alkaloid bio synthesis

8/3/2019 Alkaloid Bio Synthesis

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The Plant Cell, Vol. 7, 1059-1070, uly 1995 O 1995 American Society of Plant Physiologists

Alkaloid Biosynthesis -The Basis for Metabolic Engineeringof Medicinal PlantsToni M. KutchanLaboratorium fr Molekulare Biologie, Universitat Mnchen, K arlstrasse 29, 80333 Munich, Germany

INTRODUCTION

Alkaloids-the term is linguistically derived from the Arabicword a/-qali, he p lant from wh ich soda was first obtained-are nitrogenous compounds that constitute the pharmaco log-ically active basic principles of predominan tly, although notexclusively, lowering plants. Since the identification of the firstalkaloid, morphine, from the opium poppy, Papaver somnife-rum, by Sertrner n 1806, ulO,OOO lkaloids have been isolatedand their structures elucidated (Southon and Buckingham,1989). istorically, he use of alkaloid -contain ing plant extractsas potions, medicines, and poisons can b e traced back almostto the start of civilization. Famed examples include Socratesdeath in 399 B.C. by consumption of co niine-containing hem-lock (Conium maculatum) and Cleopatras use durin g the lastcentury B.C. of atropine-containing extracts of Egyptian hen-bane (Hyoscyamusmuficus) to dilate her pupils an d therebyappear more alluring. Medieval European women u tilized ex-tracts of dea dly nightshade, Atropa belladonna, for the samepurpose, hence the nam e belladonna. Although co niine is tootoxic to find therapeutic use today outside of homeopathy,tropicamide, an an ticholinergic that is a synthetic derivativeof atropine, is routinely used in eye exam inations to dilate thepupil. Tropicamide has also recently shown promise as an earlydiagnostic tool in th e d etection of Alzheime rs disease (Scintoet al., 1994).A tonic prepared from the bark of Cinchonaofficinalis that contains the antimalarial drug q uinine g reatlyfacilitated Eu ropean exploration and inha bitation of the trop-ics during the past two centuries.In otal, -~13,000lant species are known o have been usedas drugs througho ut he world (Tyler, 1994). pproximately 25%of contemporary ma teria me dica is derived from plants andused either as pure compoun ds (such as the narcotic analge-sic mo rphine, the an algesic and an titussive codeine, and thechemotherape utic agents vincristine and vinblastine; Figure1)or as teas a nd extracts. Plant constituents have also servedas mod els for m odern synthetic drugs, such as a tropine fortropicamide, quinine for chloroquine, and cocaine for procaineand tetracaine. In act, active plant extract screening programscontinue to result in new drug discoveries. The most recentexamples of anticancer alka loids are taxo1 (clinically a vailablesince 1994) rom th e w estern yew, Taxusbrevifolia, and camp -tothecin (and derivatives currently in clinical trials) from theChinese happy tree, xi shu (Camptotheca acuminata), both

of which were originally isolated and assayed for biologicalactivity in the 1960s n the laboratory of M.E. Wall. In otherareas, there are intense searches for nove1 antivirals andantimalarials.We also encounter alkaloids as the stimulants caffeine incoffee and tea an d nicotine in cigarettes. Although a wealthof information is available on the p harmacological effects ofthese compounds, surprisingly little is known about how plantssynthesize these substances, and almost nothing is knownabout how th is synthesis is regulated. This is due, in p art, tothe complex chemical structures of many alkaloids, which con-tain multiple asymmetric centers. For example, althoughnicotine (one asymm etric center) was discovered in 1828, tsstructure was not known un til it was synthesized in 1904, ndthe structure of morphine (five asymm etric centers) was notunequivocally elucidated un til 1952, 46 years after its isola-tion. Beginning n the late 1950s,adiolabeled precursors werefed to plants and the resultant radioactive alkaloids were chem -ically degraded o identify he position of the label. This openedthe field of alkaloid b iosynthesis to experimen tation. As ana-lytical nstrumentationbecame more sophisticated, precursorslabeled with stable isotopes were fed to plants and the prod -ucts analyzed by nuclear magnetic resonance spectroscopy.No real progress was made in iden tifying alkaloid biosyntheticenzymes un til the u se of plant cell cultures as experimentalsystems was introduced i n the 1970s.Since then, on the or-der of 80 ew enzymes that catalyze steps in he biosynthesisof the indole, isoquinoline, tropane, pyrrolizidine, and purin eclasses of alkaloids have been discovered and partiallycharacterized.Alkaloids belong to the broad category of secondary me-tabolites. This class of molecule ha s historically been definedas naturally occurring sub stances that are not vital to the or-ganism that produces them. Alkaloids have traditionally be enof interest only due to their p ronounced an d various physio-logical activities in animals and h umans. A picture has nowbegun to em erge that alkaloids do have important ecochem i-cal functions in the defense of the plant against pa thogenicorganisms a nd herbivores or, as in the case of pyrrolizidinealkaloids, as pro-toxins for insects, which further modifiy thealkaloids and then incorporate them into their own defensesecretions (reviewed n Hartmann, 1991).Alkaloids have now


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1060 The Plant Cell

HO :NCH,

Morphine, CodeinePapever somnllerum L

StrychnineStrychnos nux-vomica L.

ConilneCon/urn macula tum L.

MeIMNicotine

Nicotiana tabacum L.

OA c

TaxolTaxua brevi/olia N uttall

.. O C O C H ,| HO C O j C H jC H 3

VinblastineCatharanthus roaeua (L.)G.Don.

AjmalineRauvolf la serpentine (L.)Benth.

H i PilocarpinePilocarpus jaborand i Holmes

NxCH

|/ *A CHjOHO O C C HC H 2O H H

O O C C HC 6H S . C H 30

jfScopolamine, AtroplneHyoscyamus niger L.

. COOCH ,

NQuinine

Cinchona olficlnalls L

HCocaine

Erytnroxylcn coca Lam.

EmetineUragoga ipecacuanha (Brot.)Balll.

SanguinarlneEschacholtzla calllornica Cham.

