ALKALOID BIOSYNTHESIS IN PLANTS Biochemistry, Cell Biology

38 Pages·2001·0.28 MB·English

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Annu.Rev.PlantPhysiol.PlantMol.Biol.2001.52:29–66
Copyright(cid:176)c 2001byAnnualReviews.Allrightsreserved
A B P :
LKALOID IOSYNTHESIS IN LANTS
Biochemistry, Cell Biology, Molecular
Regulation, and Metabolic Engineering
Applications
Peter J Facchini
DepartmentofBiologicalSciences,UniversityofCalgary,Calgary,AlbertaT2N1N4,
Canada;e-mail: [email protected]
KeyWords metabolicengineering,generegulation,secondarymetabolism,
signaltransduction,subcellularcompartmentation
n Abstract Recentadvancesinthecell,developmental,andmolecularbiologyof
alkaloidbiosynthesishaveheightenedourappreciationforthecomplexityandimpor-
tance of plant secondary pathways. Several biosynthetic genes involved in the for-
mationoftropane,benzylisoquinoline,andterpenoidindolealkaloidshavenowbeen
isolated. Theearlyeventsofsignalperception, thepathwaysofsignaltransduction,
andthefunctionofgenepromotershavebeenstudiedinrelationtotheregulationof
alkaloidmetabolism. Enzymesinvolvedinalkaloidbiosynthesisareassociatedwith
diversesubcellularcompartmentsincludingthecytosol,vacuole,tonoplastmembrane,
endoplasmicreticulum,chloroplaststroma,thylakoidmembranes,andperhapsunique
“biosynthetic”ortransportvesicles. Localizationstudieshaveshownthatsequential
alkaloid biosynthetic enzymes can also occur in distinct cell types, suggesting the
intercellulartransportofpathwayintermediates. Isolatedgeneshavealsobeenused
to genetically alter the accumulation of specific alkaloids and other plant secondary
metabolites. Metabolicmodificationsincludeincreasedindolealkaloidlevels,altered
tropanealkaloidaccumulation,elevatedserotoninsynthesis,reducedindoleglucosi-
nolate production, redirected shikimate metabolism, and increased cell wall–bound
tyramineformation. Thisreviewdiscussesthebiochemistry,cellbiology,molecular
regulation,andmetabolicengineeringofalkaloidbiosynthesisinplants.
CONTENTS
INTRODUCTION :::::::::::::::::::::::::::::::::::::::::::::::: 30
BIOCHEMISTRYANDCELLBIOLOGYOFALKALOIDPATHWAYS:::::::: 31
TerpenoidIndoleAlkaloids:::::::::::::::::::::::::::::::::::::::: 31
BenzylisoquinolineAlkaloids :::::::::::::::::::::::::::::::::::::: 36
TropaneAlkaloidsandNicotine :::::::::::::::::::::::::::::::::::: 41
PurineAlkaloids ::::::::::::::::::::::::::::::::::::::::::::::: 43
1040-2519/01/0601-0029$14.00 29
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SubcellularCompartmentationofAlkaloidBiosyntheticEnzymes :::::::::::: 43
REGULATIONOFALKALOIDBIOSYNTHETICGENES:::::::::::::::::: 45
DevelopmentalRegulationandTissue-SpecificLocalization :::::::::::::::: 45
SignalTransductionandInducibleExpression :::::::::::::::::::::::::: 47
PromoterAnalysis :::::::::::::::::::::::::::::::::::::::::::::: 49
METABOLICENGINEERINGAPPLICATIONS ::::::::::::::::::::::::: 51
TerpenoidIndoleAlkaloids:::::::::::::::::::::::::::::::::::::::: 51
TropaneAlkaloids,Nicotine,andOtherPolyamineDerivatives :::::::::::::: 53
OtherAlkaloids :::::::::::::::::::::::::::::::::::::::::::::::: 53
OtherUsesofAlkaloidBiosyntheticGenesinGeneticEngineering ::::::::::: 54
FUTUREPROSPECTS :::::::::::::::::::::::::::::::::::::::::::: 55
INTRODUCTION
Alkaloidsareadiversegroupoflow-molecular-weight,nitrogen-containingcom-
poundsfoundinabout20%ofplantspecies. Manyofthe»12,000alkaloidsfor
which structures have been described function in the defense of plants against
herbivoresandpathogens(19,178). Thepotentbiologicalactivityofsomealka-
loids has also led to their exploitation as pharmaceuticals, stimulants, narcotics,
and poisons. Plant-derived alkaloids currently in clinical use include the anal-
gesicsmorphineandcodeine,theanticanceragentsvinblastineandtaxol,thegout
suppressant colchicine, the muscle relaxant (C)-tubocurarine, the antiarrythmic
ajmaline,theantibioticsanguinarine,andthesedativescopolamine. Otherimpor-
tantalkaloidsofplantoriginincludecaffeine,nicotine,cocaine,andthesynthetic
O,O-acetylatedmorphinederivativeheroin.
