ANRV342-PP59-29 ARI 29March2008 2:13 Alkaloid Biosynthesis: Metabolism and Trafficking Jo¨rg Ziegler and Peter J. Facchini g or DepartmentofBiologicalSciences,UniversityofCalgary,Calgary,AlbertaT2N1N4, ws. Canada;email:[email protected];[email protected] e alrevionly. nnuuse w.aal won m wpers oaded fro1/11. For wnl1/0 Don 1 9. y o 5-76ersit Annu.Rev.PlantBiol.2008.59:735–69 KeyWords 3v 9:7Uni TheAnnualReviewofPlantBiologyisonlineat benzylisoquinolinealkaloids,cellularcompartmentalization, Plant Biol. 2008.5Washington State pT1CA0llohal.np1irst1yi.gar4ahrin6gtt/inhscautlrneae(cid:2)n’clssrueedrr2voev0iviee0:.dwa8rspb.olyarnAgtn.5n9u.a0l3R26e0vi7e.w09s.2730 saAAellkbckaosalnltoordiidaadscrs,ytmrmeepetrtaeabsboeonliltcismaenh,gmiignhoelneyoritdneigrvpeersneoigdroinudpoolefaclokmalopiodusn,dtrsotphaanteare ev. by 1543-5008/08/0602-0735$20.00 relatedonlybytheoccurrenceofanitrogenatominaheterocyclic R u. ring.Plantsareestimatedtoproduceapproximately12,000different n An alkaloids,whichcanbeorganizedintogroupsaccordingtotheircar- bonskeletalstructures.Alkaloidbiosynthesisinplantsinvolvesmany catalytic steps, catalyzed by enzymes that belong to a wide range of protein families. The characterization of novel alkaloid biosyn- thetic enzymes in terms of structural biochemistry, molecular and cellbiology,andbiotechnologicalapplicationshasbeenthefocusof researchoverthepastseveralyears.Theapplicationofgenomicsto thealkaloidfieldhasacceleratedthediscoveryofcDNAsencoding previously elusive biosynthetic enzymes. Other technologies, such as large-scale gene expression analyses and metabolic engineering approacheswithtransgenicplants,haveprovidednewinsightsinto theregulatoryarchitectureofalkaloidmetabolism. 735 ANRV342-PP59-29 ARI 29March2008 2:13 sons. Unlike most other types of secondary Contents metabolites,themanyclassesofalkaloidshave unique biosynthetic origins. Despite the di- ALKALOIDMETABOLISM....... 736 versityofmetabolicpathways,severaltechni- AlkaloidBiosyntheticPathways... 736 cal breakthroughs have recently contributed Structure-FunctionRelationships to impressive advancements in our under- inAlkaloidBiosynthetic standing of alkaloid biosynthesis. Recent Enzymes...................... 747 applications of genomics-based technolo- REGULATIONOFALKALOID gies, such as expressed sequence tag (EST) BIOSYNTHETICPATHWAYS.. 748 databases, DNA microarrays, and proteome TranscriptomeandMetabolome analysis,haveacceleratedthediscoveryofnew Analyses...................... 748 componentsandmechanismsinvolvedinthe GeneRegulationandSignal assembly of alkaloids in plants. Our present Transduction.................. 749 org TransgenicApproaches........... 749 ability to investigate secondary metabolism ws. fromacombinedbiochemical,molecular,cel- e ALKALOIDTRAFFICKING....... 751 alrevionly. CellType–SpecificLocalization lpurloavr,edanodurpahpypsiroelcoigaticioanl pfoerrstpheectcivoemhpalesximbi-- ww.annuonal use SuboEcfneAlzlyulkmlaaelrosCi.do..Bm.i.po.as.yr.tnm.t.he.en.t.tia.cl.i.z.a.ti.o.n.. 751 odlioscguyssofreaclkenaltoaiddvpaantchewsaiyns.oIunrtuhnisderresvtiaenwd,iwnge m wpers ofAlkaloidBiosynthetic ofthemetabolismandtraffickingofalkaloids. Downloaded fron 11/01/11. For PEREnSzPECyEnhmCzaeynTmnCIeeVolssmE....p...l..e...x...e...s....a...n....d.....M.......e...t..a...b....o....l.i...c... 777555756 AMmlooknnaooltoteeirdrppeBennoioiodsidynitnhidneodtloeiclePaalkatahllkowaildaosyidss(.MTIhAe) 9. y o comprise a family of structurally and phar- 76sit maceutically diverse alkaloids. Some of the 5-er ALKALOIDMETABOLISM 3v approximately 2,000 known compounds 008.59:7State Uni Amloklaelcouidlasr-wareeighat,ndiitvreorgseen-cgoronutapinionfgcloomw-- wfoirdethlyeutrseeadtminenmteodficcianneceinrcalnuddeajvminablilnaestifnoer