-basedassayrevealedthatthe
aThekineticparameterswerenotdetected,duetothetraceamountoftheprod- ADS W332Q mutant enzyme enhanced the abilities to form
uct.bND,notdetected. boththeC AD-CoA5(6intheTLC-basedassay)andtheC
8 8
2ATL 19, as compared with those of the wild type (Fig. 8B).
1.1and0.7–1.1ÅforADSandAQS,respectively)andexhibited Furthermore,themutantenzymeincreasedtheC 2ATL18-
10
r.m.s.d.valuesof1.0and1.0ÅwithMsCHS(PDBcode1BI5) andC AD-CoA23-formingactivities(24intheTLC-based
12
(10),1.1and0.8ÅwithRpBAS(PDBcode3A5Q)(17),0.8and assay)(Fig.8,CandD).
0.7ÅwithCmQNS(PDBcode3WD8)(11),and1.1and1.1Å Similar results were also obtained in the ADS W332I,
withClCURS1(PDBcode3OV2)(18),respectively. W332V,W332A,andW332Gmutantenzymereactions,using
theC –C fattyacyl-CoAs2,11,12,and13,respectively,as
8 14
Active-sitestructureandstructure-basedmutagenesisstudies
thestartersubstrates.TheADSW332I,W332V,W332A,and
ofADS
W332G mutant enzymes further enhanced the 19-forming
In the absence of the structure of the AD-CoA-producing activity, as compared with that of the ADS W332Q mutant
typeIIIPKS,suchasClDCS,wecomparedtheactive-sitestruc- enzyme,withthelossofthe5-formingactivity(6intheTLC-
tures of ADS and the aromatic diketide-producing RpBAS. based assay) (Fig. 8B). The 18-forming activity was also
Mostoftheactive-siteresiduesofADSandRpBAS,including enhanced in the ADS W332I, W332V, W332A, and W332G
the catalytic triad, were superimposable in nearly identical mutant enzymes, in this order (Fig. 8C). In contrast, the
positions(Fig.12,AandB).However,thesidechainsofTrp- 21-forming activity (the C AD acid 22 in the TLC-based
10
332andMet-257inADSprotrudedmoretowardtheactive-site assay) tended to decrease, depending on the substitutions
cavity, as compared with those of Ser-331 and Leu-256 in towardthelessbulkyresidues(Fig.8C).The23-formingactiv-
RpBAS,respectively.Thus,thesidechainsofTrp-332andMet- ity (the C AD acid 24 in the TLC-based assay) was also
12
257blockedtheaccessofCys-191,correspondingtoCys-190at decreasedwiththeADSW332Imutantenzyme,butthismuta-
thebottomwallintheactive-sitecavityofRpBAS,totheactive- tion conferred the ability to form 2-undecyltriketide lactone
site cavity of ADS. The substitutions of Ser-332 and Leu-257 (31) (Fig. 8D). In contrast, the ADS W332V, W332A, and
withTrpandMetinADS,respectively,thusnarrowedandshal- W332G mutant enzymes lost the 23-forming activity and
lowedtheactive-sitecavityofADS,ascomparedwiththatof afforded31asthesingleproduct(Fig.8D).Amazingly,theADS
RpBAS. W332G mutant enzymes newly accepted the C fatty acyl-
14
Interestingly,incontrasttotheside-chainorientationofthe CoA 13 as the starter substrate, and generated myris-
gatekeeper Phe-258 in RpBAS, the side chain of the corre- toyldiketide-CoA(32)(26intheTLC-basedassay)and2-tride-
spondingresidue,Tyr-259,ofADSprotrudedtowardtheoppo- cyltriketide lactone (33) (Fig. 8E). The ADS W332A mutant
sitesideoftheactive-sitecavity.Thisconformationalchangeis enzyme also exhibited the 33-forming activity, although the
presumably caused by the simultaneous small to large L209F activity was significantly decreased, as compared with that of
andF259YsubstitutionsinADS.Asaresult,thesidechainof the ADS W332G mutant enzyme. These observations sug-
Phe-209,neighboringTyr-259inADS,protrudedmoretoward gestedthatthemutantenzymesincreasedtheactive-sitecavity
the active-site cavity, as compared with that of Leu-208 of volume and thereby enhanced the 2ATL-forming activity.
RpBAS, and restricted part of the active-site cavity of ADS. The crystal structures of the ADS W332Q and W332G
Thus, the active-site cavity of ADS, including its entrance, is mutantenzymesindeedindicatedtheexpansionofthetotal
muchnarrowerthanthatofRpBAS,suggestingthattheselec- active-site cavity volume, due to the loss of the planar and
tivestartersubstratespecificityforthefattyacyl-CoAofADS bulkierindoleringatthisposition(Fig.12,A,C,andD).The
arisesfromthischaracteristicactive-sitearchitecture.Thetotal estimated total cavity volumes of the ADS W332Q and
active-sitecavityvolume(200Å3)andtheactive-siteentrance W332Gmutantenzymeswere240and295Å3,whichare1.2
area (21 Å2) of ADS were apparently smaller than those of and1.5timeslarger,respectively,thanthatofthewildtype
RpBAS(350and34Å2)(Figs.11(CandD)and12(AandB)).A (200Å3).
docking study predicted that the active-site cavity of ADS is Unexpectedly, the ADS W332I, W332V, W332A, and
largeenoughtoaccommodateanenzyme-boundmonoketide W332Gmutantenzymesnewlyaccepted3asthestartersub-
intermediatewithuptoaC length(Fig.11G).Consideringthe strate with 1 to produce 8 (Fig. 8G). In contrast, none of the
10
9126 J.Biol.Chem.(2017)292(22)9117–9135