The Plant Cell, Vol.
7,1059-1070,July 1995 O 1995 American Society of Plant Physiologists
Alkaloid Biosynthesis-The Basis for Metabolic Engineering
of Medicinal Plants
Toni M. Kutchan
Laboratorium fr Molekulare Biologie, Universitat Mnchen, Karlstrasse 29,80333 Munich, Germany
INTRODUCTION
Alkaloids-the term is linguistically derived from the Arabic
word a/-qali, the plant from which soda was first obtained-
are nitrogenous compounds that constitute the pharmacolog-
ically active basic principles of predominantly, although not
exclusively, flowering plants. Since the identification of the first
alkaloid, morphine, from the opium poppy, Papaver somnife-
rum, by Sertrner in 1806,.ulO,OOOalkaloids have been isolated
and their structures elucidated (Southon and Buckingham,
1989).Historically,the use of alkaloid-containing plant extracts
as potions, medicines, and poisons can be traced back almost
to the start of civilization. Famed examples include Socrates
death in 399 B.C. by consumption of coniine-containing hem-
lock (Conium maculatum) and Cleopatras use during the last
century B.C. of atropine-containing extracts of Egyptian hen-
bane (Hyoscyamusmuficus) to dilate her pupils and thereby
appear more alluring. Medieval European women utilized ex-
tracts of deadly nightshade, Atropa belladonna, for the same
purpose, hence the name belladonna. Although coniine is too
toxic to find therapeutic use today outside of homeopathy,
tropicamide, an anticholinergic that is a synthetic derivative
of atropine, is routinely used in eye examinations to dilate the
pupil. Tropicamide has also recently shown promise as an early
diagnostic tool in the detection of Alzheimers disease (Scinto
et al., 1994).A tonic prepared from the bark of Cinchona
officinalis that contains the antimalarial drug quinine greatly
facilitated European exploration and inhabitation of the trop-
ics during the past two centuries.
In total, -~13,000 plant species are known to have been used
as drugs throughoutthe world (Tyler, 1994).Approximately 25%
of contemporary materia medica is derived from plants and
used either as pure compounds (such as the narcotic analge-
sic morphine, the analgesic and antitussive codeine, and the
chemotherapeutic agents vincristine and vinblastine; Figure
1) oras teas and extracts. Plant constituents have also served
as models for modern synthetic drugs, such as atropine for
tropicamide, quinine for chloroquine, and cocaine for procaine
and tetracaine. In fact, active plant extract screening programs
continue to result in new drug discoveries. The most recent
examples of anticancer alkaloids are taxo1(clinically available
since 1994)from the western yew, Taxusbrevifolia, and camp-
tothecin (and derivatives currently in clinical trials) from the
Chinese happy tree, xi shu (Camptotheca acuminata), both
of which were originally isolated and assayed for biological
activity in the 1960s in the laboratory of M.E. Wall. In other
areas, there are intense searches for nove1 antivirals and
antimalarials.
We also encounter alkaloids as the stimulants caffeine in
coffee and tea and nicotine in cigarettes. Although a wealth
of information is available on the pharmacological effects of
these compounds, surprisingly little is known about how plants
synthesize these substances, and almost nothing is known
about how this synthesis is regulated. This is due, in part, to
the complex chemical structures of many alkaloids, which con-
tain multiple asymmetric centers. For example, although
nicotine (one asymmetric center) was discovered in 1828,its
structure was not known until it was synthesized in 1904,and
the structure of morphine (five asymmetric centers) was not
unequivocally elucidated until 1952,146 years after its isola-
tion. Beginning in the late 1950s,radiolabeledprecursorswere
fed to plants and the resultant radioactive alkaloidswere chem-
ically degradedto identify the position of the label. This opened
the field of alkaloid biosynthesis to experimentation. As ana-
lytical instrumentationbecame more sophisticated, precursors
labeled with stable isotopes were fed to plants and the prod-
ucts analyzed by nuclear magnetic resonance spectroscopy.
No real progress was made in identifying alkaloid biosynthetic
enzymes until the use of plant cell cultures as experimental
systems was introduced in the 1970s.Since then, on the or-
der of 80 new enzymes that catalyze steps in the biosynthesis
of the indole, isoquinoline, tropane, pyrrolizidine, and purine
classes of alkaloids have been discovered and partially
characterized.
Alkaloids belong to the broad category of secondary me-
tabolites. This class of molecule has historically been defined
as naturally occurring substances that are not vital to the or-
ganism that produces them. Alkaloids have traditionally been
of interest only due to their pronounced and various physio-
logical activities in animals and humans. A picture has now
begun to emerge that alkaloids do have important ecochemi-
cal functions in the defense of the plant against pathogenic
organisms and herbivores or, as in the case of pyrrolizidine
alkaloids, as pro-toxins for insects, which further modifiy the
alkaloids and then incorporate them into their own defense
secretions (reviewed in Hartmann, 1991).Alkaloids have now
1060 The Plant Cell

HO
:NCH,

Strychnine
Strychnos nux-vomica L.
Morphine, Codeine
Papever somnllerum L
Conilne
Con/urn maculatum L.

Me OAc
IM

Nicotine Taxol
Nicotiana tabacum L. Taxua brevi/olia Nuttall

.. ^ OCOCH,
| HO COjCHj
CH3
Vinblastine
H
Catharanthus roaeua (L.)G.Don. i
Ajmaline Pilocarpine
Rauvolfla serpentine (L.)Benth. Pilocarpus jaborandi Holmes

CH
Nx

|/ *A CHjOH
OOCCH

CH2OH H
OOCCH
C6HS . CH30
jfScopolamine, Atroplne N'
Hyoscyamus niger L. Quinine
Cinchona olficlnalls L

.COOCH,

Emetine
H
Uragoga ipecacuanha (Brot.)Balll.
Cocaine
Erytnroxylcn coca Lam.

