The Alkaloids: Chemistry and Biology 63

364 Pages·2006·4.44 MB·English

by  Geoffrey A. Cordell (Eds.)

THE ALKALOIDS

Chemistry and Biology

Edited by
Geoffrey A. Cordell
College of Pharmacy

University of Illinois at Chicago

Chicago, Illinois

VOLUME 63
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CONTRIBUTORS
Numbers in parentheses indicate the pages on which the authors’ contributions begin.
JAUME BASTIDA (87), Departament de Productes Naturals, Facultat de
Farma` cia, Universitat de Barcelona, 08028 Barcelona, Spain
YEUN-MUN CHOO (181), Department of Chemistry, University of Malaya,
50603 Kuala Lumpur, Malaysia
PETER J. FACCHINI (1), Department of Biological Sciences, University of
Calgary, Calgary, AB, Canada
TOH-SEOK KAM (181), Department of Chemistry, University of Malaya,
50603 Kuala Lumpur, Malaysia
RODOLFO LAVILLA (87), Parc Cientı´fic de Barcelona, Universitat de
Barcelona, 08028 Barcelona, Spain
DANIEL G. PANACCIONE (45), Division of Plant and Soil Sciences,
West Virginia University, Morgantown, WV 26506-6108, USA
CHRISTOPHER L. SCHARDL (45), Department of Plant Pathology, University of
Kentucky, Lexington, KY 40546-0312, USA
PAUL TUDZYNSKI (45), Institut fu¨ r Botanik, Westfa¨ lische Wilhelms
Universita¨ t Mu¨ nster, Mu¨ nster D-48149, Germany
FRANCESC VILADOMAT (87), Departament de Productes Naturals, Facultat de
Farma` cia, Universitat de Barcelona, 08028 Barcelona, Spain
vii
PREFACE