(+)-Tubocura/)lChondodendmn tomentoaum R.&P.

CamptotheclnCamptotheca acumln t ta Descne.

Figure 1. Some Physiologically Active Alkaloids and Plants That Produce Them.


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Alkaloids and Their Biosynthesis 1061

been isolated from such diverse organisms as frogs, ants(pheromones), butterflies (defense), marine bacteria, sponges,fungi, spiders (venom neurotoxin), beetles (defense), and mam -mals, although is not yet clear whether de novo alkaloidbiosynthesis occurs in each organism.With the introduction of m olecular biology into the plant al-kaloid field, induction of alkaloid biosynthesis in response toexposure to wounding or to elicitors can be analyzed at theleve1 of gen e activation, an d gene expression patterns in theplant can be determ ined an d interpreted as a first indicationof possible function. We also now have the capability to alterthe pattern of alkaloid accumulation in plants for the purposeof studying the biological function of alkaloids, for engineer-ing tailor-made plants that accumulate increased quantitiesof desired pharmaceuticals, or for producing foodstuff plantswith lower alkaloid content (for example, coffee without caf-feine). Plants are some of natures very be st chemists, andsophisticated structures such as codeine, vinblastine, taxol,and camp tothecin remain well beyond the reach of commer-cially feasible total chemical syntheses. With the ability toexpress alkaloid b iosynthetic enzymes heterologously in or-ganisms with better fermentation characteristics than plants,we can achieve unlimited quantities of these biocatalystsfor use in syntheses of important drugs. In this review, I dis-cuss the progress that has been made in these areas as aresult of the fusion of alkaloid chemistry, enzymology, andmolecular biology, as well as some perspectives for future de -velopments in this field.

MONOTERPENO ID INDOLE ALKALOIDS

The m onoterpenoid indole alkaloids comprise a large familyof alkaloids, with over 1800 mem bers of rich structural diver-sity. M any of these natural products are physiologically activein mamm als. Amo ng the mon oterpenoid indole alkaloid phar-maceuticals that are still commercially isolated from plantmaterial are the antimalarial drug qu inine from C. officinalis, theantineoplastic drug camptothecin from C.acuminata, he ratpoison and hom eopathic drug strychnine from Strychnos nux-vomica,and the a ntineoplastic chemotherapeutic agents vin-cristine and vinblastine from Catharanthusm e u s periwink le)

(Figure 1).Total chemical syntheses of these complex alkaloidswould be of academic interest but due to low yields are notlikely to be applied commercially. To develop nove1 sourcesof these drugs, two options are available. The cDNAs for en-zymes that catalyze those biosynthetic steps that are difficultto achieve by chemical m eans can be isolated and heterolog-ously expressed or use inbiornimetic syntheses. Alternatively,instead of single transformation steps, microorganisms cou ldbe engineered to express short pathways, thus producing anend-product alkaloid of interest. Alkalo id biosynthe tic pathwaysthat are to0 long to be ntroduced into a single m icroorganismcould be mod ified in the parent plant using antisense or co-suppression technologies suc h that a desired alkaloid can beaccumulated by blocking side pathways or catabolic steps(schematically depicted in Figure 2).All of these approaches require thorough knowledge of thealkaloid biosynthetic pathway and the enzymes that catalyzethe individual transformation steps. Progress toward identify-ing the enzymes of mono terpenoid ndole alkaloid biosynthesishas been made primarily n the laboratory of J. Stockigt usingRauvolfiaserpentinacell suspension cultures in studies of thebiosynthesis of the antiarrythmic drug a jmaline and in the lab-oratory of V. De L uca us ing C. Toseus cell suspension culturesand plants to study the biosynthesis of vindoline, a precursorto the antineoplastics vincristine a nd vinblastine (reviewed nHerbe rt, 1994; Kutchan, 1994). The first successfu l cDN A clon-ing experiments in the alkaloid field were ach ieved with twocDNAs encoding enzymes hat catalyze early steps in he bio-synthetic pathway that leads to all monoterpenoid indolealkaloids, tryptophan decarboxylase (De Luca et al., 1989) andstrictosidine sy nthase (Kutc han et al., 1988) (Figure 3).

Tryptophan DecarboxylaseTryptophan decarboxylase (aromatic L-amino-aciddecarbox-ylase; EC4.1.1.28) catalyzes the deca rboxylation of the am inoacid L-tryptophan o the protoalkaloid tryptamine. Tryptaminecan then serve as substrate for the enzyme strictosidine syn-thase (Stockigt and Zenk, 1977), which catalyzes the firstcomm itted step in m onoterpenoid indole alkaloid biosynthe-sis. The cDNA clone en coding tryptophan decarboxylase was

Figure 1. (continued).Ajmaline, antiarrythmic that functions by inhib ition of glucose uptake by heart tissue mitochondria; atropine ([*I-hyoscyamine),anticholinergic,antidote to nerve gas poisoning; caffeine, widely used central nervous system stimulant; camptothecin, potent anticancer agent; cocaine, topicalanesthetic, potent central nervous system stimulant, and adrenergic blocking agent, drug of abuse; codeine, relatively nonaddictive analgesicand antitussive; coniine, first alkaloid to be synthesized, extremely toxic, causes paralysisof motor nerve endings, used in homeopathy; emetine,orally active emetic, amoebicide; morphine, powerful narcotic analgesic, addictive drug of abuse; nicotine, highly toxic, causes respiratory paraly-sis, horticultural nsecticide; pilocarpine, peripheral stimulant of the parasympathetic system,used to treat glaucoma; quinine, raditional antimalarial,important in treating Plasmodium alcipafum strains that are resistantto other antimalarials; sanguinarine, antibacterial showing antiplaque activ-ity, used in toothpastes and oral rinses; scopolamine, powerful narcotic, used as a sedative for motion sickness; strychnine, violent tetanic poison,rat poison, used in homeopathy; taxol, antitumor agent; (+)-tubocurarine,nondepolarizingmuscle relaxant producing paralysis, adjuvant to anesthesia;vinblastine, antineoplastic that is used to treat Hodgkins disease and other lymphomas.