(+)-Tubocura/)l
Sanguinarlne Chondodendmn tomentoaum R.&P.
Eschacholtzla calllornica Cham. Camptothecln
Camptotheca acumlntta Descne.

Figure 1. Some Physiologically Active Alkaloids and Plants That Produce Them.
 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 alkaloid
biosynthesis occurs in each organism.
With the introduction of molecular biology into the plant al-
kaloid field, induction of alkaloid biosynthesis in response to
exposure to wounding or to elicitors can be analyzed at the
leve1 of gene activation, and gene expression patterns in the
plant can be determined and interpreted as a first indication
of possible function. We also now have the capability to alter
the pattern of alkaloid accumulation in plants for the purpose
of studying the biological function of alkaloids, for engineer-
ing tailor-made plants that accumulate increased quantities
of desired pharmaceuticals, or for producing foodstuff plants
with lower alkaloid content (for example, coffee without caf-
feine). Plants are some of natures very best chemists, and
sophisticated structures such as codeine, vinblastine, taxol,
and camptothecin remain well beyond the reach of commer-
cially feasible total chemical syntheses. With the ability to
express alkaloid biosynthetic enzymes heterologously in or-
ganisms with better fermentation characteristics than plants,
we can achieve unlimited quantities of these biocatalysts
for use in syntheses of important drugs. In this review, I dis-
cuss the progress that has been made in these areas as a
result of the fusion of alkaloid chemistry, enzymology, and
molecular biology, as well as some perspectives for future de-
velopments in this field.
MONOTERPENOID INDOLE ALKALOIDS
(Figure 1). Total chemical synthesesof these complex alkaloids
would be of academic interest but due to low yields are not
likely to be applied commercially. To develop nove1 sources
of these drugs, two options are available. The cDNAs for en-
zymes that catalyze those biosynthetic steps that are difficult
to achieve by chemical means can be isolated and heterolog-
ously expressed for use in biornimetic syntheses. Alternatively,
instead of single transformation steps, microorganisms could
be engineered to express short pathways, thus producing an
end-product alkaloid of interest. Alkaloid biosynthetic pathways
that are to0 long to be introduced into a single microorganism
could be modified in the parent plant using antisense or co-
suppression technologies such that a desired alkaloid can be
accumulated by blocking side pathways or catabolic steps
(schematically depicted in Figure 2).
All of these approaches require thorough knowledge of the
alkaloid biosynthetic pathway and the enzymes that catalyze
the individual transformation steps. Progress toward identify-
ing the enzymes of monoterpenoid indole alkaloid biosynthesis
has been made primarily in the laboratory of J. Stockigt using
Rauvolfia serpentina cell suspension cultures in studies of the
biosynthesisof the antiarrythmic drug ajmaline and in the lab-
oratory of V. De Luca using C. Toseus cell suspension cultures
and plants to study the biosynthesis of vindoline, a precursor
to the antineoplastics vincristine and vinblastine (reviewed in
Herbert, 1994; Kutchan, 1994). The first successful cDNA clon-
ing experiments in the alkaloid field were achieved with two
cDNAs encoding enzymes that catalyze early steps in the bio-
synthetic pathway that leads to all monoterpenoid indole
alkaloids, tryptophan decarboxylase (De Luca et al., 1989) and
strictosidine synthase (Kutchan et al., 1988) (Figure 3).

The monoterpenoid indole alkaloids comprise a large family

of alkaloids, with over 1800 members of rich structural diver-
sity. Many of these natural products are physiologically active
in mammals. Among the monoterpenoid indole alkaloid phar-
maceuticals that are still commercially isolated from plant
material are the antimalarial drug quinine from C. officinalis, the
antineoplastic drug camptothecin from C.acuminata, the rat
poison and homeopathic drug strychnine from Strychnos nux-
vomica, and the antineoplastic chemotherapeutic agents vin-
cristine and vinblastine from Catharanthus m e u s (periwinkle)
Tryptophan Decarboxylase
Tryptophan decarboxylase (aromatic L-amino-aciddecarbox-
ylase; EC 4.1.1.28) catalyzes the decarboxylation of the amino
acid L-tryptophanto the protoalkaloid tryptamine. Tryptamine
can then serve as substrate for the enzyme strictosidine syn-
thase (Stockigt and Zenk, 1977), which catalyzes the first
committed step in monoterpenoid indole alkaloid biosynthe-
sis. The cDNA clone encoding tryptophan decarboxylasewas

Figure 1. (continued).

Ajmaline, antiarrythmic that functions by inhibitionof glucose uptake by heart tissue mitochondria; atropine ([*I-hyoscyamine),anticholinergic,
antidote to nerve gas poisoning; caffeine, widely used central nervoussystem stimulant; camptothecin, potent anticancer agent; cocaine, topical
anesthetic, potent central nervous system stimulant, and adrenergic blocking agent, drug of abuse; codeine, relatively nonaddictive analgesic
and antitussive; coniine, first alkaloid to be synthesized, extremely toxic, causes paralysis of 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 insecticide; pilocarpine, peripheralstimulant of the parasympathetic system, usedto treat glaucoma; quinine,traditionalantimalarial,
important in treating Plasmodiumfalcipafum strains that are resistant to other antimalarials; sanguinarine, antibacterial showing antiplaque activ-
ity, used in toothpastes and oral rinses; scopolamine, powerful narcotic, used as a sedativefor 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.
1062 The Plant Cell

Microorganisms Plants
Plant pathway: >^ F
D => E Heterologous Normal Antisense or
G, G3 Gene Pattiway Cosuppression