Biol. 2ngton EseSqTue:necxeptraegssed pseocuonnddsardyemriveetdabmoloitsetslyfofuronmdinamapinporoaxcimidas.teAlys asonutiracrersyftohrmthicesehecaormtpdoiusonrddsearrse.CTathhearamntahjours Plant Washi MmoInAo:terpenoid 20%ofplantspecies,alkaloidsarepurported roseusandRauvolfiaserpentina.MIAsarecon- ev. by indolealkaloid toplayadefensiveroleagainstherbivoresand densation products of a nitrogen-containing R pathogens. Owing to their potent biological indole moiety derived from tryptamine nu. activity, many of the approximately 12,000 and a monoterpenoid component derived n A knownalkaloidshavebeenexploitedasphar- from the iridoid glucoside secologanin maceuticals, stimulants, narcotics, and poi- (Figure1).IntheMIAbiosyntheticpathway, −−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→ Figure1 Biosynthesisofthemonoterpenoidindolealkaloidsvinblastineandajmaline.Enzymesforwhich correspondingmolecularcloneshavebeenisolatedareshowninbold.Abbreviations:AAE,acetylajmalan esterase;ANAMT,acetylnorajmalanmethyltransferase;Cyp72A1,secologaninsynthase;Cyp76B6, geraniol10-hydroxylase;D4H,desacetoxyvindoline4-hydroxylase;DAT,deacetylvindoline 4-O-acetyltransferase;DHVR,dihydrovomileninereductase;LAMT,loganicacidmethyltransferase; MAT,minovincinineacetyltransferase;NAMT,norajmalanmethyltransferase;NMT, N-methyltransferase;PER,peroxidase;PNAE,polyneuridinealdehydeesterase;RG,raucaffricine O-β-glucosidase;SBE,sarpaganbridgeenzyme;SGD,strictosidineβ-d-glucosidase;STR,strictosidine synthase;T16H,tabersonine16-hydroxylase;TDC,tryptophandecarboxylase;VH,vinorine hydroxylase;VR,vomileninereductase;VS,vinorinesynthase. · 736 Ziegler Facchini ANRV342-PP59-29 ARI 29March2008 2:13 Geraniol Cyp76B6 LAMT 7-Deoxyloganic acid Loganic acid Loganin L-Tryptophan Cyp72A1 TDC 10-Hydroxygeraniol LAMT 7-Deoxyloganin Secologanin Tryptamine 10-Oxogeraniol 7-Deoxyloganetic acid STR org SGD s. w e alrevionly. 16-Methoxytabersonine 16-Hydroxytabersonine Strictosidine aglycoside Strictosidine nnuuse T16H w.aal m wwperson SBE PNAE oaded fro1/11. For 163--Mhyedthrooxxyyt-a2b,3e-rdsoihnyidnreo- Tabersonine Dehydrogeissoschizine Polyneuridine aldehyde epi-VelloVsimSine Downln 11/0 NMT VH 9. y o 76sit Vomilenine Vinorine 9:735-Univer Lochnericine 6,7-DehydroMmAinTovincinine VR v. Plant Biol. 2008.5y Washington State DesacetoxyvDin4dHoline Hörhammericine 6,7-Dehydroechitovenine 1,2-DihydrovomileninDeHAVAREAcetylnoraAjmNaAlinMeT Reb MAT u. Deacetylvindoline n n A DAT Norajmaline Acetylajmaline 19-O-Acetylhörhammericine NAMT AAE Vindoline Cantharanthine PER RG Ajmaline Raucaffricine Vinblastine www.annualreviews.org • AlkaloidBiosynthesis 737 ANRV342-PP59-29 ARI 29March2008 2:13 tryptophan decarboxylase (TDC) converts quently converted via several unstable in- tryptophantotryptamine;thegeneencoding termediatestodehydrogeissoschizine,which this enzyme has been cloned from different represents a key branchpoint intermediate MIA-accumulating plants (25, 89, 178). that leads to several diverse MIA pathways. Incorporation experiments with isotopically The branch pathways proceeding through labeled glucose in C. roseus cell cultures tabersonineandpolyneuridinealdehydehave showed that the initial steps for the biosyn- been characterized most thoroughly. Other thesisofthemonoterpenoidskeletonproceed branch pathways, such as that leading to via the triose phosphate/pyruvate pathway catharanthine,remainpoorlyunderstood. (22).CognatecDNAsfor1-deoxy-d-xylulose Hydroxylation of tabersonine at position 5-phosphate reductoisomerase and 2C- 16 represents the first reaction leading methyl-d-erythritol 2,4-cyclodiphosphate to vindoline (Figure 1). The responsi- synthase