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1062 The Plant Cell

M icroorganismsPlant pathway: >^ F

D => EG, G 3

Single geneexpression forbiomimetic syntheses C D

HeterologousGeneAINc1

PlantsNormalPattiway AntisenseorCosuppression

1E\

AIB1Ci

AIBICD\iE

Expression ofshort pathways

Figure 2. Potential Biotechnological Exploitation of Alkaloid Biosynthetic Genes.Plant alkaloid genes can be functionally expressed in microorganisms to produce either single biotransformation steps or short biosynthetic path-ways. Likewise, using overexpression or antisense or cosuppression technologies, medicinal plants can be tailored to produce important pharmaceuticalalkaloids by introducing side pathways, eliminating side pathways, or acc umulating biosynthetic intermediates. Fo r example, a known plant biosyn-thetic pathway contains an enzyme encoded by gene G3 that cata lyzes a transformation step, of compound C to alkaloid D, which is difficultto achieve by chemical synthesis. Th e G3 gene can be heterologously expressed in a microorganism and the gene product used in a biomimeticsynthesis of alkaloid D (that is, the microorganism is supplied with compound C and produces alkaloid D). Likewise, a short pathway consistingof enzymes encoded by genes G,, G2, 63, an d G,, couldbe expressed in a microorganism to produce alkaloidE directly from precursor A. Manyalkaloid biosynthetic pathways involve 20 to 30 enzymes. O ur curren t knowledge of these pathways indicates tha t the genes encoding the biosyn-thetic enzymes are neither clustered nor coordinately controlled by one operon. Ex pression of an entire, long alkaloid pathway in a single microorganismis currently beyond our technic al capability. In this case, it may be possible to alter the pathway in the plant ce ll and produce the desired alkaloideither in culture or in the field. F or example, to accumulate alkaloid Z, which is not normally produced in a particular plant species, a transgene(from another plant or a microorganism) can be introduced. To accumulate alkaloid E, a side pathway that a lso uses precursor D may have tobe blocked. If intermediate alkaloid C is the target for accumulation, catabolism to D could be interrupted.isolated from C . roseus by antibody screening of a cDNA ex -pression library prepared from poly(A)+ RNA of developingseedlings (De Luca et al., 1989). The tryptophan decarboxy-lase amino acid sequence shows similarities with an aromaticL-aminoacid decarboxylase from Drosophi la m elanogastar andwith L-amino acid decarboxylases from diverse animal origins.The tryptophan decarboxylase transcript was shown to ac-cumulate in C. roseus cell suspension cultures exposed to avariety of biotic elicitors (Pasquali et al., 1992; Roewer et al.,1992). Auxin reduces transcription of the gene, as demon-strated by run-off transcription experiments with C. roseusnuclei (Goddijn et al., 1992).A main point of interest in C. roseusand the likely reasonfor the recent increase in the number of researchers workingon thisplant- s that itproduces thedimeric alkaloidsvinblastine

and vincristine. It has been well established that cell culturesof C. roseus do not produce these commercially interestingand valuable alkaloids because the cultures lack vindoline,one component of both dimers. Induction of the last two en-zymes of vindoline biosynthesis in C. roseus seedlings,2-oxoglutarate-/V(1)-methyl-16-methoxy-23-dihydro-3-hydroxyta-bersonine4-hydroxylase and 17-O-deacytylvindolineO-acetyl-transferase, appears to be regulated by phytochrome (Aertsand De Luca, 1992), but red light-induced accumulation ofat least the acetyl transferase has not been observed in cellcultures. Thebiotechnologically most important experiments,those that may potentially result in the production of theanti-neoplastic dimeric alkaloids in cell culture, thus await theisolation of these final genes of vindoline biosynthesis.

The tryptophan decarboxylase cDNA from C. roseus has


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Monoterpenoid Indole Alkaloids

3n(S)-Slnclosidme

Benzylisoquinoline Alkaloids

EnzymeOCH3

(S)-Reticuline (S)-Scoulerine

Bisbenzylisoquinoline AlkaloidsH3CO

Berberine Alkaloids

Protopine Alkaloids

BenzophenanthridineAlkalloids

ObamegineBerbamine


Tropane and Nicotine AlkaloidsPutrescine HaN NHS

PutrescmeA/-Methyl transferaseW-Methyl- J [putrescine H;N NHCH3*

^NCH,

1-Melhyl-i'Ncotinicacid

/pyrrolinium /cation /Tropinone/Reduclase-l

nNNcotine

H OTropine

CH,OHHOOCCQ

Tropic acid

CH,OH

Hyoscyamine ][ H [I0Iyoscyamme6fi-Hydroxylase,

(fl)-W -Methylcoclaurine R, pH, R2 CH3(S)-N -Methylcoclaur ine R, - aH, R; - CH3

Figure 3. Reactions Catalyzed by Alkaloid Biosynthetic Enzymes for which cDNAs Have Been Isolated.been heterologously expressed in tobacco plants (Songstadet al., 1990,1991), and it increased their levels of tryptaminean d tyramine, the product of u-tyrosine decarboxylation. A fineexample of what metabolic engineering ca n achieve in second-ary metabolism ha s been provided by the transformation of8rass/ca napus with the C . roseus tryptophan decarboxylasecDNA (Chavadej et al., 1994). The seed of this oil-producingcrop ha s limited use as animal feed due in part to the presenceof indoleglucosinolates, which make the protein meal less pal-atable. The introduced cDNA for tryptophan decarboxylaseredirects tryptophan pools away from indoleglucosinolatepro-duction and into tryptamine. The mature seed of the transgenic8. napus plants contain reduced levels of indole glucosinatesbut no tryptamine, achieving a potentially economically use-fu l product.