A A A
I I I

Single gene
expression for
biomimetic syntheses
N
c
B
1
C
I
C
B

C D 1 i
\ D
\
1 i
E E

Expression of
short 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 pharmaceutical
alkaloids by introducing side pathways, eliminating side pathways, or accumulating biosynthetic intermediates. For example, a known plant biosyn-
thetic pathway contains an enzyme encoded by gene G3 that catalyzes a transformation step, of compound C to alkaloid D, which is difficult
to achieve by chemical synthesis. The G3 gene can be heterologously expressed in a microorganism and the gene product used in a biomimetic
synthesis of alkaloid D (that is, the microorganism is supplied with compound C and produces alkaloid D). Likewise, a short pathway consisting
of enzymes encoded by genes G,, G2, 63, and G,, could be expressed in a microorganism to produce alkaloid E directly from precursor A. Many
alkaloid biosynthetic pathways involve 20 to 30 enzymes. Our current knowledge of these pathways indicates that the genes encoding the biosyn-
thetic enzymes are neither clustered nor coordinately controlled by one operon. Expression of an entire, long alkaloid pathway in a single microorganism
is currently beyond our technical capability. In this case, it may be possible to alter the pathway in the plant cell and produce the desired alkaloid
either in culture or in the field. For 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 also uses precursor D may have to
be 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 developing
seedlings (De Luca et al., 1989). The tryptophan decarboxy-
lase amino acid sequence shows similarities with an aromatic
L-amino acid decarboxylase from Drosophila melanogastar and
with 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 a
variety 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. roseus
nuclei (Goddijn et al., 1992).
A main point of interest in C. roseusand the likely reason
for the recent increase in the number of researchers working
on this plant- is that it produces the dimeric alkaloids vinblastine
and vincristine. It has been well established that cell cultures
of C. roseus do not produce these commercially interesting
and 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-
bersonine 4-hydroxylase and 17-O-deacytylvindoline O-acetyl-
transferase, appears to be regulated by phytochrome (Aerts
and De Luca, 1992), but red light-induced accumulation of
at least the acetyl transferase has not been observed in cell
cultures. The biotechnologically most important experiments,
those that may potentially result in the production of the anti-
neoplastic dimeric alkaloids in cell culture, thus await the
isolation of these final genes of vindoline biosynthesis.
The tryptophan decarboxylase cDNA from C. roseus has
 Alkaloids and Their Biosynthesis 1063

Monoterpenoid Indole Alkaloids Tropane and Nicotine Alkaloids

Putrescine HaN NHS

Putrescme
A/-Methyltransferase

W-Methyl- J [
putrescine H;N NHCH3

*
^
^N'
3n(S)-Slnclosidme CH,
Nicotinic
acid 1-Melhyl-i'
pyrrolinium /
Benzylisoquinoline Alkaloids cation / Tropinone
/ Reduclase-l
/
Berberine Alkaloids
n CH,OH^

HOOC-C^Q
Enzyme Protopine Alkaloids N
HO
Nicotine Tropine Tropic acid
OCH3
Benzophenanthridine
(S)-Reticuline (S)-Scoulerine Alkalloids

CH,OH
Bisbenzylisoquinoline Alkaloids
H3CO Hyoscyamine ][ H [I
0

Obamegine
Berbamine I Hyoscyamme
6fi-Hydroxylase

,
(fl)-W -Methylcoclaurine R, pH, R 2 CH3
(S)-N -Methylcoclaurine R, - aH, R; - CH3

Figure 3. Reactions Catalyzed by Alkaloid Biosynthetic Enzymes for which cDNAs Have Been Isolated.