Research in the field of plant alkaloid biochemistry began with the isolation
ofmorphinein1806. Remarkably,thestructureofmorphinewasnotelucidated
until1952owingtothestereochemicalcomplexityofthemolecule. Sincethen,
threemajortechnicaladvanceshaveledtosubstantialprogressinourunderstand-
ing of plant alkaloid formation. The first was the introduction in the 1950s of
radiolabeledprecursorsthatallowedthechemicalelucidationofalkaloidbiosyn-
theticpathways. Thesecondinvolvedtheincreaseduseduringthe1970sofplant
cell cultures as an abundant source of biosynthetic enzymes that could be iso-
lated,purified,andcharacterized. Finally,thewidespreadapplicationinthe1990s
of molecular techniques to the alkaloid field facilitated the isolation of several
genes involved in indole, tropane, and benzylisoquinoline alkaloid biosynthesis
(Table1). Theearlyeventsofsignalperception,thepathwaysofsignaltransduc-
tion,andthefunctionofgenepromotershavesincebeeninvestigatedinrelation
totheregulationofalkaloidmetabolism. Tissue-specificlocalizationstudieshave
shownthatsequentialbiosyntheticenzymescanoccurindistinctcelltypes. The
predicted translocation of pathway intermediates between cells further demon-
stratestheintricatecellbiologyofalkaloidbiosynthesis.Isolatedgeneshavealso
been used to genetically engineer the accumulation of alkaloids and other sec-
ondarymetabolitesinplants. Inthisreview,recentadvancesinthebiochemistry,
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ALKALOIDBIOSYNTHESISINPLANTS 31
cell biology, molecular regulation, and metabolic engineering of plant alkaloid
pathwaysarediscussed.
BIOCHEMISTRYANDCELLBIOLOGY
OFALKALOIDPATHWAYS
TerpenoidIndoleAlkaloids
Terpenoid indole alkaloids (TIAs) comprise a family of »3000 compounds that
includestheantineoplasticagentsvinblastineandcamptothecin,theantimalarial
drug quinine, and the rat poison strychnine. Some TIAs have been proposed to
playaroleinthedefenseofplantsagainstpestsandpathogens(93). TIAsconsist
of an indole moiety provided by tryptamine and a terpenoid component derived
fromtheiridoidglucosidesecologanin. Tryptophanisconvertedtotryptamineby
tryptophandecarboxylase(TDC;Figure1),whichisencodedbyasinglegenein
Catharanthusroseus(31,58)andbytwoautonomouslyregulatedgenesinCamp-
totheca acuminata (91). The C. roseus TDC gene exhibits both developmental
andinducibleregulation. Incontrast,C.acuminataTDC1isexpressedintissues
containinghighlevelsofcamptothecinincludingtheshootapexandbark,butthe
geneisnotinducedinresponsetoelicitortreatment. However,TDC2isinducedin
elicitor-treatedC.acuminatacellcultures,butisnotdevelopmentallyexpressed.
ThedifferentialregulationofTDCgenesinC.acuminatasuggeststhatonepartici-
patesinadevelopmentallycontrolleddefensepathway,whiletheotherisinvolved
inaninducibledefensemechanism.
The first committed step in secologanin biosynthesis is the hydroxylation of
geraniolto10-hydroxygeraniol. Theenzymegeraniol10-hydroxylase(G10H)was
characterizedasaP450monooxygenasebecauseitismembranebound,dependent
on NADPH and O , and displays light-reversible CO inhibition (106). G10H is
2
specificfortheC-10positionandexhibitssimilaraffinityforgeraniolandnerol,
the cis-isomer of geraniol. The conversion of loganin to secologanin represents
the last step in the pathway and is also catalyzed by a P450-dependent enzyme
(180). The production of terpenoid precursors might play a regulatory role in
TIA biosynthesis since the addition of secologanin or loganin to C. roseus cell
culturesincreasesalkaloidaccumulation(111,113). Moreover,thelevelofG10H
activity positively correlates with the accumulation of alkaloids when C. roseus
cell cultures are transferred to alkaloid production medium (148). The enzyme
3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), which is involved
in the biosynthesis of mevalonate, was cloned and characterized from C. roseus
(97) and C. acuminata (16,99). The differential expression of HMGR genes in
responsetowoundingandmethyljasmonate(MeJA)wassuggestedtocontribute
totheregulationofTIAbiosynthesis. However,secologaninwasrecentlyshownto
bederivedfromthetriosephosphate/pyruvatepathway(22);thus,thecorrelation
betweenHMGRexpressionandTIAaccumulationislikelycoincidental.