Strictosidine SynthaseStrictosidine synthase (EC 4.3.3.2) catalyzes the stereospecificcondensation of the primary amino group of tryptamine an dthe aldehyde moiety of the iridoid glucoside secologanin toform thefirst monoterpenoid indole alkaloid, 3a(S)-strictosidine.Strictosidine synthase is of biotechnological interest in the bio-mimetic syntheses of monoterpenoid indole alkaloids because,

although the condensation of tryptamine and secologanin canbe achieved chemically, the reaction product is a mixture ofthe diastereomers vincoside and Strictosidine. Only Strictosi-dine with the 3a(S) configuration, th e exclusive product of theenzymatic reaction, can serve as a precursor to the monoterpe-noid indole alkaloids (Stockigt and Zenk, 1977). Theenzymeis stable, requires no cofactor addition, and is readily immobi-lized (Pfitzner and Zenk, 1987) for producing Strictosidine.The cDNA encoding Strictosidine synthasewa s first isolatedfrom R . serpentine (Kutchan et al., 1988), a plant used todayas thecommercialsource of antihypertensiveindolealkaloidssuch as ajmaline and ajm alicine; a Strictosidine synthase cDNAwa s subsequently isolated from C . roseus (McKnight et al.,1990). These twoenzymes show 80% amino acid homology,which probably reflects the fac t that both plants are membersof the Apocynaceae. DMA ge l blot analysis using the R. ser-pent ina cDNA as aprobe shows that not all species that containthe enzyme contain a similar gene, possibly due to differencesin codon usage (Kutchan, 1993a). Strictosidine synthase fromR. serpentine ha s been functionally expressed in Escher ich iaco// and Saccha romyces cerev is iae, and in insect cells usinga baculovirus vector (Kutchan, 1989; Kutchanet al., 1994). TheC . roseus enzyme has been expressed n tobacco and co//(McKnight et al., 1991; Roessner et al., 1992). A comparisonof these as production systemsha s been reviewedby Kutchan(1994).


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1064 The Plant Cell

The gene for strictosidine synthase, str l, has been isolatedfrom R. serpentina (Bracher and Kutchan, 1992). A sin gle geneappears to code for the enzyme in both R. serpentina andC. mseus(Pasqua1i et al., 1992). In both species, the tr ansc riptaccum ulates predom inantly n the roots and leaves of matureplants, although it can also be detecte d n flowers and stems.Levels of C mseus strictosidine synthase transcript can beincrea sed n cell culture (Pas quali et al., 1992; Roewer et al.,1992) and during a very na rrow ime period n developing seed-lings (Aerts et al., 1994) by treatmen t with various elicitors.However, the h igh levels of transcrip t present n mature plantssuggest that sfrl is not one of the regula ting genes of indolealkaloid biosynthesis.

TROPANE AND NlCOTlNE ALKALOIDS

The tropane class of alkaloids, wh ich are found m ainly in theSolanaceae, contains the anticholinergic drugs hyoscyam ine(the racemate of which is called atropine) and scopolamine.Solanaceous plants have been used traditionally or their m e-dicina l, hallucinogenic, and poisonous properties, which aredue to their tropan e alkaloids. Th e narcotic topical anestheticand central nervo us system stimulant cocaine is a tropane al-kaloid found ou tside of the Solanaceae in Erythmxylon coca.What is a u seful medicinal n small, controlled doses is poten-tially lethal when abused. Hence, scopolamine isolated fromDuboisia s commonly used today in he form of a transdermalpatch to combat motion sickness, but Datura leaves are smokedfor the hallucinoge niceffects of this alkaloid. Although coca inehas served as a lead, a starting structure that medicinalchemists modify to design an optimized drug, for the develop-ment of synthe tic topical anesthetics, this alkaloid is illicitlyapplie d to m ucus mem branes for its addictive stimulato ry ef-fects. Metabolic engineering of the plants that serve ascomm ercial sources of nicotine or scopolamine could enha nceclassic breeding in the effort to develop plants with optimalalkaloid patterns, that is, that serve as improve d sources ofpharmaceuticals.A study of the bios ynthesis of tropane alkaloids implies astudy of n icotine biosynthesisalso, because the N-me thyl-A-pyrrolin ium cation is a precursor to bo th classes of alkaloids.Analyses of nicotine biosynthesis n tobacco and cocaine bio-synthesis in coca were pioneere d n the laboratoryof E. Leete.The enzym ology and molecular biology of scopolamine bio-synthesis in Hyoscyamus have been studied principally byT. Hashim oto and Y. Yamada. The cDNA s encoding hyoscya-mine 6p-hydroxylase (Matsuda et ai., 1991) and tropinonereductase (Nakajima et al., 1993), both of w hich are enzyme sof scopolam ine biosynthesis n Hyoscyamus niger, have beencloned (Figure 3). In addition, a tobacco cDNA for pu trescineN-methyltransferase, an enzyme involved n the biosynthesisof the N-me thyl-A-pyrroliniumcation, has been isolated (Hibiet al., 1994).

Putrescine N-MethyltransferaseCuban ciga r tobacco varieties discovered in the 1930s to havelow nicotine content were used in backcrosse s o e stablish agenetically stable breeding ine with a low alkaloid content forproducing ow n icotine cigarettes. This comm ercial low nico-tine variety, LA Bu rley 21, and the parenta1 strain , Bu rley 21,which is isogenic at all but two low nicotine biosynthesis oci,were used to isolate a cDNA for an enzym e of nicotin e biosyn-thesis, putrescine N-me thyltransferase (EC 2.1.1.53). Thisenzym e catalyzes the transfer of a me thyl grou p from S-ade-nosyl-L-m ethionine o an am ino group of putrescine , which isthe first committed step in the biosynthesisof nicotine and tro-pane alkaloids. The availability of the near-isogenjc BurleyStrains made it possible o use subtractive hybridization o iso-late a cDNA encoding this enzym e (Hibi et al., 1994). Thededuce d amino acid sequence of one of the clones obtainedby subtractive hybridization s homo logous o sperm idine syn-thase from human (73% iden tical), mouse(70% identical), andE. coli(58% dentical).Heterologousexpression of the tobac cocDNA in an E. colideletion mutant acking he spermidine syn-thase gene resulted in accumulation of N-methyl putrescine,confirming the ide ntity of the cDNA . Transcripts for putrescineN-methyltransferaseaccumulate predom inantly, f not exclu-sively, in root tissue of the wild-type obacco plant, sugges tingthat this organ is the m ajor site of nico tine biosynthes is. Thiscorrespo nds o the site of tropane alkaloid biosynthesis in othermem bers of th e Solanaceae (Hashimotoet al., 1991). The log-ical progression of this work i st o solate a putrescineN-methyl-transferase gene from a plant that produce s ropane alkaloids.