been heterologously expressed in tobacco plants (Songstad
et al., 1990,1991), and it increased their levels of tryptamine
and tyramine, the product of u-tyrosine decarboxylation. A fine
example of what metabolic engineering can achieve in second-
ary metabolism has been provided by the transformation of
8rass/ca napus with the C. roseus tryptophan decarboxylase
cDNA (Chavadej et al., 1994). The seed of this oil-producing
crop has limited use as animal feed due in part to the presence
of indole glucosinolates, which make the protein meal less pal-
atable. The introduced cDNA for tryptophan decarboxylase
redirects tryptophan pools away from indole glucosinolate pro-
duction and into tryptamine. The mature seed of the transgenic
8. napus plants contain reduced levels of indole glucosinates
but no tryptamine, achieving a potentially economically use-
ful product.
Strictosidine Synthase
Strictosidine synthase (EC 4.3.3.2) catalyzes the stereospecific
condensation of the primary amino group of tryptamine and
the aldehyde moiety of the iridoid glucoside secologanin to
form the first 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 can
be achieved chemically, the reaction product is a mixture of
the diastereomers vincoside and Strictosidine. Only Strictosi-
dine with the 3a(S) configuration, the exclusive product of the
enzymatic reaction, can serve as a precursor to the monoterpe-
noid indole alkaloids (Stockigt and Zenk, 1977). The enzyme
is stable, requires no cofactor addition, and is readily immobi-
lized (Pfitzner and Zenk, 1987) for producing Strictosidine.
The cDNA encoding Strictosidine synthase was first isolated
from R. serpentine (Kutchan et al., 1988), a plant used today
as the commercial source of antihypertensive indole alkaloids
such as ajmaline and ajmalicine; a Strictosidine synthase cDNA
was subsequently isolated from C. roseus (McKnight et al.,
1990). These two enzymes show 80% amino acid homology,
which probably reflects the fact that both plants are members
of the Apocynaceae. DMA gel blot analysis using the R. ser-
pentina cDNA as a probe shows that not all species that contain
the enzyme contain a similar gene, possibly due to differences
in codon usage (Kutchan, 1993a). Strictosidine synthase from
R. serpentine has been functionally expressed in Escherichia
co// and Saccharomyces cerevisiae, and in insect cells using
a baculovirus vector (Kutchan, 1989; Kutchan et al., 1994). The
C. roseus enzyme has been expressed in tobacco and co//
(McKnight et al., 1991; Roessner et al., 1992). A comparison
of these as production systems has been reviewed by Kutchan
(1994).
1064 The Plant Cell
The gene for strictosidine synthase, strl, has been isolated
from R. serpentina (Bracher and Kutchan, 1992). A single gene
appears to code for the enzyme in both R. serpentina and
C. mseus(Pasqua1i et al., 1992). In both species, the transcript
accumulates predominantly in the roots and leaves of mature
plants, although it can also be detected in flowers and stems.
Levels of C. mseus strictosidine synthase transcript can be
increased in cell culture (Pasquali et al., 1992; Roewer et al.,
1992) and during a very narrowtime period in developing seed-
lings (Aerts et al., 1994) by treatment with various elicitors.
However, the high levels of transcript present in mature plants
suggest that sfrl is not one of the regulating genes of indole
alkaloid biosynthesis.
TROPANE AND NlCOTlNE ALKALOIDS
The tropane class of alkaloids, which are found mainly in the
Solanaceae, contains the anticholinergic drugs hyoscyamine
(the racemate of which is called atropine) and scopolamine.
Solanaceousplants have been used traditionally for their me-
dicinal, hallucinogenic, and poisonous properties, which are
due to their tropane alkaloids. The narcotic topical anesthetic
and central nervous system stimulant cocaine is a tropane al-
kaloid found outside of the Solanaceae in Erythmxylon coca.
What is a useful medicinal in small, controlled doses is poten-
tially lethal when abused. Hence, scopolamine isolated from
Duboisia is commonly used today in the form of a transdermal
patch to combat motion sickness, but Datura leaves are smoked
for the hallucinogeniceffects of this alkaloid. Although cocaine
has served as a lead, a starting structure that medicinal
chemists modify to design an optimized drug, for the develop-
ment of synthetic topical anesthetics, this alkaloid is illicitly
applied to mucus membranes for its addictive stimulatory ef-
fects. Metabolic engineering of the plants that serve as
commercial sources of nicotineor scopolaminecould enhance
classic breeding in the effort to develop plants with optimal
alkaloid patterns, that is, that serve as improved sources of
pharmaceuticals.
A study of the biosynthesis of tropane alkaloids implies a
study of nicotine biosynthesisalso, because the N-methyl-A-
pyrrolinium cation is a precursor to both classes of alkaloids.
Analyses of nicotine biosynthesis in tobacco and cocaine bio-
synthesis in coca were pioneered in the laboratory of E. Leete.
The enzymology and molecular biology of scopolamine bio-
synthesis in Hyoscyamus have been studied principally by
T. Hashimotoand Y. Yamada. The cDNAs encoding hyoscya-
mine 6p-hydroxylase (Matsuda et ai., 1991) and tropinone
reductase(Nakajima et al., 1993), both of which are enzymes
of scopolamine biosynthesis in Hyoscyamus niger, have been
cloned (Figure 3). In addition, a tobacco cDNA for putrescine
N-methyltransferase, an enzyme involved in the biosynthesis
of the N-methyl-A-pyrroliniumcation, has been isolated (Hibi
et al., 1994).
Putrescine N-Methyltransferase
Cuban cigar tobacco varieties discovered in the 1930s to have
low nicotine content were used in backcrosses to establish a
genetically stable breeding line with a low alkaloid content for
producing low nicotine cigarettes. This commercial low nico-
tine variety, LA Burley 21, and the parenta1strain, Burley 21,
which is isogenic at all but two low nicotine biosynthesis loci,
were used to isolate a cDNA for an enzyme of nicotine biosyn-
thesis, putrescine N-methyltransferase (EC 2.1.1.53). This
enzyme catalyzes the transfer of a methyl group from S-ade-
nosyl-L-methionineto an amino group of putrescine, which is
the first committed step in the biosynthesisof nicotine and tro-
pane alkaloids. The availability of the near-isogenjc Burley
Strains made it possible to use subtractive hybridizationto iso-
late a cDNA encoding this enzyme (Hibi et al., 1994). The
deduced amino acid sequence of one of the clones obtained
by subtractivehybridization is homologousto spermidine syn-
thase from human (73% identical), mouse (70% identical), and
E. coli(58% identical).Heterologousexpression of the tobacco
cDNA in an E. colideletion mutant lackingthe spermidine syn-
thase gene resulted in accumulation of N-methyl putrescine,
confirming the identity of the cDNA. Transcripts for putrescine
N-methyltransferaseaccumulate predominantly, if not exclu-
sively, in root tissue of the wild-type tobacco plant, suggesting
that this organ is the major site of nicotine biosynthesis.This
correspondsto the site of tropane alkaloid biosynthesis in other
members of the Solanaceae (Hashimotoet al., 1991). The log-
ical progression of this work isto isolate a putrescineN-methyl-
transferase gene from a plant that produces tropane alkaloids.
Tropinone Reductase
Along the biosynthetic pathway that leads specifically to the tro-
pane alkaloid scopolamine,tropinone reductase I (EC 1.1.1.236)
converts the 3-keto group of tropinone to the 3a-hydroxyl of
tropine. The cDNA encoding this enzyme was isolated from
Datura stramonium (jimsonweed)by screening a cDNA library
preparedfrom hairy roots with oligonucleotides based on pep-
tide amino acid sequences from purified native tropinone
reductase I (Nakajimaet al., 1993). The cDNA was expressed
as a p-galactosidasefusion protein in E. coli and found to have
tropinone reductase I activity. DNA gel blot analysis identified
homologous DNA fragments in the nuclear DNA of the tro-
pane alkaloid-producing species H. niger and A. belladonna,
but not in tobacco, a species in which tropane alkaloids are
not biosynthesized.
A cDNA for a second reductase, tropinone reductase II, was
also isolated in these experiments. Tropinone reductase I1
reduces tropinone with a stereospecificity opposite to that of
tropinone reductase I. Hence, the 3-keto group of tropinone
is converted to the 3p-hydroxyl of pseudotropine. The deduced
amino acid sequences of the two cDNAs are 64% identical.
In a series of elegant experiments, various domains of these