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e
c
n
e
r
e
Ref 31 91 104 84 55 151 176 164 38 114 114 49
e e
e-ase ent e-ase ne-ansferas ne-ansferas ne-nsferase
ncloned Type 0Pyridoxal-5-phospahatdependentdecarboxyl Vacuolarglycoprotein Membrane-associatedglucosidase P450-dependentmonooxygenase 2-Oxoglutarate-dependdioxygenase AcetylCoA-dependentacetyltransferase 0-phospahatPyridoxal-5dependentdecarboxyl -methioniS-Adenosyl-LdependentO-methyltr S-Adenosyl--methioniLdependentO-methyltr -methioniS-Adenosyl-LdependentO-methyltra
e
e
b
e
av a
correspondinggenesh Species Catharanthusroseus Camptothecaacuminat Catharanthusroseus Rauwolfiaserpentina Catharanthusroseus Catharanthusroseus Catharanthusroseus Catharanthusroseus Papaversomniferum Coptisjaponica Coptisjaponica Thalictrumtuberosum
e
h
oidbiosyntheticenzymesforwhicht Function dolealkaloidbiosynthesisTryptophandecarboxylase Strictosidinesynthase (cid:12)Strictosidine- -glucosidaseD Tabersonine16-hydroxylase Desacetoxyvindoline4-hydroxylase Deacetylvindolineacetyltransferase ealkaloidbiosynthesisTyrosinedecarboxylase Norcoclaurine6-O-methyltransferase03-Hydroxy-N-methylcoclaurine04-O-methyltransferase Norcoclaurine6-O-methyltransferase
Alkal noidin uinolin 1-4
TABLE1 Enzyme MonoterpeTDC STR SGD T16H D4H DAT BenzylisoqTYDC 6OMT 0OMT4 OMTII;
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ALKALOIDBIOSYNTHESISINPLANTS 33
2
8
1
1 33 2, 4 1 1 71 73 12 67 1 19 19 01 68
8 1 7 3 4 2 1 1 1 1 7 1 1 1 1 –
e e e
as as as
P450-dependentmonooxygenase P450-dependentmonooxygenase Flavinylatedoxidoreductase -methionine-S-Adenosyl-LdependentO-methyltransferAldo/ketoreductase 0Pyridoxal-5-phospahate-dependentdecarboxylaseS-Adenosyl--methionine-LdependentN-methyltransfer Short-chaindehydrogense Short-chaindehydrogenase 2-Oxoglutarate-dependentdioxygenase S-Adenosyl--methionine-LdependentN-methyltransfer
a a
c c
Berberisstolonifera Eschscholziacaliforni Papaversomniferum Eschscholziacaliforni Papaversomniferum Berberisstolonifera Coptisjaponica Papaversomniferum Daturastamonium Atropabelladonna Nicotinatabacum Daturastramonium Daturastramonium Hyoscyamusniger Atropabelladonna Camelliasinensis
Berbamuninesynthase 0(cid:13)-N-Methylcoclaurine3-hydroxylase Berberinebridgeenzyme Scoulerine9-O-methyltransferaseCodeinonereductase dandnicotinebiosynthesisOrnithinedecarboxylase PutrescineN-methyltransferase Tropinonereductase-I Tropinonereductase-II Hyoscyamine(cid:12)6-hydroxylase biosynthesisCaffeinesynthase
YP80A1 YP80B1 BE OMT OR anealkaloiDC MT R-I R-II 6H nealkaloidS
C C B S C TropO P T T H PuriC
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Figure 1 Reactions catalyzed by enzymes involved in monoterpenoid indole alkaloid
biosynthesisforwhichthecorrespondinggeneshavebeencloned. TDC,tryptophandecar-
boxylase;STR,strictosidinesynthase;SGD,strictosidinefl-D-glucosidase;T16H,taber-
sonine16-hydroxylase;D4H,desacetoxyvindoline4-hydroxylase;DAT,deacetylvindoline
4-O-acetyltransferase.