Tropinone ReductaseAlong the biosynthetic pathway that leads specifically to the tro-pane alkaloid scopolamine, ropinone reductase (EC 1.1.1.236)converts the 3-keto group of tropinone to th e 3a -hydroxyl oftropine. The cDN A encoding this enzyme was isolated fromDatura stramonium (jimsonweed) by screen ing a cDNA libraryprepared rom ha iry roots with oligonucleotide s basedon pep-tide amino acid sequences from purified native tropinonereductase I (Nak ajima et al., 1993). The cDNA was expressedas a p-galactosida se usion protein n E. coli and found to havetropinone reductase Iactivity. DNA gel blot analysis dentifie dhomologous DNA fragments in the nuclear DNA of the tro-pane alkaloid-producing species H. niger and A. belladonna,but not i n tobacco, a sp ecies in which tropane alkaloids arenot biosynthesized.

A cDNA for a second reductase, tropinone reductase II, wasalso isolated in these experiments. Tropinone reductase I1reduces tropinone with a stereo specificity opposite to tha t oftropinone reductase I. Hence, the 3-keto group of tropinoneis converted to the 3p-hydroxyl of pseudo tropine. The deducedamino acid sequences of the two cDNAs are 64% identical.In a series of elegant experiments, various d oma ins of these


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two reductases were exchanged and the chimeric enzymeswere expresse d in E. coli to identify the dom ain that confersthe stereospecificityof the reaction (Nakajima et al., 1994). Inthis manner, it was dem onstrated hat a 120-amino a cid resi-due pe ptide at the C terminus of each reductase determinesthe s tereospe cificity of the reaction it catalyzes.Hyoscyamine 6p-HydroxylaseThe final two steps in the biosynthetic pathway leading fromhyoscyamine o the m edicinally important alkaloid scopo lamineis catalyzed by a 2-oxoglutarate-dependent enzyme, hyoscya-mine 6P-hydroxylase (hyoscyamine [6S]-dioxygenase; EC1.14.11.11). Th is dio xyge nas e first hydroxylates hy oscyaminein the 6s position and subsequently catalyzes epoxide for-mation. M olecular cloning and heterologous expres sion ofhyoscyamine 6P-hydroxylase have demonstrated that thisenzyme catalyzes both the hyd roxylation reaction and the intra-molecular epoxidation reaction (Mats udaet al., 1991). RNA ge lblot ana lysis of th e hyoscyamine 6P-hydroxylase gene hascorroborated the results obtained by imm unoh istochem icalo-calizat ion of the enzyme (Hashim oto et al., 1991), wh ich showedthat hyoscyam ine 6P-hydroxylase s loca lized to the root peri-cycle in scop olamin e-produc ing species of Hyoscyamus,Atropa, and Duboisia. Sim ilarly, he hyoscyamine 6P-hydroxy-lase transcript is loc alized o roots and cu ltured roots of H. nigerbut is not found in stem, leaves, or cultu red cells of th is samespecies. These results explain why it has not been p ossibleto produ ce substan tial quantities of tropane alkaloids in cellculture. The gene encoding hyoscyamine 6P-hydroxylaseshould help with the elucida tionof the underlying mechan ismsof tissue- and ce ll-spec ific expression of tropa ne alkaloids inthe S olanaceae.The current comm ercial source of scopolam ine s Duboisia,which was cultivated origina lly in Australia. Ce rtain tropanealkaloid-producing species, such as Atropa, accumulatehyoscyamine instead of scopolamine as the major alkaloid.To ask whether expression of a transg ene n a me dicina l plantcould alter the alkaloid pattern such that more of the phar-maceu tically useful alkaloid, scopolamine, could be produced,the cDNA encoding hyoscyamine 6P-hydroxylase rom H. nigerwas introduced ntoA. belladon na using either Agrobacferiumtumefaciens- or Agrobacterium rhizogenes-mediated trans-formation. The resultant transge nic plants (Yun et al., 1992)and h airy roots (Hashimoto et al., 1993) each con tainedelevated levels of scopolam ine.Each of these two successful transformation experimentshas a distinct implica tion or the future of m etabolic engine er-ing of medicinal plants. First, the transgenic A. belladonnaplants, although not neces sarily com mercially useful, providethe first example of how me dicina l plants can be successfullyaltered using molecular genetic techniques to produce in-creased quantities of a medicinally important alkaloid. Thefuture of this field is completely dependent upon a thoroug h

knowledge of the alkaloid biosyntheticpathway at the enzym elevel so that m eaningful transformation experiments can bedesigned. It is also limited by our ability to transform andregenerate me dicina l plants. To date, expe rtise in this im por-tant area lags well beh ind hat for tobacco, petunia, and cerealcrops, among others. For example, in the area of tropanealkaloids, transformation and regeneration of Duboisia, a plantfor which plantation, harvesting, and purification techniqueshave already bee n comm ercially established , will have to bedeveloped before any potential comme rcialization. The se c-ond implication is that m etabolic engineering may make itpossible to produce alkaloids in cultured cells. The produc-tion of alkaloids in tissue and cell culture, such as n ha iry roots,is an area that rece ived much attention in the past becauseit promisedan alternative source of pharmaceuticals hat wouldbe independentof weather, bligh t, and politics. It has becom eclear with time, however, that many important compounds, suchas vincristine, vinblastine, pilocarpine, morphine, and codeine,are not synthesized o any app reciable extent in culture. Thereason is thought to be tissue-specific expression of alkaloidbiosynthe tic genes, beca use in some cases, plants regener-ated from nonprod ucingcallus cells contain he same alkaloidprofile as the parent plant. For certain costly alkaloid s (for ex-ample, taxol), cell culture production at a commercial levelwould be worthwhile establishing. The increased accumula-tion of scopolam ine n hairy roots that contain an hyoscyam ine6P-hydroxylase ransgen e s an elegant demonstration that thisapproach remains a viable one.