two reductases were exchanged and the chimeric enzymes
were expressed in E. coli to identify the domain that confers
the stereospecificityof the reaction (Nakajima et al., 1994). In
this manner, it was demonstratedthat a 120-amino acid resi-
due peptide at the C terminus of each reductase determines
the stereospecificity of the reaction it catalyzes.
Hyoscyamine 6p-Hydroxylase
The final two steps in the biosynthetic pathway leading from
hyoscyamine to the medicinally important alkaloid scopolamine
is catalyzed by a 2-oxoglutarate-dependent enzyme, hyoscya-
mine 6P-hydroxylase (hyoscyamine [6S]-dioxygenase; EC
1.14.11.11). This dioxygenase first hydroxylates hyoscyamine
in the 6s position and subsequently catalyzes epoxide for-
mation. Molecular cloning and heterologous expression of
hyoscyamine 6P-hydroxylase have demonstrated that this
enzyme catalyzes both the hydroxylation reaction and the intra-
molecular epoxidation reaction (Matsudaet al., 1991). RNA gel
blot analysis of the hyoscyamine 6P-hydroxylase gene has
corroborated the results obtained by immunohistochemicallo-
calization of the enzyme (Hashimoto et al., 1991),which showed
that hyoscyamine 6P-hydroxylaseis localized to the root peri-
cycle in scopolamine-producing species of Hyoscyamus,
Atropa, and Duboisia. Similarly,the hyoscyamine 6P-hydroxy-
lase transcript is localizedto roots and cultured roots of H. niger
but is not found in stem, leaves, or cultured cells of this same
species. These results explain why it has not been possible
to produce substantial quantities of tropane alkaloids in cell
culture. The gene encoding hyoscyamine 6P-hydroxylase
should help with the elucidationof the underlying mechanisms
of tissue- and cell-specific expression of tropane alkaloids in
the Solanaceae.
The current commercialsource of scopolamine is Duboisia,
which was cultivated originally in Australia. Certain tropane
alkaloid-producing species, such as Atropa, accumulate
hyoscyamine instead of scopolamine as the major alkaloid.
To ask whether expressionof a transgene in a medicinal plant
could alter the alkaloid pattern such that more of the phar-
maceutically useful alkaloid, scopolamine, could be produced,
the cDNA encoding hyoscyamine 6P-hydroxylasefrom H. niger
was introduced intoA. belladonna using either Agrobacferium
tumefaciens- or Agrobacterium rhizogenes-mediated trans-
formation. The resultant transgenic plants (Yun et al., 1992)
and hairy roots (Hashimoto et al., 1993) each contained
elevated levels of scopolamine.
Each of these two successful transformation experiments
has a distinct implicationfor the future of metabolic engineer-
ing of medicinal plants. First, the transgenic A. belladonna
plants, although not necessarilycommercially useful, provide
the first example of how medicinal plants can be successfully
altered using molecular genetic techniques to produce in-
creased quantities of a medicinally important alkaloid. The
future of this field is completely dependent upon a thorough
Alkaloids and Their Biosynthesis 1065
knowledge of the alkaloid biosyntheticpathway at the enzyme
level so that meaningful transformation experiments can be
designed. It is also limited by our ability to transform and
regenerate medicinal plants. To date, expertise in this impor-
tant area lags well behind that for tobacco, petunia, and cereal
crops, among others. For example, in the area of tropane
alkaloids, transformation and regeneration of Duboisia, a plant
for which plantation, harvesting, and purification techniques
have already been commercially established, will have to be
developed before any potential commercialization. The sec-
ond implication is that metabolic engineering may make it
possible to produce alkaloids in cultured cells. The produc-
tion of alkaloids in tissue and cell culture, such as in hairy roots,
is an area that received much attention in the past because
it promisedan alternative source of pharmaceuticals that would
be independentof weather, blight, and politics. It has become
clear with time, however, that many important compounds, such
as vincristine, vinblastine, pilocarpine, morphine, and codeine,
are not synthesized to any appreciable extent in culture. The
reason is thought to be tissue-specific expression of alkaloid
biosynthetic genes, because in some cases, plants regener-
ated from nonproducingcallus cells contain the same alkaloid
profile as the parent plant. For certain costly alkaloids (for ex-
ample, taxol), cell culture production at a commercial level
would be worthwhile establishing. The increased accumula-
tion of scopolaminein hairy roots that contain an hyoscyamine
6P-hydroxylasetransgene is an elegant demonstration that this
approach remains a viable one.
BENZYLISOQUINOLINEALKALOIDS
The benzylisoquinoline alkaloids are avery large and diverse
class of alkaloids. This family contains such varied physiolog-
ically active members as emetine (an antiamoebic), colchicine
(a microtubuledisrupter and gout suppressant), berberine(an
antimicrobial against eye and intestinal infections), morphine
(a narcotic analgesic), codeine (a narcotic analgesic and an-
titussive), and sanguinarine (an antimicrobial used in oral
hygeine). As with the pharmacologically active members of
the indole and tropane classes of alkaloids, those of phar-
maceutical interest from the benzylisoquinoline class are
largely still isolated from plants, again due to the complexity
of their structures. One notable exception to this is the ben-
zylisoquinoline alkaloid berberine, which is isolated in Japan
from cell suspension cultures of Copfisjaponica (summarized
in Zenk et al., 1988).
Benzo(c)phenanthridinealkaloids, a subclass of the benzyl-
isoquinolines,are produced in a numberof species within the
Papaveraceae, including Sanguinaria canadensis (bloodroot),
I? somniferum (opium poppy), and Eschscholtzia californica
(California poppy, used as a sedative by Native Americans).
Extracts of bloodroot that are rich in the intensely red ben-
zophenanthridine alkaloid sanguinarine are currently added
1066 The Plant Cell
to toothpastes and oral rinses in'the United States because
of this alkaloid's antiplaque properties. Benzophenanthridine