This volume of The Alkaloids: Chemistry and Biology is comprised of four very
different chapters; a reflection of the diverse facets that comprise the study of
alkaloids today. As awareness of the global need for natural products which can be
made available as drugs on a sustainable basis increases, so it has become increas­
ingly important that there is a full understanding of how key metabolic pathways
can be optimized. At the same time, it remains important to find new biologically
active alkaloids and to elucidate the mechanisms of action of those that do show
potentially useful or novel biological effects.
Facchini, in Chapter 1, reviews the significant studies that have been
conducted with respect to how the formation of alkaloids in their various diverse
sources are regulated at the molecular level.
The history of the ergot alkaloids and their biological effects is a very rich
and fascinating one. In Chapter 2, advances in the biosynthetic pathway of these
alkaloids are discussed, the genes involved are reviewed, and their biological and
clinical significance are presented by Schardl, Panaccione, and Tudzynski.
In Chapter 3, Bastida, Lavilla, and Viladomat provide a classical review
of the isolation, structure elucidation, biosynthesis, synthesis, and biology of the
Narcissus alkaloids. This group of alkaloids is receiving a great deal of attention at
the present time because of the significant biological activities observed for some of
these metabolites.
It has been over 25 years since the broad area of the bisindole alkaloids was
reviewed in this series. In the concluding chapter, Kam and Choo have taken on
this formidable task and provide a very comprehensive summation of tremendous
advances in the structural, synthetic, and biological aspects of more than 30 basic
alkaloid types represented.
Geoffrey A. Cordell
University of Illinois at Chicago
ix
—C 1—
HAPTER
REGULATION OF ALKALOID BIOSYNTHESIS IN
PLANTS
PETER J. FACCHINI
Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada
I. Introduction
II. Alkaloid Biosynthetic Pathways
III. Regulation ofAlkaloid Biosynthesis
IV. Applications ofGenomics to the Studyof Alkaloid Biosynthesis
V. Metabolic Engineering of Alkaloid Pathways
VI. Future Prospects
Acknowledgments
References
I. Introduction
Several key technical breakthroughs over the last half-century have contrib-
uted to an impressive advancement in our understanding of alkaloid biosynthesis in
plants. The use of radiolabeled precursors in the 1950s allowed the elucidation of
several biogenic pathways. The widespread application of plant cell cultures during
the1970sprovidedarichsourceofbiosyntheticenzymesandencouragedworkonthe
elucidation of signal transduction mechanisms that activate alkaloid pathways. The
introductionofmoleculartechniquesinthe1990spromptedtheisolationofnumerous
molecular clones involved in alkaloid biosynthesis (1), which have been used to de-
termine the tissue-specific localization of alkaloid biosynthetic enzymes and gene
transcripts, and functionally analyze the corresponding promoters. Recent applica-
tionsofgenomics-basedtechnologies,suchasexpressedsequencetag(EST)databas-
es,DNAmicroarrays,andproteomeanalysis,haveshownthepotentialtoaccelerate
the discovery of new components and mechanisms involved in the assembly and
function of plant alkaloids. Our emerging ability to investigate alkaloid metabolism
from a combined biochemical, molecular, cellular, and physiological perspective has
greatly improved our appreciation for the complex regulation of diverse biosynthetic
pathways. Unlike other types of secondary metabolites, the different structural cat-
egoriesofalkaloidshaveuniquebiosyntheticorigins.Thisreviewwillfocusonrecent
advances in our understanding of the metabolic regulation involved in the biosyn-
thesisofsixgroupsofalkaloids–benzylisoquinoline,monoterpenoidindole,tropane,
purine, pyrrolizidine, and quinolizidine alkaloids – for which biosynthetic and reg-
ulatory genes have been reported and characterized.
THEALKALOIDS,Vol.63 Copyrightr2006ElsevierInc.
1
ISSN: 1099-4831 Allrightsreserved
DOI: 10.1016/S1099-4831(06)63001-0
2 FACCHINI
II. Alkaloid Biosynthetic Pathways
A. BENZYLISOQUINOLINE ALKALOIDS
Manybenzylisoquinolinealkaloids(BAs)areusedaspharmaceuticalsdueto
theirpotentpharmacologicalactivity,whichisoftenanindicationoftheirbiological
function.Forexample,theeffectivenessofmorphineasananalgesic,colchicineasa
microtubule disrupter, and (+)-tubocurarine as a neuromuscular blocker suggests
thatthesealkaloidsfunctionasherbivoredeterrents.Theantimicrobialpropertiesof
sanguinarine and berberine suggest that they confer protection against pathogens
(2). BAs occur mainly in basal angiosperms, including the members of Ran-