BENZYLISOQUINOLINE ALKALOIDS

The benzylisoquinoline alkaloids are ave ry large and d iverseclass of alkaloids. This fam ily contains such varied physiolog-ically active mem bers as emetine (an antiamoebic), colchicine(a microtub uledisrupter and gout suppressant), berberine anantimicrobial against eye and intestinal nfections), morphine(a narcotic analgesic), codeine (a narcotic analgesic and an-titussive), and sanguinarine (an antimicrobial used in oralhygeine). As with th e p harm acologically active mem bers ofthe indole and tropane classes of alkaloids, those of phar-maceutical interest from the benzylisoquinoline class arelargely still isolated from plants, again due to the com plexityof their structures. One notable exception to this is the be n-zylisoquinoline alkaloid berberine, which is iso lated in Japanfrom cell suspension cultures of C opfisjapon ica (summ arizedin Zenk et al., 1988).Benzo(c)phenanthridinealkaloids, a su bclass of the ben zyl-isoquinolines,are produced n a numb erof species within thePapaveraceae, ncluding Sanguinaria canadensis (bloodroot),I? somniferum (opium poppy), and Esch scholtzia californica(California poppy, used as a sedative by Na tive Americans).Extracts of bloo droot that are rich in the inten sely red ben-zophenanthridine alkaloid sanguinarine are currently added


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1066 The Plant Cell

to toothpastes and oral rinses in'the United States becauseof this alkaloid's antiplaque properties. Benzophenan thridinealkaloids have been shown to accum ulate in cell cultures ofE. californica in resp onse o funga1 attack an d to treatment withnume rous elicitor substances (Schumacher et al., 1987). Thisphenom enon, when explo ited n cell suspe nsion cultures, pro-vided the main experimental system w ith which the entirebiosyntheticpathway eading rom two molec ules of L-tyrosineto the most highly oxidized benzophe nanthridine alkaloid,macarpine, was elucidated in the laboratory of M.H. Zenk. Thispathway, enco mpa ssing 21 con version s, 20 of wh ich areenzymatic and one of which is a spontaneous skeletal rear-rangem ent, s the sing le longest secondary me tabolic pathwayto have been comp letely elucida ted at the enz yme eve1 (sum-marize d in Kutchan and Zenk, 1993; Kamm erer et al., 1994).A cDNA encod ing one of seven indu cible enzymes from thispathway, the berberine bridge enzyme (Figure 3), has beenisolated (Dittrich and Kutchan, 1991).

Berber ine Br idge EnzymeThe berbe rine brid ge enzym e ([SI-reticu1ine:oxygen oxidore -ductas e [methylene bridg e forming]; EC 1.5.3.9) ca talyzes hestereospecific conversion of the N-methyl group of (S)-reticulineinto the berberine bridge carbon, C-8,f (S)-scoulerine.Thereare two main reason s why this p articu lar enzyme is of interest.First, the reaction catalyzed by the b erberine bridge enzymeis very elegant from the chem ist's po int of view. The direct con-version of the N -methyl group into a me thylene bridge m oietyis not presently achievable by synthetic chemica l methods andexists nowhere else in n ature. It is of biochem ical interest toknow how an enzyme catalyzes a reaction hat we as chemistscannot. The se cond po int of interest is that the E. californicaberberine bridge enzyme is elicitor inducible. This implies hatregulation of be rberine bridge enzyme transcript accumu la-tion may regulate benz ophe nanthridin e lkaloid accumulation.From an analysis of the prom oter of the bb e l gene, details cantherefore be learned about how alkaloid biosynthesis s regu-lated in response to pathoge n attack.The cDNA enco ding the berberine bridge enzyme has beenisolated from a cDNA b ank p repared from elicited cell sus-pension cultures of E. californica using oligonucleotidesbasedon p eptide amino acid sequences of the purified native en-zyme (Dittrich and Kutchan, 1991). Translation of the n ucleotidesequence confirmed he presence of a signal peptide (by com-parison with the experimentally determined sequence of themature protein N terminus) that directs the enzyme into theendoplasmic reticulum and then into the smooth vesicles inwhich it accum ulates (Am ann et al., 1986). Hetero logou s ex-pression of this cDNA in nsect cell culture using a baculovirusexpression vector resulted in the produ ctionof sufficient quan-tities (4 mg of hom ogen eous enzym e per liter of insect cellculture medium) for a b iochem ical analysis of the be rberinebridge enzyme (Kutchan et al., 1994). A series of spectraldeterminations and modified-substrate conversion-ratemea-surements has demonstrated that the berberine b ridge enzyme

is covale ntly flavinylated and that closu re of the ring systemto form (S)-scou lerineduring the co urse of the enzymatic reac-tion proceeds via an ionic mechanism with the methyleneimin ium ion as a reac tion interm ediate (T.M. Kutchan , unpub-lished data).An ana lysis of the time course of the elicitation process usingthe cDNA clone as a hyb ridization probe in RNA ge l blot ex-perimen ts revealed that the be rberine brid ge enzyme transcriptreaches maximal levels within 6 hr after add ition of a yeastelicitor to E. californica cultures. Enzyme activity increases un til17 to 22 hr after elicitation, and total benzophenanthridinealkaloids continue o accumu late or severa1days after elicita-tion (Dittrich and Kutchan, 1991). This scenario is indicativeof de novo transcription of phytoalexin biosynthetic genes,which also occurs durin g stress-induced phen ylpropa noidme-tabolism (see Dixon and Paiva, 1995, this issue). Methyljasmonate nduce s he accumulation of low molecular weightcompounds in a large number of plant species in culture(Gun dlach et al., 1992), and b oth meth yl jasmo nate and thejasmonic acid biosynthetic precursor 124x0 -phytodienoic acidinduce transcript ion of the single b be l gene in E. californicacell cultures (Kutchan, 1993b; Kutchan and Zenk, 1993). It hasrecently been shown that the Pseudomonas syringae von Hallphytotoxin, coronatine, mimics 124x0 -phytod ienoic acid in tsability to induce a series of physiolog ical responses such astendril coiling, secondary metabolite accumulation n cell cul-ture, and bb e l transcription in E. californica (Weiler et al., 1994).Unlike various abiotic and biotic elicito rs Gun dlach et al., 1992;Mue ller et al., 1993), coron atine affects ge ne transc riptionwithout inducing endogenous 12-0~0 -phytodieno ic cid andjasmonic acid accumulation. lndu ction of the be rberine bridgeenzyme transcript with trihomo-jasmona te has dem onstrated