alkaloids have been shown to accumulate in cell cultures of
E. californica in responseto funga1attack and to treatment with
numerous elicitor substances (Schumacher et al., 1987). This
phenomenon, when exploited in cell suspensioncultures, pro-
vided the main experimental system with which the entire
biosyntheticpathway leading from two moleculesof L-tyrosine
to the most highly oxidized benzophenanthridine alkaloid,
macarpine, was elucidated in the laboratory of M.H. Zenk. This
pathway, encompassing 21 conversions, 20 of which are
enzymatic and one of which is a spontaneous skeletal rear-
rangement,is the single longest secondary metabolic pathway
to have been completely elucidated at the enzyme leve1(sum-
marized in Kutchan and Zenk, 1993; Kammerer et al., 1994).
A cDNA encoding one of seven inducible enzymes from this
pathway, the berberine bridge enzyme (Figure 3), has been
isolated (Dittrich and Kutchan, 1991).
Berberine Bridge Enzyme
The berberine bridge enzyme ([SI-reticu1ine:oxygenoxidore-
ductase [methylene bridge forming]; EC 1.5.3.9) catalyzes the
stereospecific conversion of the N-methyl group of (S)-reticuline
into the berberine bridge carbon, C-8, of (S)-scoulerine.There
are two main reasons why this particular enzyme is of interest.
First, the reaction catalyzed by the berberine bridge enzyme
is very elegant from the chemist's point of view. The direct con-
version of the N-methyl group into a methylene bridge moiety
is not presently achievable by synthetic chemical methods and
exists nowhere else in nature. It is of biochemical interest to
know how an enzyme catalyzes a reactionthat we as chemists
cannot. The second point of interest is that the E. californica
berberine bridge enzyme is elicitor inducible. This implies that
regulation of berberine bridge enzyme transcript accumula-
tion may regulate benzophenanthridinealkaloid accumulation.
From an analysis of the promoter of the bbel gene, details can
therefore be learned about how alkaloid biosynthesis is regu-
lated in response to pathogen attack.
The cDNA encoding the berberine bridge enzyme has been
isolated from a cDNA bank prepared from elicited cell sus-
pension cultures of E. californica using oligonucleotidesbased
on peptide amino acid sequences of the purified native en-
zyme (Dittrich and Kutchan, 1991). Translation of the nucleotide
sequence confirmed the presence of a signal peptide (by com-
parison with the experimentally determined sequence of the
mature protein N terminus) that directs the enzyme into the
endoplasmic reticulum and then into the smooth vesicles in
which it accumulates (Amann et al., 1986). Heterologous ex-
pression of this cDNA in insect cell culture using a baculovirus
expression vector resulted in the productionof sufficient quan-
tities (4 mg of homogeneous enzyme per liter of insect cell
culture medium) for a biochemical analysis of the berberine
bridge enzyme (Kutchan et al., 1994). A series of spectral
determinations and modified-substrateconversion-ratemea-
surements has demonstrated that the berberine bridgeenzyme
is covalently flavinylated and that closure of the ring system
to form (S)-scoulerineduring the course of the enzymatic reac-
tion proceeds via an ionic mechanism with the methylene
iminium ion as a reaction intermediate (T.M. Kutchan, unpub-
lished data).
An analysis of the time course of the elicitation process using
the cDNA clone as a hybridization probe in RNA gel blot ex-
periments revealed that the berberine bridge enzyme transcript
reaches maximal levels within 6 hr after addition of a yeast
elicitor to E. californica cultures. Enzyme activity increases until
17 to 22 hr after elicitation, and total benzophenanthridine
alkaloids continue to accumulate for severa1days after elicita-
tion (Dittrich and Kutchan, 1991). This scenario is indicative
of de novo transcription of phytoalexin biosynthetic genes,
which also occurs during stress-induced phenylpropanoidme-
tabolism (see Dixon and Paiva, 1995, this issue). Methyl
jasmonate induces the accumulation of low molecular weight
compounds in a large number of plant species in culture
(Gundlach et al., 1992), and both methyl jasmonate and the
jasmonic acid biosynthetic precursor 124x0-phytodienoic acid
induce transcription of the single bbel gene in E. californica
cell cultures (Kutchan, 1993b; Kutchan and Zenk, 1993). It has
recently been shown that the Pseudomonas syringae von Hall
phytotoxin, coronatine, mimics 124x0-phytodienoic acid in its
ability to induce a series of physiological responses such as
tendril coiling, secondary metabolite accumulation in cell cul-
ture, and bbel transcription in E. californica (Weiler et al., 1994).
Unlike various abiotic and biotic elicitors(Gundlachet al., 1992;
Mueller et al., 1993), coronatine affects gene transcription
without inducing endogenous 12-0~0-phytodienoicacid and
jasmonic acid accumulation. lnduction of the berberine bridge
enzyme transcript with trihomo-jasmonatehas demonstrated
that P-oxidation is not necessary for gene activation (Blechert
et al., 1995). Analysis of the cis and rrans elements necessary
for elicitor-inducedtranscription of bbel and of other induc-
ible genes along the benzophenanthridinealkaloid biosynthetic
pathway should help to elucidate the complex defense re-
sponse signal transduction chain.
BISBENZYLISOQUINOLINE ALKALOIDS
The bisbenzylisoquinolineclass of alkaloids contains over 270
members, all of which are dimers of tetrahydrobenzylisoquino-
linesconnectedby one to three ether linkagesformed by phenol
coupling. The structure of a prototype of these natural prod-
ucts,(+)-tubocurarine, which is the muscle relaxant isolated
from tube-curare (a preparation of Chondodendmn romenro-
sum in wooden tubes), contains two ether linkages with the
tetrahydrobenzylisoquinoline moieties combined in a head-to-
tail orientation (Figure 1). Tube-curare has been traditionally
used by South American lndians as an arrow poison. In modern
medicine, tubocurarine chloride is used as a neuromuscular
blocking agent to secure muscular relaxationin surgical oper-
ations. It is of interest that only one plant, C. romenrosum, is
known to contain (+)-tubocurarine.