were isolated from C. roseus and ble enzyme, tabersonine 16-hydroxylase org the corresponding gene transcripts were (T16H, Cyp71D12), belongs to the P450 ws. upregulated in MIA-producing cell cul- monooxygenase family, and the correspond- e alrevionly. tsuecreoslog(1a6n3in). bTihoesynfitrhsetsiscomismictatteadlyzsetedp biny ienngcodcDinNgAP45w0a-sdeipdeenndteifinetdenfzryommesainsduubcseedt nnuuse the cytochrome P450 monooxygenase by the treatment of C. roseus cultures with ww.aonal geraniol-10-hydroxylase (Cyp76B6), which light (140). O-Methylation at position 16, m wpers was purified and cloned from C. roseus (20). reduction of the 2,3-double bond, and oaded fro1/11. For ShaveaavvieelarabnliolitstytebpoesefnCin.chrotahsreeaucstseEercSizoTeloddg,aaantlaitbnhaospueagsth(h1w0tha8ye) NhAyl-tdmhrooeutxhgyythalabcteDiorNsnoArneissnuhelatvtioenntdhoeetsacbcoeenetvnoexriyssvoioilnandteoodfli1nfo6e-r. wnl1/0 has led to the recent discovery of loganic the enzymes that participate in these steps, 769. Dosity on 1 apceirdsomneatlhcyolmtramnusfneircaasteio(nL)A.TMhTe)fi(Vna.lDreeaLctuicoan, tbheeenNc-haarancdterOiz-emdeitnhypllaatnintgexatrcaticvtisti(e2s7,h3a7v)e. 5-er ofthisearlybranchofthepathwayinvolvesan The 2-oxoglutarate–dependent dioxygenase 3v 9:7Uni oxidativeC-Ccleavagecatalyzedbyasecond desacetoxyvindoline 4-hydroxylase (D4H) 008.5State P450monooxgenase(Cyp72A1),resultingin producesdesacetylvindoline,whichisfurther Biol. 2ngton theSctornicvteorssiidoinneo,fltohgeancionmtmosoencoploregcaunrinso(r70t)o. csaocnevteyrlvteinddboylinthee4-aOce-taycletCyoltAra–ndsefpereansdee(nDtAdTe-) Plant Washi aclolndMenIAsast,ioins ofofrtmryepdtambyinae aPnidctseet-cSoploegnagnleinr tboeevnincdloonliendea(n1d50c,h1ar6a2c)t.eBriozethdfernozmymCe.srohseauvse. v. y eb (Figure1). Strictosidine synthase (STR) DATbelongstotheBAHD(benzylalcoholO- R u. from R. serpentina was the first cDNA in- acetyl-, anthocyanin-O-hydroxycinnamoyl-, n An volved in alkaloid biosynthesis to be cloned anthranilate-N-hydroxycinnamoyl/benzoyl-, and functionally expressed in microorgan- and deacetylvindoline 4-O-acetyltransferase) isms.STRorthologsfromC.roseusandOphi- family of acyl-CoA dependent acyltrans- orrhiza pumila have also been isolated (79, ferases;theseproteinsparticipateinavariety 99, 178). The strictosidine glucose moiety of plant secondary metabolic pathways. is subsequently removed by a family 1 glu- Ultimately, vindoline is coupled to catha- cosyl hydrolase enzyme, strictosidine β-d- ranthine by a nonspecific peroxidase to glucosidase (SGD); a cDNA for SGD was form vinblastine (145). A cDNA with 78% functionallycharacterizedfromC.roseusand amino acid identity to DAT was obtained R. serpentina. SGD from R. serpentina shows from C. roseus and encodes minovincinine exclusivesubstratespecificitytowardstricto- 19-O-acetyltransferase, yielding echitove- sidine and closely related analogs (46, 50). nine, which is a second pathway originating The strictosidine-derived aglycone is subse- from the tabersonine branch (80). Whereas · 738 Ziegler Facchini ANRV342-PP59-29 ARI 29March2008 2:13 minovincinineacetyltransferase(MAT)shows thoughtheglucosylatingactivityhasnotbeen residual activity toward the DAT substrate, reported, researchers found a cDNA encod- deacetoxyvindoline, DAT accepts only its ingtheenzymeraucaffricineO-β-glucosidase BIA: naturalsubstrate. (RG),responsiblefordeglycosylationofrau- benzylisoquinoline Thepolyneuridinealdehydebranchofthe caffricine (168). RG shares 60% amino acid alkaloid MIApathwayisinitiatedbytheformationof identitywithSGDandacceptsstrictosidineas Sanguinarine: an a bond, known as the sarpagan bridge, be- asubstrate,whereasSGDisnotactiveagainst