that P-oxidation s not necessary for gene activation (Blech ertet al., 1995). Analys is of the cis and rrans elements necessaryfor elicitor-induced ra nscription of bb e l and of other induc-ible genes along the benzo phenan thridinealkaloid biosyntheticpathway should h elp to elucidate the complex defense re-sponse signal transduc tion chain.

BISBENZYLISOQUINOLINE ALKALO IDS

The bisbenzylisoquinolineclass of a lkaloids contains over 270mem bers, all of wh ich are dime rs of tetrahydrobenzylisoquino-lines connectedby one to three ether linkages ormed by pheno lcoupling. The structure of a prototype of these natural prod-ucts, +)-tubocurarine, which is the muscle relaxant isolatedfrom tube-curare (a preparation of Chondodendmn romenro-sum in wooden tubes), contains two ether linkages with thetetrahydrobenzylisoquinolinemoieties combined in a head-to-tail orientation (Figure 1). Tube-curare has been trad itiona llyused by South American ndians asan arrow poison. In modernmedicine, tubocurarine chloride is used as a neuromuscularblocking agent to secure muscular relaxation n surgical oper-ations. It is of intere st that on ly one pla nt, C romenrosum, isknown to contain (+)-tubocurarine.


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The bisbenzylisoquino line family of alkaloids is rich in phar-macologically active constituents that range in activity fromcytotoxins to antihypertensives o antima larials. The structuralvariations n these alkaloids include substitutions on the phenylrings, the regiospecificity of the ether linkages, and thestereospecificity of the isoquinoline moieties. The best under-stood biosynthesis s that of the dimeric alkaloids berbamunineand guattegaumerine, which are produced in cell suspensioncultures of Berberisstolonifera(barberry). The single m ost im-portant step in the biosynthesis of this category of alkaloidsis ether linkage formation through p henol coupling. Any bio-technological production of the pharmaceutically importantbisbenzylisoquinolines requires correct stereo- and regio-selectivity of ether bond formation. It has only recently beenshown that the enzymes that catalyze either carbon-carbonor carbon-oxygen pheno l coupling in plants are not nonspecificperoxidases or laccases but rather high ly regio- and stereo-selectke substrate-specific cytochrome P-450-dependentoxidases (Zenk et al., 1989; Stad ler and Zenk, 1993). The reac-tion catalyzed by these enzymes s nove1 or cytochrom es P-450in that it proceeds without incorporation of oxygen nto the prod-uct. In other words, this cytochrome P-450 functions as anoxidase rather than as a mono oxygenase. t would be mecha-nistically interesting to discern how these enzymes act onmolecular oxygen differently from th e m ore common hydrox-ylases. Studies of the enzyma tic mechanism require he abilityto isolate large quantities of functional enzyme a nd to alterthe enzyme's structure and observe the types of changes thatoccur in the enzymatic reaction. These approaches are, ofcourse, dependent on molec ular genetic manipulations. TO thisend, the first cDNA known to encode a plant cytochrome P-450unequivocally involved in alkaloid biosynthesis has recentlybeen cloned. This cDNA encodes berbamunine synthase (Fig-ure 3) (Kraus and Kutchan, 1995).

Berbamunine SynthaseBerbamunine synthase (N-methylcoclaurine,NADPH:oxygenoxidoreductase carbon-oxygen phenol coupling]; EC 1.1.3.34;CYP80) catalyzes formation of the ethe r linkage betw een onemolecule of (R)-N-methylcoclaurine and one molecule of(S)-N-methylcoclaurine to form the bisbenzylisoquinoline al-kaloid berbamunine. This cytochrome P-450 enzyme alSOcouples two m olecules of (R)-N-methylcoclaurine o form guat-tegaumerine. The cDNA encoding berbamunine synthase wasisolated from a B. stolonifera cell suspension culture cDNAlibrary (Kraus and Kutchan, 1995). An oligonucleotide prim erbased on the N-terminal amino acid sequence of the purifiednative oxidase was used as a screening probe. Translationofthe nucleotide sequence of the clone revealed at least onenotable amino acid residue difference in berbamunine syn-thase as compared with the sequences of cloned cytochromeP-450 monooxygenases. Structure-function studies on bac-teria1 P-450cam suggest that three amino acid residues of thedista1 helix (he lix I),Gly-248, Gly-249, and Thr-252, are essen-tia1 for formation of the molecular oxygen binding pocket

(Poulos et al., 1985). Mutation of Thr-25 2 to Ala or Val abol-ishes insertion of oxygen into the substrate (Imai et al., 1989).Thr-252 is present in berbamunine synthase, although thereis no monooxygenation reaction in the carbon-oxygen phe-no1 coupling process. However, the essen tial Ala o r Gly at theposition corresponding to 248 in P-450cam is replaced by aPro in berbamunine synthase. Because this amino acid resi-due is one of three h ighly conserved residues of the oxygenbinding pocket that are thought to be required for insertionof oxygen into the substrate, this m ay be a first indication ofhow berbamunine synthase functions as an oxidase.Berbamunine synthase has been expressed in functionalform in insect cell culture using a baculovirus expression vec-tor (Kraus and Kutchan, 1995). The oxidase accepts electronsfrom either insect cell, porcine, or Berberis cytochrome P-450reductases. Heterologously expressed berbamun ine synthasecan be obtained in near-homogeneous orm and in large quan-tities (5m gl l) after insect cell microsome solubilization followedby a single column-chromatograph y fractionation step. To ob-tain this amount of enzyme from B. stolonifera cells, 18,000L of suspension culture would have to be extracted. A singlegene probably codes for berbamunine synthase in the B. stolo-nifera genome, although two more weakly hybridizing DNAfragments are also present that may represent genes encod-ing other phenol coupling cytochromes.