unculaceae, Papaveraceae, Berberidaceae, Menispermaceae, and Magnoliaceae.
BA biosynthesis begins with the conversion of tyrosine into both dopamine
and 4–hydroxyphenylacetaldehyde by a lattice of decarboxylations, ortho-hydro-
xylations, and deaminations (3). The aromatic amino acid decarboxylase (TYDC)
that converts tyrosine and dopa into their corresponding amines has been purified,
and several cDNAs have been cloned (Scheme 1) (4–6).
A family of (cid:1)15 genes, which can be divided into two subgroups based on
sequence identity, encodes TYDC in opium poppy (Papaver somniferum) (5). The
catalyticpropertiesofthevariousproteinisoformsaresimilardespitethedifferential
developmental and inducible expression of the TYDC gene family (5,7–9). Dopa-
mine and 4–hydroxyphenylacetaldehyde are condensed by norcoclaurine synthase
(NCS) to yield (S)-norcoclaurine (Scheme 1), the central precursor to all BAs in
plants (10,11). Owing to the inability of NCS to discriminate between 4–hydroxy-
phenylacetaldehyde and 3,4–dihydroxyphenylacetaldehyde, and the nonspecificity
of the subsequent methyltransferase reactions, it was originally thought that the
tetrahydroxyBA (S)-norlaudanosoline was the precursor (12). However, only nor-
coclaurine has been found to occur in plants. NCS has been purified (13,14) and
correspondingmolecularcloneshavebeenisolatedfromThalictrumflavum(15)and
P. somniferum (16). NCS is ancestrally related to the pathogenesis-related (PR) 10
and Bet v 1 protein families, which include the enzyme (HYP1) from St. John’s
wort (Hypericum perforatum) responsible for the biosynthesis of the bioactive
naphthodianthronehypericin(17).ThenovelcatalyticfunctionsofNCSandHYP1
define a new class of plant secondary metabolic enzymes.
(S)-Norcoclaurine is converted into (S)-reticuline by a 6–O-methyltransferase
(6OMT), an N-methyltransferase (CNMT), a P450 hydroxylase (CYP80B1), and a
40-O-methyltransferase(40OMT)(Scheme1)(18–21).6OMTand4’OMTwerepurified
(18),andcorrespondingcDNAsisolatedand characterizedfromCoptisjaponica (22),
T. flavum (23), and P. somniferum (24–26). Each enzyme exhibits unique substrate
specificity and a different reaction mechanism despite extensive homology. A previ-
ously reportedfamily ofcatechol O-methyltransferases that accepts (S)-norcoclaurine
asa substrate isprobablynot involved inalkaloidbiosynthesis(27). CNMThasalso
been purified (28), and the corresponding cDNA isolated from C. japonica (29), T.
flavum (23), and P.somniferum (24). The aromaticring hydroxylation involvedin the
conversion of (S)-norcoclaurine into (S)-reticuline was once thought to proceed via a
nonspecific phenol oxidase (30). However, a P450-dependent monooxygenase
(CYP80B1) was shown to have a lower K for (S)-N-methylcoclaurine than the
m
REGULATIONOFALKALOIDBIOSYNTHESIS 3
Scheme 1. Biosynthesis of the benzylisoquinoline alkaloids berberine, morphine, and sang-
uinarine.Enzymesforwhichcorrespondingmolecularcloneshavebeenisolatedareshownin
bold.Abbreviations:40OMT,30-hydroxy-N-methylcoclaurine40-O-methyltransferase;6OMT,
norcoclaurine 6-O-methyltransferase; 7OMT, reticuline 7-O-methyltransferase; BBE,
berberinebridgeenzyme;CFS,cheilanthifolinesynthase;CNMT,coclaurineN-methyltransf-
erase; COR, codeinone reductase; CYP719A1, canadine synthase; CYP80A1, berbamunine
synthase; CYP80B1, N-methylcoclaurine 30-hydroxylase; DBOX, dihydrobenzophenanthri-
dine oxidase; DRR, 1,2-dehydroreticuline reductase; DRS, 1,2-dehydroreticuline synthase;
MSH, N-methylstylopine 14-hydroxylase; NCS, norcoclaurine synthase; P6H,
(contd)
4 FACCHINI
phenolase and is now accepted as the enzyme catalyzing the conversion of
(S)-N-methylcoclaurine into (S)-3’-hydroxy-N-methylcoclaurine (Scheme 1) (21).
Molecular clones encoding CYP80B1 have been isolated from California poppy
(Eschscholzia californica) (21), P. somniferum (31,32), and T. flavum (23).
(S)-Reticuline pathway intermediates also serve as precursors to the bis-
benzylisoquinoline alkaloids, such as (+)-tubocurarine (Scheme 1). A phenol-
coupling P450-dependent oxidase (CYP80A1) was purified, and the corresponding
cDNA was isolated from Berberis stolonifera (33,34). CYP80A1 couples two mol-
eculesof(R)-N-methylcoclaurineoroneeachof(R)-and(S)-N-methylcoclaurineto
form(R,R)-guattegaumerineor(R,S)-berbamunine,respectively(Scheme1).Phenyl
ring substitutions, regiospecificity, number of ether linkages, and monomer stere-
ospecificityadd additional diversity to thebis-benzylisoquinolinealkaloids. Acyto-
chrome P450 reductase (CPR) was purified from opium poppy, and corresponding
cDNAs were isolated from opium poppy and E. californica (35).
(S)-Reticulineisabranch-pointintermediateinthebiosynthesisofmostBAs
and many substituted derivatives are produced. For example, a molecular clone