antimicrobial tweenC5andC16ofstrictosidine-derivedin- raucaffricine. benzylisoquinoline termediate compounds (Figure 1). The en- alkaloidsometimes usedinoralhygiene zymecharacterizedfromcrudeextractsofR. Benzylisoquinoline alkaloids. Benzyliso- products serpentinashowsNADPHandoxygendepen- quinoline alkaloids (BIA) represent approxi- dence,andissensitivetoP450inhibitors,sug- mately2,500elucidatedstructuresandpossess gestingitisaP450monooxygenase(139).The potentpharmacologicalproperties.Themost org resulting polyneuridine aldehyde undergoes prominent compounds are the narcotic ws. desterificationtoyieldepi-vellosimine,which analgesic morphine, the cough suppressant e alrevionly. insanatcpeotylylanteeudridtoinefoarldmehvyidneoersitneer.asRe(ePcoNmAbEi-) cthoedeainnet,imthicermobuisaclleagreelnatxsanstanpgapuainvaerriinnee,aanndd nnuuse fromR.serpentinashowsesteraseactivityonly berberine.Collectively,thesealkaloidsoccur ww.aonal to polyneuridine aldehyde and not to struc- mainly in the Papaveraceae, Ranunculaceae, m wpers turallyrelatedesters,andisamemberofthe Berberidaceae,andMenispermaceae;Papaver oaded fro1/11. For αitmo/βaDtheAlyyTd3ro0al%nadseidMseunpAteTirt,fyavmoinnilotyrh(ien29ea)m.sWyinnoitthhaacasiepdpl(reVovSxe-)l fasoorrmenntchiiafee,rTmumhoaslt(icoetprxuituemmnssipvpeeoclpyiepisyn,)va,enEsdtsicCghaostcphetdoislzsjiapapeoccnaieilcisa-. wnl1/0 fromR.serpentinaalsobelongstotheBAHD BIA biosynthesis, which fundamentally Don 1 familyofacyltransferases(11).Acetylajmaline involves the condensation of two tyrosine 769. sity o is subsequently formed via a series of reac- derivatives, begins with the decarboxylation 5-er tions that includes hydroxylation, two dou- oftyrosinetotyramineorofdihydroxypheny- 3v 9:7Uni ble bond reductions, and an N-methylation. lalaninetodopaminebytyrosinedecarboxy- 008.5State The enzymes involved in these steps have lase(TYDC).TYDCconstitutesalargegene Biol. 2ngton bdeeepnenbdioecnhtemmoicnaololyxcyhgaernaacsteerviizneodrainsethheyPd4r5o0x-- finamoiplyiu;mappproopxpimya(t3e3l)y.1D5ompaemmibneersisartehefopurned- Plant Washi yNlaAsDe(PVHH-)d,ewphenicdhenytielrdesdvuocmtaisleesninvoem,ainledntihnee c4u-hrsyodrrofoxyrpthheenyisloacqeutianlodleihnyedme,oreiestuyl,tiwnghefrroemas v. y eb reductaseand1,2-dehydrovomileninereduc- thedeaminationoftyramine,isincorporated R u. tase (39, 44, 166). Acetylajamaline esterase as the benzyl component (Figure 2). As is n An (AAE)ultimatelyhydrolyzesthe17-O-acetyl the case in MIA biosynthesis, BIA conden- group to produce ajmaline. Active recombi- sationisaPictet-Spengler–typereactionand nantAAE,whichcouldbeproducedonlyvia is catalyzed by the first committed step of a virus expression system in Nicotiana ben- the pathway, norcoclaurine synthase (NCS). thamiana, shows slight substrate preference The enzyme has been purified from Thalic- for acetylajmaline compared with norajma- trumflavumandcorrespondingcDNAshave line. The latter compound might represent been isolated and functionally characterized anintermediateinaparallelajamalinebiosyn- from opium poppy and T. flavum (87, 136, theticpathwayinwhichN-methylationoccurs 137). NCS is related to the pathogenesis after deesterification (134). Unlike PNAE, relatedprotein(PR)10andBetv1allergen AAEbelongstotheGDSLlipasefamily.Rau- proteinfamilies.However,homologousPR10 caffricine, a glucosylated derivative of vom- proteins from opium poppy are not catalyt- ilenine,isfoundinR.serpentinacultures.Al- icallyactive.Biochemicalcharacterizationof www.annualreviews.org • AlkaloidBiosynthesis 739 ANRV342-PP59-29 ARI 29March2008 2:13 TYDC NCS L-Dopa Dopamine (S)-Norcoclaurine 4-Hydroxyphenylacetaldehyde 6OMT Papaverine CoOMT Palmatine Columbamine (S)-Coclaurine