ECOCHEMICAL FUNCTION OF ALKALOIDS

Their medicinal and toxicological properties clearly makealkaloids important to peop le, but their role in plants has beena longstanding question. Why sh ould a plant invest so muchnitrogen in synthesizing such a large number of alkaloids ofsuch diverse structure?C, meus alone contains over 100 differ-ent m onoterpenoid indole alkaloids. That m any alkaloids arecytotoxic provides insight into their p otential function in thechemical defense arsenal of the plant. It needs to b e system-atically addressed whether alkaloids function as phytoalexins,as nitrogen storage forms, as UV protectants, or i n any com bi-nation of these and other potential functions. The question ofalkaloid function in plants and the relevanceof that functionhave been reviewed (Hartmann, 1991; Caporale, 1995).One alkaloid whose ecochemical functions have been thor-oughly analyzed in recent years is nicotine, a highly toxicalkaloid that has been identified n the leaves, stems, and rootsof a num ber of Nicotiana species. Nicotine sulfate, a byproductof the tobacco industry, serves commercially as a very effec-tive insecticide and fumigant. To date, no insect has evolveda resistance mechanism against nicotine. In m amm als, n ges-tion of nicotine results in nausea, vomiting, diarrhea, mentalconfusion, convulsions, and respiratory paralysis. With suc hstrong and varied physiological responses, nicotine wouldseem to b e an effective deterrent against insect attack andherbivore grazing. This is indee d he case. A series of studiesin the labo ratory of I.T. Baldw in has demonstrated that nico-tine, which is known o b e synthesized n roots and transported


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1068 The Plant Cell

to leaves, is synthesized d e n ovo from (15N)-nitrate in re-sponse to wound ing of leaves of hydroponically grown N. sy/-vesfris (Baldwin et al., 1994a). The am oun t of alkaloid that canbe a ccumulated in th e leaf is sufficient to reduce larva1 growthof the tobacco hornworm Manducasexta (Baldwin, 1988).Mostimportantly, field tests with native populations of the summ erannual N. artenuata demo nstrated a systemic alkaloid induc-tion in response to simulated leaf herbivory and browsing(Baldwin and Ohnmeiss, 1993).Nicotine is clearly synthesized n response towounding, butdoes the alkaloid have additional functions, for example, asa nitrogen storage form o ra s a U V protectant? Although exog-enously adm inistered nicotine is catabolized to n ornicotine andmyosm ine, most likely as a result of a detoxification mecha -nism, de novo-synthesized nicotine is not turned over. Theplant does n ot recover nicotine nitrogen and reinvest it in othermetabolic processes, even under conditions of nitrogen-limitedgrowth, im plyin g that nicotine is no t used as a nitrogen stor-age form (Baldwin and Ohnmeiss, 1994). Nicotine suppliedexogenously to D. sframonium, a species that accumulates ro-pane alkaloids bu t not nicotine, does not result in increasedUV protection, although nicotine has a hig h molar extinctioncoefficient (2695M-l cm-l) at 262 nm (Baldwin and Huh,1994). Taken together, these results suggest that nicotine func -tions as a phytoalexin in tobacco and has n o additional roleas a storage form of nitrogen or as a filter for UV radiation.These results raise an important question: How does leafwounding indu ce nicotine biosynthesis n the root? Jasmonatesare known to indu ce accumulation of secondary m etabolitesin plant cell culture, and endogeno us asmona te pools ncreaserapidly in response to treatment of ce lls with a yeast elicitor(Gun dlach et al., 1992). Leaf damag e to N. sy/vesfris producesa similar increase n endogenous asmon ic acid pools n shootswithin 30 m in, and root pools of this signaling molecule in-crease within 2 hr (Baldwin et al., 1994b). In addition,application of m ethyl jasmonate a s a lanolin paste to leavesresults in increases n both endogenous asm onic acid in rootsand de novo nicotine biosynthesis. Jasmonate and 12-0x0-phytodienoic ac id function a s inducers of alkaloid biosynthe-sis both in culture an d in plants. The next question that needsto be addressed is whether jasmonate is the systemic signal-ing molecule hat is transported from the wounded leaf to roots,where it then indu ces transcription of the alkaloid biosyntheticgenes.

PERSPECTIVES

Although the alkaloid field is a very old one, it is still in its in-fancy with regard to being fully unde rstood, and our exploitationof the b iotechnological potential of alkaloid biosynthesis hasonly just begun. We are still a long way from understandinghow most alkaloids are synthesized in plants and how this bio-synthesis is regulated. We also have m uch to learn about thechemical ecology of alkaloids so that we can better understand

why these so phisticated and diverse structures evolved. Whatis clear is that alkaloids as integral components of med icina lplants have enjoyed a long and imp ortant history in raditionalmedicine. Our first drug s originated n he form of plant extracts,and some of our most important contemp orary pharma ceuti-cals are either still isolated from p lants or structurally de rivedfrom na tural products. New antineoplastic, antiviral, a nd an-timalarial alkaloids are still be ing discovered in plants. As weaccumulate the tools (biochemical knowledge, clones, andtransformation and regen eration echniques) necessa ry for fu-ture developments in this field, we can look forward togenetically engineered microorgan ismsand eukaryotic cell cul-tures that produce medicinal alkaloids, to m edicinal plants withopitimized alkaloid spe ctra, and even to the produ ction of im-portant pharmaceuticals in transgenic plant cell cultures.

ACKNOWLEDGMENTS

Our molecular genetic research on strictosidine synthase, he berber-ine bridge enzyme, and berbamunine synthase has been supportedby a grant from the Bundesminister ijr Forschung und Technologie,Bonn, and by the Deutsche Forschungsgemeinschaft Grant No. SFB369), Bonn.

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