encoding (R,S)-reticuline 7–O-methyltransferase (7OMT), which catalyzes the con-
versionof(R,S)-reticulineintolaudanine,hasbeenidentified(25).Muchofthework
has focused on branch pathways leading to the benzophenanthridine (e.g., sang-
uinarine), protoberberine (e.g., berberine), and morphinan (e.g., morphine and co-
deine) alkaloids (36). A multitude of relevant enzymes have been isolated, many
have been purified, and an impressive number of corresponding cDNAs have been
cloned (36). The first committed step in benzophenanthridine and protoberberine
alkaloidbiosynthesisinvolvestheconversionof(S)-reticuline into(S)-scoulerineby
the berberine bridge enzyme (BBE) (Scheme 1). BBE was purified from Berberis
beaniana(37),correspondingcDNAswereclonedfromB.stolonifera,E.californica,
and T. flavum (23,38,39), the recombinant enzyme was characterized (40,41), and
BBE genes were isolated from P. somniferum and E. californica (42,43). Interest-
ingly, BBE belongs to the same FAD-dependent oxidoreductase family as D1-tetra-
hydrocannabinolic acid synthase, which is involved in the biosynthesis of the psy-
choactive compound D1-tetrahydrocannabinol in Cannabis sativa (44).
Benzophenanthridine alkaloid biosynthesis requires the conversion of
(S)-scoulerine into (S)-stylopine by two P450-dependent oxidases, (S)-chelanthifo-
line synthase (CFS) and (S)-stylopine synthase (SPS), resulting in the formation
of two methylenedioxy bridges (45,46), (S)-Stylopine is N-methylated by
tetrahydroprotoberberine-cis-N-methyltransferase, which has been isolated from
E. californica and Corydalis vaginans cells (47), and purified from Sanguinaria can-
adensis cultures (48). A P450-dependent monooxygenase, (S)-cis-N-methylstylopine
14–hydroxylase(MSH),thencatalyzestheformationofprotopine(49).AnotherP450-
dependent enzyme, protopine-6–hydroxylase (PPH) followed by a spontaneous intra-
molecular rearrangement converts protopine into dihydrosanguinarine (50). The oxi-
dationofdihydrosanguinarinetosanguinarineoccursviadihydrobenzophenanthridine
protopine 6-hydroxylase; SAT, salutaridinol 7-O-acetyltransferase; SOMT, scoulerine 9-
O-methyltransferase; SOR, salutaridine:NADPH 7-oxidoreductase; SPS, stylopine synthase;
STOX, (S)-tetrahydroxyprotoberberine oxidase; STS, salutaridine synthase; TNMT, tetra-
hydroprotoberberine cis-N-methyltransferase; TYDC, tyrosine decarboxylase.
REGULATIONOFALKALOIDBIOSYNTHESIS 5
oxidase (DBOX) (51), a cytosolic enzyme purified from S. canadensis cultures (52).
Two other species-specific enzymes, dihydrochelirubine-12–hydroxylase and 12–
hydroxydihydrochelirubine-12–O-methyltransferase, catalyze the final two steps in the
biosynthesisofmacarpine,the most highlyoxidizedBA foundinnature(53).
In some plants, (S)-scoulerine is methylated, rather than oxidized, to yield
(S)-tetrahydrocolumbamine(Scheme1).Thereactioniscatalyzedbyscoulerine-9–O-
methyltransferase (SOMT) (54), which was purified, and the corresponding cDNA
isolated, from C. japonica (55,56) and T. flavum (23). The P450-dependent enzyme
canadine synthase (CYP719A) was detected in members of the genera Coptis
and Thalictrum and shown to catalyze methylenedioxy bridge formation in
(S)-tetrahydrocolumbamine (57), but not in the quaternary alkaloid columbamine
(58), showing that berberine biosynthesis cannot proceed via columbamine as once
proposed. Molecular clones for CYP719A1 have been isolated from C. japonica (59)
and T.flavum (23). (S)-Canadine,alsoknown as(S)-tetrahydroberberine,isoxidized
to berberine by either (S)-canadine oxidase (CDO) or (S)-tetrahydroprotoberberine
oxidase(STOX)(60).Theseenzymescatalyzethesamereaction,buttheirbiochemical
propertiesaredistinct.STOXfromthegenusBerberisisaflavinylatedproteinwitha
broadsubstraterange,whereasCDOfromthegeneraCoptisandThalictrumcontains
iron, proceeds via a different mechanism, and prefers (S)-canadine as a substrate.
Conversion of (S)-reticuline into its (R)-epimer is the first committed step in
morphinan alkaloid biosynthesis in certain species. The still poorly characterized en-
zymes1,2–dehydroreticulinesynthaseand1,2–dehydroreticulinereductasecatalyzethe
stereospecific reduction of 1,2–dehydroreticuline to (R)-reticuline (61,62). Intramolec-
ular carbon–carbon phenol coupling of (R)-reticuline by the P450-dependent enzyme
salutaridine synthase (STS) results in the formation of salutaridine (63). The cytosolic
enzyme, salutaridine:NADPH7–oxidoreductase (SOR),foundinPapaver bracteatum
and P. somniferum reduces salutaridine to (7S)-salutaridinol (64). Conversion of
(7S)-salutaridinolintothebainerequiresclosureofanoxidebridgebetweenC-4andC-5
byacetylcoenzymeA:salutaridinol-7–O-acetyltransferase(SAT).Theenzymewaspu-
rifiedfromopiumpoppycellculturesandthecorrespondingcDNAisolated(Scheme1)