CNMT org Cyp719A1 Cyp80A1 Bisbenzylisoquinoline s. alkaloids w e Cyp80B3 nnualreviuse only. (S)-Canadine (S)-Tetrahydrocolumbamine N-(mSe)-t3h'y-Hlcyodcrloaxuyri-ne (S)-N-Methylcoclaurine w.aal STOX SOMT 4'OMT won m wpers DRS DRR oaded fro1/11. For Berberine (S)-Scoulerine BBE (S)-Reticuline 1,2-Dehydroreticuline (R)-Reticuline wnl1/0 Don 1 CFS 7OMT Cyp719B1 9. y o 5-76ersit SalR 3v 9:7Uni Cyp719A2 008.5State (S)-Stylopine (S)-Cheilanthifoline Laudanine Salutaridinol Salutaridine Biol. 2ngton TNMT SalAT Plant Washi v. y eb R MSH nnu. (S)-cis-N-Methylstylopine Protopine Thebaine Salutaridinol-7-O-acetate A P6H Spontaneous Dihydrosanguinarine 6-Hydroxyprotopine Neopinone Oripavine Morphinone DBOX Spontaneous COR COR Sanguinarine Codeinone Codeine Morphine · 740 Ziegler Facchini ANRV342-PP59-29 ARI 29March2008 2:13 recombinant T. flavum NCS has been ac- cludes catechols and phenylpropanoids in complishedusingacontinuousenzymeassay addition to various BIA derivatives (42). based on circular dichroism spectroscopy Coclaurine N-methyltransferases (CNMT) that follows the generation of the enzyme’s have been cloned from opium poppy and chiral product. These studies revealed a C. japonica and are more closely related to reaction mechanism that involves a two- S-adenosyl-L-methionine (SAM)-dependent step cyclization with a direct electrophilic cyclopropanefattyacidsynthasesthanother aromatic substitution (91). Recently, a N-methyltransferases(19,35).Thehydroxy- second enzyme capable of producing only lation of N-methylcoclaurine is catalyzed by the (S)-norcoclaurine enantiomer was iso- aP450monooxygenaseclassifiedintheCyp lated from C. japonica; this enzyme displays subfamily80B(65,68,125,138). sequence similarity to 2-oxoglutarate de- (S)-Reticuline is the central pathway in- pendent dioxygenases (102). However, this termediate from which most BIA structural org enzyme does not possess a 2-oxoglutarate- types are derived. Only the dimeric bisben- ws. binding domain and requires ferrous ions, zylisoquinoline alkaloids are not produced e alrevionly. raactthiveirty.thRanem2a-rokxaobglyl,uttawraotepororteoinxysgeinn,tfwoor vtoiach(rSo)m-reetPic4u5li0nee.nzInymtehisCyppa8th0wAa1y,cathtaelyczye-s nnuuse different protein families can catalyze the the regio- and stereoselective oxidative C- ww.aonal same reaction in vitro. Nevertheless, the O phenol coupling of the reticuline pre- m wpers relativeparticipationofeachenzymeinBIA cursor N-methylcoclaurine to produce the oaded fro1/11. For (bSio)Ts-yrhentetihcceuosliinnsveienirnsvviiovonolvmeosfuOs(t-Sms)t-einltlhobyreclaodtcieoltaneurarmitnipneoestdio-. b(d7ios8eb)s.ennAzoyntloistroheqepurrienseoexlncinteepatinaolknianlotweihdrmerbeeedribaraetemticucunoliimnnee- wnl1/0 tion6,N-methylation,3(cid:3)-hydroxylation,and poundmightbethebiosynthesisofthesim- 769. Dosity on 1 acoscelcaounridne4(cid:3)-6O--Om-methetyhlaytlitorann(sFfeirgausree(26)O.NMoTr-) pcelentN-ddisecmoevtehryylaotefdaBI7A-Op-ampaevtehryinltera.nTshfeerarese- 35-ver and 3(cid:3)-hydroxy-N-methylcoclaurine 4(cid:3)-O- specific for norreticuline (N7OMT, nor- 9:7Uni methyltransferase(4(cid:3)OMT)arebothclassII reticuline 7-O-methyltransferase), but not 008.5State O-methyltransferases that display strict re- N-methylated analogs, suggests that (S)- Biol. 2ngton gtaiionsepdecfiofircietya.cChoegnnzaytmeceDfrNoAmshoapviuembeepnopopby- rweatyicutloinepappraevceurrinsoers(Sm.igPhietneknntye,rJt.heZipeagtlhe-r Plant Washi 6anOdMCT. jhapoomniocalog(1s07fr,o1m21T,h1a8li2c)t.ruAmddtiutiboenroa-l &tion)W. .ABsreacnodntd, Om-amnuestchryilpttranisnferapsreepathraa-t v. y eb sum were functionally characterized and ex- acts on reticuline and catalyzes the forma- R u. hibit