The bisbenzylisoquinoline family of alkaloids is rich in phar-
macologically active constituents that range in activity from
cytotoxins to antihypertensives to antimalarials. The structural
variations in these alkaloids include substitutions on the phenyl
rings, the regiospecificity of the ether linkages, and the
stereospecificity of the isoquinoline moieties. The best under-
stood biosynthesis is that of the dimeric alkaloids berbamunine
and guattegaumerine, which are produced in cell suspension
cultures of Berberis stolonifera (barberry). The single most im-
portant step in the biosynthesis of this category of alkaloids
is ether linkage formation through phenol coupling. Any bio-
technological production of the pharmaceutically important
bisbenzylisoquinolines requires correct stereo- and regio-
selectivity of ether bond formation. It has only recently been
shown that the enzymes that catalyze either carbon-carbon
or carbon-oxygen phenol coupling in plants are not nonspecific
peroxidases or laccases but rather highly regio- and stereo-
selectke substrate-specific cytochrome P-450-dependent
oxidases (Zenk et al., 1989; Stadler and Zenk, 1993). The reac-
tion catalyzed by these enzymes is nove1for cytochromes P-450
in that it proceeds without incorporation of oxygen into the prod-
uct. In other words, this cytochrome P-450 functions as an
oxidase rather than as a monooxygenase.It would be mecha-
nistically interesting to discern how these enzymes act on
molecular oxygen differently from the more common hydrox-
ylases. Studies of the enzymatic mechanism require the ability
to isolate large quantities of functional enzyme and to alter
the enzyme's structure and observe the types of changes that
occur in the enzymatic reaction. These approaches are, of
course, dependent on molecular genetic manipulations. TOthis
end, the first cDNA known to encode a plant cytochrome P-450
unequivocally involved in alkaloid biosynthesis has recently
been cloned. This cDNA encodes berbamuninesynthase (Fig-
ure 3) (Kraus and Kutchan, 1995).
Berbamunine Synthase
Berbamunine synthase (N-methylcoclaurine,NADPH:oxygen
oxidoreductase [carbon-oxygen phenol coupling]; EC 1.1.3.34;
CYP80) catalyzes formation of the ether linkage between one
molecule of (R)-N-methylcoclaurine and one molecule of
(S)-N-methylcoclaurine to form the bisbenzylisoquinoline al-
kaloid berbamunine. This cytochrome P-450 enzyme alSO
couples two molecules of (R)-N-methylcoclaurineto form guat-
tegaumerine. The cDNA encoding berbamunine synthase was
isolated from a B. stolonifera cell suspension culture cDNA
library (Kraus and Kutchan, 1995). An oligonucleotide primer
based on the N-terminal amino acid sequence of the purified
native oxidase was used as a screening probe. Translation of
the nucleotide sequence of the clone revealed at least one
notable amino acid residue difference in berbamunine syn-
thase as compared with the sequences of cloned cytochrome
P-450 monooxygenases. Structure-function studies on bac-
teria1P-450camsuggest that three amino acid residues of the
dista1 helix (helix I), Gly-248, Gly-249, and Thr-252, are essen-
tia1 for formation of the molecular oxygen binding pocket
Alkaloids and Their Biosynthesis 1067
(Poulos et al., 1985). Mutation of Thr-252 to Ala or Val abol-
ishes insertion of oxygen into the substrate (Imai et al., 1989).
Thr-252 is present in berbamunine synthase, although there
is no monooxygenation reaction in the carbon-oxygen phe-
no1coupling process. However, the essential Ala or Gly at the
position corresponding to 248 in P-450cam is replaced by a
Pro in berbamunine synthase. Because this amino acid resi-
due is one of three highly conserved residues of the oxygen
binding pocket that are thought to be required for insertion
of oxygen into the substrate, this may be a first indication of
how berbamunine synthase functions as an oxidase.
Berbamunine synthase has been expressed in functional
form in insect cell culture using a baculovirus expression vec-
tor (Kraus and Kutchan, 1995). The oxidase accepts electrons
from either insect cell, porcine, or Berberis cytochrome P-450
reductases. Heterologously expressed berbamunine synthase
can be obtained in near-homogeneousform and in large quan-
tities (5 mgll) after insect cell microsomesolubilization followed
by a single column-chromatography fractionation step. To ob-
tain this amount of enzyme from B. stolonifera cells, 18,000
L of suspension culture would have to be extracted. A single
gene probably codes for berbamuninesynthase in the B. stolo-
nifera genome, although two more weakly hybridizing DNA
fragments are also present that may represent genes encod-
ing other phenol coupling cytochromes.
ECOCHEMICAL FUNCTION OF ALKALOIDS
Their medicinal and toxicological properties clearly make
alkaloids important to people, but their role in plants has been
a longstanding question. Why should a plant invest so much
nitrogen in synthesizing such a large number of alkaloids of
such diverse structure?C, meus alone contains over 100 differ-
ent monoterpenoid indole alkaloids. That many alkaloids are
cytotoxic provides insight into their potential function in the
chemical defense arsenal of the plant. It needs to be system-
atically addressed whether alkaloids function as phytoalexins,
as nitrogen storage forms, as UV protectants, or in any combi-
