Alkaloid Biosynthesis: Metabolism and Trafficking - Washington

38 Pages·2011·1.52 MB·English

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