a broader substrate specificity that in- tion of laudanine was previously isolated n n A ←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−− Figure2 Biosynthesisofthebenzylisoquinolinealkaloidsberberine,morphine,andsanguinarine.Enzymesfor whichcorrespondingmolecularcloneshavebeenisolatedareshowninbold.Abbreviations:4(cid:3)OMT, 3(cid:3)-hydroxy-N-methylcoclaurine4(cid:3)-O-methyltransferase;6OMT,norcoclaurine6-O-methyltransferase; 7OMT,reticuline7-O-methyltransferase;BBE,berberinebridgeenzyme;CFS,cheilanthifoline synthase;CNMT,coclaurineN-methyltransferase;CoOMT,columbamineO-methyltransferase;COR, codeinonereductase;Cyp719A1,canadinesynthase;Cyp719A2,stylopinesynthase;Cyp719B1, salutaridinesynthase;Cyp80A1,berbamuninesynthase;Cyp80B3,N-methylcoclaurine3(cid:3)-hydroxylase; DBOX,dihydrobenzophenanthridineoxidase;DRR,1,2-dehydroreticulinereductase;DRS, 1,2-dehydroreticulinesynthase;MSH,N-methylstylopine14-hydroxylase;NCS,norcoclaurinesynthase; P6H,protopine6-hydroxylase;SalAT,salutaridinol7-O-acetyltransferase;SalR,salutaridine:NADPH 7-oxidoreductase;SOMT,scoulerine9-O-methyltransferase;STOX,(S)-tetrahydroxyprotoberberine oxidase;TNMT,tetrahydroprotoberberinecis-N-methyltransferase;TYDC,tyrosinedecarboxylase. www.annualreviews.org • AlkaloidBiosynthesis 741 ANRV342-PP59-29 ARI 29March2008 2:13 from opium poppy (121). (R,S)-Reticuline niumcompounds.Subsequenthydroxylation 7-O-methyltransferase(7OMT)doesnotac- by(S)-cis-N-methylstylopine14-hydroxylase ceptN-demethylatedBIAsubstrates,butisac- yields protopine, which is further hydrox- tivetowardphenoliccompounds. ylated by protopine 6-hydroxylase to dihy- A major branch pathway that gives rise drosanguinarine.Bothenzymeshavebeende- to many BIA classes begins with the forma- tected in protopine alkaloid-containing cell tionof(S)-scoulerinebytheberberinebridge cultures, and their characterization suggests enzyme (BBE) (Figure 2). This enzyme has that they are P450 monooxygenases (132, beenclonedfromseveralsources(28,36,138) 156). Dihydrobenzophenanthridine oxidase, and was recently characterized thoroughly which converts dihydrosanguinarine to san- (174, 175). BBE belongs to a novel family guinarine, has been purified from Sanguia- offlavoproteinsthatpossesstwocovalentat- nariacanadensis(7). tachmentsitesforflavinadeninedinucleotide Analternativebranchforthemetabolism org (FAD); one is histidine and the other is cys- of(S)-scoulerineinsomespeciesinvolvesthe ws. teine. The cysteinylation of the cofactor in- formationof(S)-tetrahydrocolumbamineby e alrevionly. chriegahseerstthheanmitdhpaotinotbrseedrovexdpoftoerntoiathlteoraflvaavluoe- s(Fcoiguulerreine2)9-(O15-m5)e.thTylhterancsDfeNraAse e(SnOcoMdiTng) nnuuse proteins,therebyfacilitatinghydrideabstrac- SOMT from C. japonica was the first re- ww.aonal tionof(S)-reticuline.Thissteprepresentsthe ported clone for an O-methyltransferase m wpers first half reaction toward the conversion to specifically implicated in BIA metabolism. oaded fro1/11. For (zfSoorp)m-hseactnoiuaonlnetrhiornifed.itnweToahlekmaelbotihiodysslyeinsnteihndeiistoiiaxstyeodbfbriybdetghnee-s AecoxlhlluiObm-itbmacemothninysieldtreiasrnacsbofleenrvahesoretmseidonltotohgeyc.oBTlIueAmtrpbaahatmyhdwirnoaey-, wnl1/0 resulting in (S)-cheilanthifoline and (S)- which is methylated by columbamine 769. Dosity on 1 satlyylzoepdinbey(FPi4g5u0r-ede2p)e.nBdoetnhtrmeaocntiooonxsyagreenacsaet-s Opa-lmmeatthinyeltr(a1n0s6fe).raRseeco(mCobOinManTt )CotOoMyTielids 5-er and two cDNAs coding for stylopine syn- active only against protoberberine alkaloid 3v 9:7Uni thasehavebeenclonedfromE.californicaand substrates, such as scoulerine or tetrahy- 008.5State classified as Cyp719A2 and Cyp719A3 (67). drocolumbamine, but