(65,66).Inthelaststepsofmorphinebiosynthesis,codeinoneisproducedfromthebaine
and then reduced to codeine, which is finally demethylated to yield morphine. Codei-
none reductase (COR), which reduces (–)-codeinone to (–)-codeine, has been purified
and the correspondingcDNAisolatedfromP.somniferum(Scheme1) (67,68).
B. MONOTERPENOID INDOLE ALKALOIDS
Monoterpenoid indolealkaloidsare found mainly inthe Apocynaceae, Log-
aniaceae, and Rubiaceae. Many have potent biological activities (69), including the
cerebral metabolism enhancing alkaloids of periwinkle (Vinca minor), the antima-
larial and cardiotonic alkaloids of cinchona (Cinchona ledgeriana), and the anti-
canceragentsfromChinese‘‘happy’’tree(Camptothecaacuminata).Thewell-known
centralnervousstimulantsstrychnineandyohimbinearealsomonoterpenoidindole
alkaloids. Perhaps the most important from a health perspective are the anticancer
agents vincristine and vinblastine from the Madagascar periwinkle (Catharanthus
roseus). The importance of C. roseus as a source of anticancer medicines has
prompted intensive research on alkaloid biosynthesis in this plant.
6 FACCHINI
Monoterpenoid indole alkaloids consist of an indole moiety provided by
tryptamine and a terpenoid component derived from the iridoid glucoside seco-
loganin (Scheme 2). Molecular clones forboth the a-and b-subunits of anthranilate
synthase (AS), which catalyze the first committed reaction of the indole pathway,
have been isolated from C. acuminata (70,71). Comparison of the two differentially
regulated genes encoding the AS a-subunit showed that the spatial and develop-
mentalexpressionofonlyoneparalleledthatoftheb-subunitgene,andthepattern
ofcamptothecinaccumulation.Thus,theindoleandmonoterpenoidindolealkaloid
pathways appear coordinately regulated through the duplication of specific genes,
suchasthatencodingtheASa-subunit.Tryptophanisconvertedintotryptamineby
tryptophan decarboxylase (TDC), which is encoded by a single gene in C. roseus
(72–74), and by two autonomously regulated genes in C. acuminata (75).A
molecular clone for TDC was also reported from Ophiorrhiza pumila (76).
Secologanin is formed from precursors derived from the triose phosphate/
pyruvate pathway (77). Two cDNAs encoding the enzymes 1–deoxy-D-xylulose
5–phosphatereductoisomerase(DXR)and2C-methyl-D-erythritol2,4–cyclodiphos-
phatesynthase(MECS)ofthe2C-methyl-D-erythritol4–phosphate(MEP)pathway
were isolated from C. roseus (78). The corresponding gene transcripts were induced
in C. roseus cell cultures producing monoterpenoid indole alkaloids. The first com-
mitted step in secologanin biosynthesis is the hydroxylation of geraniol to 10–hy-
droxygeraniol (79,80). A novel P450-dependent monooxygenase (CYP76B6) is
specific for the C-10 position of geraniol and exhibits similar affinity for nerol, the
cis-isomer of geraniol. The enzyme was purified and shown to contain FMN and
FAD as cofactors (81), and the corresponding cDNA was isolated (Scheme 2) (82).
Conversionofloganinintosecologaninrepresentsthelaststepinthepathwayandis
catalyzed by another P450-dependent enzyme (CYP72A1) for which the corre-
spondingcDNAhasalsobeenreported(Scheme2)(83,84).Theenzyme3–hydroxy-
3–methylglutaryl coenzyme A reductase (HMGR), which is involved in the biosyn-
thesis of mevalonate, was cloned and characterized from C. roseus (85) and C.
acuminata (86,87). The differential expression of HMGR genes in response to
wounding and methyl jasmonate (MeJA) was suggested to contribute to the reg-
ulation of terpenoid indole alkaloid (TIA) biosynthesis. However, the formation of
secologanin via the non-mevalonate terpenoid pathway (77) indicates that the cor-
relation between HMGR expression and TIA accumulation is coincidental.
Tryptamine and secologanin condense to form strictosidine, the common pre-
cursortoallmonoterpenoidindolealkaloids,by strictosidinesynthase(STR)(Scheme
2).STRcDNAshavebeenisolatedfromRauwolfiaserpentina,C.roseus,andO.pumila
(88–91).CrystallizationandpreliminaryX-rayanalysisofSTRfromR.serpentinahas
alsobeenreported(92). Strictosidineisdeglucosylatedbystrictosidineb-D-glucosidase
(SGD), which has been purified (93), and the corresponding cDNA isolated from C.
roseus(94)andR.serpentina(95)cellcultures(Scheme2).STRandSGDhavealsobeen
crystallized and preliminary X-ray analyses have been performed (96,97). Deglucosy-
lated strictosidine is converted via several unstable intermediates into 4,21–de-
hydrogeissoschizine. Although many monoterpenoid indole alkaloids are produced
from 4,21–dehydrogeissoschizine, the enzymology of the branch pathways leading to
catharanthineandmostotherproductsis poorlyunderstood.However,the final steps
of vindoline biosynthesis from tabersonine have been characterized in considerable
detail(Scheme2).