Tryptamineandsecologaninarecondensedbystrictosidinesynthase(STR)to
formstrictosidine,thecommonprecursortoallTIAs(Figure1). STRcDNAshave
beenisolatedfromRauvolfiaserpentina(84)andC.roseus(104,132). Theenzyme
isencodedbyasinglegeneinC.roseus,indicatingthatthemultipleSTRisoforms
reportedpreviouslyresultfromposttranslationalmodificationofasingleprecursor
(132). Strictosidine is deglucosylated by strictosidine fl-D-glucosidase (SGD),
which has been purified from C. roseus cell cultures (92). The native enzyme
exhibitsahighmolecularmass,suggestingthatitexistsasanaggregatecomposed
ofmultiple63-kDasubunits. DigestionofSGDwithtrypsincausesthecomplexto
disintegrate,solubilizingtheenzymewithoutlossofactivity. SGDisencodedbya
singlegeneinC.roseusthatshares»60%homologywithotherplantglucosidases
(55). Deglucosylatedstrictosidineisconvertedviaseveralunstableintermediates
to 4,21-dehydrogeissoschizine. Although several TIAs are produced from 4,21-
dehydrogeissoschizine, few of the enzymes involved have been isolated (108).
For example, none of the enzymes leading to catharanthine has been described.
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ALKALOIDBIOSYNTHESISINPLANTS 35
However, the biosynthesis of vindoline has been characterized in considerable
detail. Vindolineisultimatelycoupledtocatharanthinebyanonspecificperoxidase
toyieldvinblastine(157).
Thefirstofsixstepsinvolvedintheconversionoftabersoninetovindolinecon-
sistsofhydroxylationattheC-16positionbytabersonine16-hydroxylase(T16H;
Figure1),whichwasdetectedintotalproteinextractsofyoungC.roseusleaves
(163). BasedonitsrequirementforNADPHandO ,anditsinhibitionbyCO,cy-
2
tochromec,andspecificinhibitors,T16HwascharacterizedasaP450-dependent
monooxygenase. AT16HcDNAwasisolatedfromC.roseuscellculturesusing
a cloning strategy based on the activation of the enzyme by light (151). Sev-
eral P450 sequences were amplified by polymerase chain reaction (PCR), using
degenerate primers specific to the conserved heme-binding domain. The PCR
products were hybridized to RNA from induced and noninduced cells, and one
showedinductionkineticsconsistentwithT16H.TheisolatedcDNA,encodinga
P450homologuedesignatedCYP71D12,wasexpressedinEscherichiacoliasa
translationalfusionwithcytochromeP450reductase(CPR)fromC.roseus(107).
CYP71D12wasidentifiedasT16Hbasedonitsabilitytoconverttabersonineto
16-methoxytabersonine. GenomicDNAhybridizationanalysessuggestthepres-
ence of at least two T16H genes (151), but only a single copy of the CPR gene
(90),inC.roseus.
Subsequent to the 16-hydroxylation of tabersonine, the next three steps in
vindoline biosynthesis are now accepted as 16-O-methylation, hydration of the
2,3-double bond, and N-methylation of the indole-ring nitrogen (8,27,28). An
S-adenosyl-L-methionine(SAM)-dependentO-methyltransferase(OMT)hasbeen
reportedthatmethylates16-O-demethyl-4-O-deacetylvindoline(45). Initially,two
consecutive hydroxylations at the C-3 and C-4 positions were proposed to fol-
lowthe16-hydroxylationoftabersonine(45).However,theisolationofaSAM-
dependent N-methyltransferase (NMT) specific for the indole-ring nitrogen of
16-methoxy-2,3-dihydro-3-hydroxytabersonine indicated that theO-methylation
stepprecedesN-methylation, andthat16-hydroxytabersonineisthenaturalsub-
strateoftheOMT(28,33). Theenzymeinvolvedinhydratingthe2,3-doublebond
hasnotbeenisolated.