not against other Biol. 2ngton Bgiootshpereccifiocmitbyinfoanrtmpreothteyilnesnsdhiooxwythberisdagmeeforer-- BofIAademrievtahtyivleens.edTiohxeysubbrsiedqgueenits focarmtalaytzioend Plant Washi mchaetiiloann,thbifuotlinCeytpo71(S9A)-2styolonplyinec.oInnvecrotnstr(Sas)t-, bthyatcbaenlaodnignse tosynththeasCey,p7a19PA45fa0mielynz(y6m8)e. v. y eb Cyp719A3 also accepts compounds without Cyp719A1displayshighsubstratespecificity R u. a pre-existing methylenedioxy bridge. (S)- for tetrahydrocolumbamine and does not n An Stylopine is subsequently N-methylated to acceptcolumbamine.Assuch,aparallelpath (S)-cis-N-methylstylopine by tetrahydropro- toberberineviathiscolumbamineisunlikely. toberberine N-methyltransferase (TNMT). (S)-Canadine, or (S)-tetrahydroberberine On the basis of homology to CNMT, a as it is also called, is oxidized by either (S)- cDNA encoding TNMT has been isolated canadineoxidaseor(S)-tetrahydroberberine and functionally characterized from opium oxidase (STOX), which catalyze the same poppy(86).Theenzymeshowsanarrowsub- reaction but show substantially different strate range in that it converts only tetrahy- biochemicalproperties. droprotoberberine alkaloids with dimethoxy Whereasallpathwaysdownstreamofreti- ormethylenedioxyfunctionalgroupsatC2/3 culinebeginwiththe(S)-epimer,conversion and C9/10, respectively. TNMT is also one to the (R)-epimer of reticuline is a required of only a few plant enzymes able to cat- entrystepintothemorphinanalkaloidbiosyn- alyze the formation of quaternary ammo- theticpathway(Figure2).Theepimerization · 742 Ziegler Facchini ANRV342-PP59-29 ARI 29March2008 2:13 of reticuline is a two-step process that in- morphineconsistoftwodemethylationsand volvestheoxidationof(S)-reticulineby1,2- onereduction.Boththedemethylationfrom dehydroreticulinesynthaseandthereduction thebaine to neopinone, which isomerises to SDR: shortchain of 1,2-dehydroreticuline to (R)-reticuline. codeinone,andfromcodeinetomorphineare dehydrogenases/ Bothstepshavebeencharacterizedbiochem- notyetunderstoodandnoenzymescapableof reductases icallyandtheenzymeshavebeenpartiallypu- catalyzingeitherreactionhavebeendetected. Calystegines: rified(23,63).Intramolecularcarbon-carbon Codeinone reductase has been purified and nortropanealkaloids phenol coupling between C2 of the benzyl cloned from opium poppy, and, in contrast thoughttoserveas and C4a of the isochinoline moiety leads to SalR, belongs to the aldo-keto reductase nutritionalsources forsoil to the formation of salutaridine. This en- (AKR)family(83,160). microorganisms zyme belongs to the P450 monooxygenase family (48). The gene encoding salutaridine Tropane alkaloids and nicotine. Tropane synthase (SalSyn) was recently cloned from alkaloids are an important class of plant- org opiumpoppyonthebasisofitshigherexpres- derived anticholinergic compounds, such as ws. sioninmorphine-containingPapaverspecies hyoscyamine and scopolamine, that occur e alrevionly. aanctdertihzeeden(Azy.mGeeswealls, tFh.Cen. Hfuunacntigo,nJa.llZyicehgalerr- isnpecseievse.raClalHysytoesgcyianmesu,s,whAitcrhopfau,nacntidonDaastusrea- nnuuse &T.M.Kutchan,manuscriptinpreparation). lectiveglucosidaseinhibitors,aremorewidely ww.aonal The SalSyn protein shows high homology spreadthanhyoscyamineandscopolaminein m wpers tothemethylenedioxybridge-formingP450- the plant kingdom and occur mainly in the oaded fro1/11. For dCcaeytpapel7yn1zd9eedBn1tb.yeTnshazleyumtnaeersxi,dtisantneedpreiwdnuatschtaecslepasa(stSihfiawelRday);aiass Staicontlesa,notahnceenaaiecctoiavtniendpicCrianoccneivptoylellvciuhnloaNlcineicaeoetria(e3nc0ea)p.stpoNercsicieotso-, wnl1/0 cognate cDNA was obtained via the same cause a variety of physiological