nation of these and other potential functions. The question of
alkaloid function in plants and the relevance of that function
have been reviewed (Hartmann, 1991; Caporale, 1995).
One alkaloid whose ecochemical functions have been thor-
oughly analyzed in recent years is nicotine, a highly toxic
alkaloid that has been identified in the leaves, stems, and roots
of a number of Nicotiana species. Nicotine sulfate, a byproduct
of the tobacco industry, serves commercially as a very effec-
tive insecticide and fumigant. To date, no insect has evolved
a resistance mechanism against nicotine. In mammals, inges-
tion of nicotine results in nausea, vomiting, diarrhea, mental
confusion, convulsions, and respiratory paralysis. With such
strong and varied physiological responses, nicotine would
seem to be an effective deterrent against insect attack and
herbivore grazing. This is indeed the case. A series of studies
in the laboratory of I.T. Baldwin has demonstrated that nico-
tine, which is known to be synthesized in roots and transported
1068 The Plant Cell
to leaves, is synthesized de novo from (15N)-nitrate in re-
sponse to wounding of leaves of hydroponically grown N. sy/-
vesfris (Baldwin et al., 1994a). The amount of alkaloid that can
be accumulated in the leaf is sufficient to reduce larva1growth
of the tobacco hornwormManducasexta (Baldwin, 1988). Most
importantly, field tests with native populations of the summer
annual N. artenuata demonstrated a systemic alkaloid induc-
tion in response to simulated leaf herbivory and browsing
(Baldwin and Ohnmeiss, 1993).
Nicotine is clearly synthesized in response to wounding, but
does the alkaloid have additional functions, for example, as
a nitrogen storage form oras a UV protectant?Although exog-
enously administered nicotine is catabolized to nornicotineand
myosmine, most likely as a result of a detoxification mecha-
nism, de novo-synthesized nicotine is not turned over. The
plant does not recover nicotine nitrogen and reinvest it in other
metabolic processes, even under conditions of nitrogen-limited
growth, implying that nicotine is not used as a nitrogen stor-
age form (Baldwin and Ohnmeiss, 1994). Nicotine supplied
exogenously to D. sframonium, a species that accumulates tro-
pane alkaloids but not nicotine, does not result in increased
UV protection, although nicotine has a high molar extinction
coefficient (2695 M-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 no additional role
as a storage form of nitrogen or as a filter for UV radiation.
These results raise an important question: How does leaf
wounding induce nicotine biosynthesisin the root? Jasmonates
are known to induce accumulation of secondary metabolites
in plant cell culture, and endogenousjasmonate pools increase
rapidly in response to treatment of cells with a yeast elicitor
(Gundlach et al., 1992). Leaf damage to N. sy/vesfris produces
a similar increase in endogenous jasmonic acid pools in shoots
within 30 min, and root pools of this signaling molecule in-
crease within 2 hr (Baldwin et al., 1994b). In addition,
application of methyl jasmonate as a lanolin paste to leaves
results in increases in both endogenous jasmonic acid in roots
and de novo nicotine biosynthesis. Jasmonate and 12-0x0-
phytodienoic acid function as inducers of alkaloid biosynthe-
sis both in culture and in plants. The next question that needs
to be addressed is whether jasmonate is the systemic signal-
ing molecule that is transported from the wounded leaf to roots,
where it then induces transcription of the alkaloid biosynthetic
genes.
PERSPECTIVES
Although the alkaloid field is a very old one, it is still in its in-
fancy with regard to being fully understood, and our exploitation
of the biotechnological potential of alkaloid biosynthesis has
only just begun. We are still a long way from understanding
how most alkaloids are synthesized in plants and how this bio-
synthesis is regulated. We also have much to learn about the
chemical ecology of alkaloids so that we can better understand
why these sophisticated and diverse structures evolved. What
is clear is that alkaloids as integral components of medicinal
plants have enjoyed a long and important history in traditional
medicine. Our first drugs originated in the form of plant extracts,
and some of our most important contemporary pharmaceuti-
cals are either still isolated from plants or structurally derived
from natural products. New antineoplastic, antiviral, and an-
timalarial alkaloids are still being discovered in plants. As we
accumulate the tools (biochemical knowledge, clones, and
transformation and regeneration techniques) necessary for fu-
ture developments in this field, we can look forward to
genetically engineered microorganismsand eukaryotic cell cul-
tures that produce medicinal alkaloids, to medicinal plants with
opitimized alkaloid spectra, and even to the production of im-
portant pharmaceuticals in transgenic plant cell cultures.
ACKNOWLEDGMENTS
Our moleculargenetic researchon strictosidine synthase,the berber-
ine bridge enzyme, and berbamuninesynthase has been supported
by a grant from the Bundesministerfijr Forschung und Technologie,
Bonn, and by the DeutscheForschungsgemeinschaft(Grant No. SFB
369), Bonn.
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