The second-to-last step in vindoline biosynthesis is catalyzed by a 2-
oxoglutarate-dependent dioxygenase that hydroxylates the C-4 position of de-
sacetoxyvindoline (D4H; Figure 1) (23). The enzyme requires ferrous ions and
ascorbate, occurs as three unique charge isoforms, and exhibits an “ordered ter
ter” mechanism with 2-oxoglutarate binding first, followed by O and desace-
2
toxyvindoline (24). Degenerate primers, designed from amino acid sequences
derivedfromthepurifiedprotein,wereusedtoisolatecDNAandgenomicclones
encodingD4H(174). TwodifferentcDNAswereisolated,representingdimorphic
allelesofasingle-copygene.
The final step in vindoline biosynthesis is catalyzed by acetylcoenzyme A:
deacetylvindoline 4-O-acetyltransferase (DAT; Figure 1) (26,44). The purified
enzymeisstronglyinhibitedbytabersonineandcoenzymeA(50%inhibitionat
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36 FACCHINI
45 „M and 37 „M, respectively), and weakly inhibited by tryptamine, secolo-
ganin, and vindoline (28%, 25%, and 40% inhibition, respectively, at 500„M),
suggesting that DAT activity is modulated by pathway precursors and products
(135). The original purification of DAT led to the incorrect conclusion that the
enzymeconsistsoftwosubunitswithmolecularweightsof33and21kDa. How-
ever, the isolated DAT gene encodes a 50-kDa polypeptide, suggesting that the
proteinwascleavedasanartifactofpurification(164). Moreover,theproteinthat
cross-reactswithanti-DATantibodyinseedlingsandleavesalsohasamolecular
weightof50kDa(164).
BenzylisoquinolineAlkaloids
Benzylisoquinolinealkaloids(BIAs)arealargeanddiversealkaloidgroupwith
»2500definedstructures. ThepharmacologicalactivityofBIAsrendersmanyof
themusefulaspharmaceuticalsandisoftenacluetotheirbiologicalroleintheplant
(19). Forexample,theeffectivenessofmorphineasananalgesic,colchicineasa
microtubuledisrupter,and(C)-tubocurarineasaneuromuscularblockersuggests
thatthesealkaloidsfunctionasherbivoredeterrents. Theantimicrobialproperties
of sanguinarine suggest that it confers protection against pathogens. The BIAs
berberine,sanguinarine,andpalmatinewerespecificallyshowntoconferprotec-
tionagainstherbivoresandpathogens(149).
BIA biosynthesis begins with a metabolic lattice of decarboxylations, ortho-
hydroxylations, anddeaminationsthatconverttyrosinetobothdopamineand4-
hydroxyphenylacetaldehyde(142). Theonlyenzymeinvolvedintheseearlysteps
that has been purified (100), and for which the corresponding cDNA has been
cloned(38,98),isthearomaticL-aminoaciddecarboxylase(TYDC)thatconverts
tyrosine and dopa to their corresponding amines (Figure 2). TYDC is encoded
by a family of »15 genes in Papaver somniferum (opium poppy) that can be
dividedintotwosubgroupsbasedonsequenceidentity(38). Althoughthecatalytic
properties of the isoforms are similar, each TYDC subfamily exhibits a distinct
developmental and inducible expression pattern (38,40). TYDC cDNAs have
alsobeenreportedfromparsley(80)andArabidopsisthaliana(172),whichdonot
accumulatetyrosine-derivedalkaloids. TYDCmRNAswereshowntoberapidly
induced in response to elicitor treatment (40,80,172) and pathogen challenge
(150)invariousplants. InductionofTYDCmRNAsinparsleyandArabidopsis
suggests that tyramine serves as the precursor to a ubiquitous class of defense-
responsemetabolites,inadditiontoBIAs. Recentstudiessuggestthatthesynthesis
and deposition in the cell wall of amides, composed of hydroxycinnamic acid-
derivativesandtyramine,iscentraltothedefense-responseofmanyplants(105).
Amides,togetherwithotherphenolics,arebelievedtoreducecellwalldigestibility.
ThedualroleoftyramineasaprecursorforBIAandhydroxycinnamicacidamide
biosynthesissuggeststhattheTYDCgenefamilyinopiumpoppyencodesTYDC
isoformswithdiversemetabolicroles.