effects, in- Don 1 approach used for SalSyn (183). Functional cludingaddiction.Tropanealkaloidandnico- 769. sity o characterization of the recombinant enzyme tine biosynthesis begin with the methyla- 35-ver showed the stereospecific reduction of the tion of putrescine to N-methylputrescine 9:7Uni ketogroupto7(S)-salutaridinol,whichisalso by putrescine N-methyltransferase (PMT) 008.5State reportedforthepurifiedenzymefromopium (Figure3). The isolation of the cDNA en- Biol. 2ngton piloypopfysh(4o9r)t.cThhaeinednezhymyderobgeelonnagsesst/oretdhuecftaamse-s cfiorsdtinegxaPmMplTesfroofmthteobsuaccccoesswfuasl ionnteegoraftitohne Plant Washi (tShDisRf)a,mbiluyt iutnleixkheibmitasnya ohtihgehrerenmzyomleecsulianr oasf amesttarabtoelgitye taondisogleantee ceDxpNreAsssioinmpplriocafitleeds v. y eb weightandismonomeric.Thestereospecific in plant secondary metabolite biosynthe- R u. reduction of salutaridine is required for the sis (61). Homologous cDNAs from other n An next step, which is catalyzed by salutaridi- plants that produce tropane alkaloids, such nol 7-O-acetyltransferase (SalAT). This en- as Hyoscyamus niger, Atropa belladonna, and zyme specifically acetylates the 7(S)-epimer Solanum tuberosum, have also been isolated of salutaridinol to salutaridinol-7-O-acetate (147, 153, 157). The second step in the (82). With considerable sequence homology pathway is the oxidative deamination of N- to the acetylating enzymes from the MIA methylputrescine to 4-methylaminobutanal pathway, SalAT also belongs to the BAHD byN-methylputrescineoxidase(MPO).This family of acetyltransferases (52). The in- enzyme belongs to a class of amine ox- troduced acetyl group is eliminated sponta- idases that require copper as a cofactor. neously,leadingtotheformationofanoxide This property was exploited in a homology- bridgebetweenC-4andC-5toyieldthebaine, based cloning strategy to isolate the MPO the first pentacyclic alkaloid of the pathway cDNAfromtobacco(59).Thecentralinter- (82).Thefinalstepstowardthebiosynthesisof mediate N-methyl-(cid:3)1-pyrrolium cation for www.annualreviews.org • AlkaloidBiosynthesis 743 ANRV342-PP59-29 ARI 29March2008 2:13 ODC Putrescine Ornithine PMT Arginine NAD biosynthesis N-Methylputrescine intermediate 3,6-Dihydronicotinone MPO Spontaneous g 4-Methylaminobutanal N-Methyl-∆1-pyrrolium or cation Nicotine s. w Figure3 alrevieonly. Btrioopsaynnethaelskiaslooifdtshe Cyp82E4 nnuuse hyoscyamineand TR-II w.aal scopolamine,the won calystegines,and m wpers nicotine. Hygrine Tropinone Pseudotropine Nornicotine oaded fro1/11. For Mhfoarovealelblcueeenlanzryicmslooelnasteesd TR-I wnl1/0 shown. Don 1 Abbreviations: 9. y o Cyp80F1,littorine 76sit mutase/ 35-ver monooxygenase; Tropine Littorine 9:7Uni Cyp82E4,nicotine 008.5State NH-6dHem,heythosyclaysaem;ine Cyp80F1 Biol. 2ngton 6MβP-hOy,droxylase; Plant Washi moxeidthayselp;uOtrDesCc,ine Calystegine A3 v. y ornithine Hyoscyamine Hyoscyamine aldehyde eb R decarboxylase; nu. PMT,putrescine H6H n A N- methyltransferase; TR-I,tropinone Calystegine B1 Calystegine B2 reductaseI;TR-II, tropinone reductaseII. Scopolamine tropane alkaloid and nicotine biosynthesis NicotinecanbeN-demethylatedtoformnor- results from the spontaneous cyclization of nicotine, which is an undesirable derivative 4-methylaminobutanal. For nicotine, the owingtoitsroleastheprecursorofthecar- cationiscondensedwithnicotinicacidtoform cinogenN(cid:3)-nitrosonornicotine.Onthebasis 3,6-dihydronicotine,whichsubsequentlyun- of the differential abundance of transcripts dergoes dehydrogenation to nicotine by en- corresponding to several P450-encoding zymes that remain poorly characterized. cDNAs in tobacco varieties acccumulating · 744 Ziegler Facchini