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Dopamine and 4-hydroxyphenylacetaldehyde are condensed by norco-
claurine synthase (NCS) to yield the trihydroxybenzylisoquinoline alkaloid (S)-
norcoclaurine, which is the central precursor to all BIAs in plants (Figure 2)
(158,159). DuetotheinabilityofNCStodiscriminatebetween4-hydroxyphenyl-
acetaldehydeand3,4-dihydroxyphenylacetaldehyde,andthenonspecificityofthe
Figure 2 Reactions catalyzed by enzymes involved in benzylisoquinoline alkaloid biosyn-
thesis for which the corresponding genes have been cloned. TYDC, tyrosine/dopa decar-
0 0
boxylase;6OMT,norcoclaurine6-O-methyltransferase;4OMT,3-hydroxy-N-methylcoclaurine
0
4-O-methyltransferase;OMTII-1,O-methyltransferaseII-1;CYP80A1,berbamuninesynthase,
0
CYP80B1, (S)-N-methylcoclaurine 3-hydroxylase; BBE, berberine bridge enzyme; SOMT,
scoulerineN-methyltransferase;COR,codeinonereductase.
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subsequentmethyltransferasereactions,itwasoriginallythoughtthatthetetrahy-
droxybenzylisoquinolinealkaloid(S)-norlaudanosolinewastheprecursortoBIAs
(141). However,onlynorcoclaurinehasbeenfoundtooccurinplants.
(S)-Norcoclaurineisconvertedto(S)-reticulinebya6-O-methyltransferase(49,
147),anN-methyltransferase(47),aP450hydroxylase(133),anda40-O-methyl-
transferase (48,147). The SAM-dependent 6-O- and 40-O-methyltransferases
(6OMTand40OMT,respectively)havebeenpurifiedfromculturedCoptisjapon-
ica cells (147), and the corresponding cDNAs isolated and characterized (114).
Althoughthetwoenzymesdisplaysimilarenzymologicalproperties,theyexhibit
distinctsubstratespecificities.Moreover, the6OMTfollowsa“ping-pongbibi”
mechanism,whereasthe40OMTcatalyzesan“orderedbibi”reaction(114). Four
homologous O-methyltransferase cDNAs (OMT II;1-4) have also been isolated
from MeJA-treated Thalictrum tuberosum cell cultures (49). Heterologous ex-
pression of the OMT II;1-4 cDNAs showed that homodimers and various het-
erdimeric combinations of the four isoforms exhibit broad substrate specificity.
The O-methylated substrates included simple catechols, phenylpropanoids, and
various BIAs, suggesting that some of the isoforms are involved in both BIA
andphenylpropanoidmetabolism. Forexample,thehomodimerofOMTII;1ef-
ficiently O-methylates (R,S)-norcoclaurine (Figure 2) and various catechol and
caffeic acid derivatives. Remarkably, OMT II;4 differs from OMT II;1 by only
one amino acid, but its homodimer does not catalyze the alkaloid methylations.
Boththe6OMTand40OMTfromC.japonicashowrelativelylowidentity(24and
35%, respectively) to the various catechol OMT II isoforms (114). The in vivo
contribution, if any, of the OMT II enzymes to BIA biosynthesis remains to be
established.
Originally,thearomatic-ringhydroxylationinvolvedintheconversionof(S)-
norcoclaurineto(S)-reticulinewasthoughttobecatalyzedbyanonspecificphenol
oxidase(89). However,aP450-dependentmonooxygenase(CYP80B1;Figure2)
isolatedfromEschscholziacalifornica(133)andopiumpoppy(72,182)exhibits
a K for (S)-N-methylcoclaurine 39-fold lower than that of the phenolase; thus,
m
CYP80B1isnowknowntoconvert(S)-N-methylcoclaurineto(S)-30-hydroxy-N-
methylcoclaurine. CYP80B1isencodedbytwotothreegenesinE.californica
(133)andopiumpoppy(72,182).
Intermediates of the (S)-reticuline pathway also serve as the precursors
to »270 dimeric bisbenzylisoquinoline alkaloids such as berbamunine and
(C)-tubocurarine. A phenol-coupling P450-dependent oxidase berbamunine
synthase(CYP80A1)hasbeenpurified(160),andthecorrespondingcDNAiso-
lated(81),fromBerberisstolonifera. CYP80A1couplestwomoleculesof(R)-N-
methylcoclaurineoroneeachof(R)-and(S)-N-methylcoclaurinebyanetherlink-
agetoform(R,R)-guattegaumerineor(R,S)-berbamunine,respectively(Figure2).
Additional variations in bisbenzylisoquinoline alkaloid structure include phenyl
ringsubstitutions,thenumberofetherlinkages,andregio-andstereoselectionof
monomers. CPRhasalsobeenpurifiedfromopiumpoppy,andthecorresponding
cDNAs isolated from opium poppy and E. californica (139). The CPR proteins