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Alkaloid Biosynthesis:
Metabolism and Trafcking
Ziegler and Peter J. Facchini
Jorg
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
735
ANRV342-PP59-29 ARI 29 March 2008 2:13
Figure 1
Biosynthesis of the monoterpenoid indole alkaloids vinblastine and ajmaline. Enzymes for which
corresponding molecular clones have been isolated are shown in bold. Abbreviations: AAE, acetylajmalan
esterase; ANAMT, acetylnorajmalan methyltransferase; Cyp72A1, secologanin synthase; Cyp76B6,
geraniol 10-hydroxylase; D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline
4-O-acetyltransferase; DHVR, dihydrovomilenine reductase; LAMT, loganic acid methyltransferase;
MAT, minovincinine acetyltransferase; NAMT, norajmalan methyltransferase; NMT,
N-methyltransferase; PER, peroxidase; PNAE, polyneuridine aldehyde esterase; RG, raucaffricine
O--glucosidase; SBE, sarpagan bridge enzyme; SGD, strictosidine -d-glucosidase; STR, strictosidine
synthase; T16H, tabersonine 16-hydroxylase; TDC, tryptophan decarboxylase; VH, vinorine
hydroxylase; VR, vomilenine reductase; VS, vinorine synthase.
736 Ziegler Facchini
ANRV342-PP59-29 ARI 29 March 2008 2:13
Geraniol
Cyp76B6 LAMT
Cyp72A1 TDC
10-Hydroxygeraniol LAMT
SGD
T16H
SBE PNAE
NMT
VH
Vomilenine Vinorine
Lochnericine 6,7-Dehydrominovincinine
VR
MAT
Desacetoxyvindoline
DHVR
D4H
1,2-Dihydrovomilenine Acetylnorajmaline
Hrhammericine 6,7-Dehydroechitovenine
ANAMT
AAE
MAT
Deacetylvindoline
DAT
Norajmaline Acetylajmaline
19-O-Acetylhrhammericine
NAMT AAE
Vindoline Cantharanthine
RG Ajmaline
PER
Raucaffricine
Vinblastine
tryptophan decarboxylase (TDC) converts quently converted via several unstable in-
tryptophan to tryptamine; the gene encoding termediates to dehydrogeissoschizine, which
this enzyme has been cloned from different represents a key branchpoint intermediate
MIA-accumulating plants (25, 89, 178). that leads to several diverse MIA pathways.
Incorporation experiments with isotopically The branch pathways proceeding through
labeled glucose in C. roseus cell cultures tabersonine and polyneuridine aldehyde have
showed that the initial steps for the biosyn- been characterized most thoroughly. Other
thesis of the monoterpenoid skeleton proceed branch pathways, such as that leading to
via the triose phosphate/pyruvate pathway catharanthine, remain poorly understood.
(22). Cognate cDNAs for 1-deoxy-d-xylulose Hydroxylation of tabersonine at position
5-phosphate reductoisomerase and 2C- 16 represents the rst reaction leading
methyl-d-erythritol 2,4-cyclodiphosphate to vindoline (Figure 1). The responsi-
synthase were isolated from C. roseus and ble enzyme, tabersonine 16-hydroxylase
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
the corresponding gene transcripts were (T16H, Cyp71D12), belongs to the P450
upregulated in MIA-producing cell cul- monooxygenase family, and the correspond-
tures (163). The rst committed step in ing cDNA was identied from a subset
by Washington State University on 11/01/11. For personal use only.
minovincinine acetyltransferase (MAT) shows though the glucosylating activity has not been
residual activity toward the DAT substrate, reported, researchers found a cDNA encod-
deacetoxyvindoline, DAT accepts only its ing the enzyme raucaffricine O--glucosidase
BIA:
natural substrate. (RG), responsible for deglycosylation of rau- benzylisoquinoline
The polyneuridine aldehyde branch of the caffricine (168). RG shares 60% amino acid alkaloid
MIA pathway is initiated by the formation of identity with SGD and accepts strictosidine as Sanguinarine: an
a bond, known as the sarpagan bridge, be- a substrate, whereas SGD is not active against antimicrobial
tween C5 and C16 of strictosidine-derived in- raucaffricine. benzylisoquinoline
termediate compounds (Figure 1). The en- alkaloid sometimes
used in oral hygiene
zyme characterized from crude extracts of R. Benzylisoquinoline alkaloids. Benzyliso-
products
serpentina shows NADPH and oxygen depen- quinoline alkaloids (BIA) represent approxi-
dence, and is sensitive to P450 inhibitors, sug- mately 2,500 elucidated structures and possess
gesting it is a P450 monooxygenase (139). The potent pharmacological properties. The most
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
nant polyneuridine aldehyde esterase (PNAE) the antimicrobial agents sanguinarine and
from R. serpentina shows esterase activity only berberine. Collectively, these alkaloids occur
to polyneuridine aldehyde and not to struc- mainly in the Papaveraceae, Ranunculaceae,
turally related esters, and is a member of the Berberidaceae, and Menispermaceae; Papaver
/ hydrolase superfamily (29). With approx- somniferum (opium poppy), Eschscholzia cali-
imately 30% identity on the amino acid level forncia, Thalictrum species, and Coptis japonica
to DAT and MAT, vinorine synthase (VS) are the most extensively investigated species.
from R. serpentina also belongs to the BAHD BIA biosynthesis, which fundamentally
family of acyltransferases (11). Acetylajmaline involves the condensation of two tyrosine
is subsequently formed via a series of reac- derivatives, begins with the decarboxylation
tions that includes hydroxylation, two dou- of tyrosine to tyramine or of dihydroxypheny-
ble bond reductions, and an N-methylation. lalanine to dopamine by tyrosine decarboxy-
The enzymes involved in these steps have lase (TYDC). TYDC constitutes a large gene
been biochemically characterized as the P450- family; approximately 15 members are found
dependent monooxygenase vinorine hydrox- in opium poppy (33). Dopamine is the pre-
ylase (VH), which yields vomilenine, and the cursor for the isoquinoline moiety, whereas
NADPH-dependent reductases vomilenine 4-hydroxyphenylacetaldehyde, resulting from
reductase and 1,2-dehydrovomilenine reduc- the deamination of tyramine, is incorporated
tase (39, 44, 166). Acetylajamaline esterase as the benzyl component (Figure 2). As is
(AAE) ultimately hydrolyzes the 17-O-acetyl the case in MIA biosynthesis, BIA conden-
group to produce ajmaline. Active recombi- sation is a Pictet-Spenglertype reaction and
nant AAE, which could be produced only via is catalyzed by the rst committed step of
a virus expression system in Nicotiana ben- the pathway, norcoclaurine synthase (NCS).
thamiana, shows slight substrate preference The enzyme has been puried from Thalic-
for acetylajmaline compared with norajma- trum avum and corresponding cDNAs have
line. The latter compound might represent been isolated and functionally characterized
an intermediate in a parallel ajamaline biosyn- from opium poppy and T. avum (87, 136,
thetic pathway in which N-methylation occurs 137). NCS is related to the pathogenesis
after deesterication (134). Unlike PNAE, related protein (PR) 10 and Bet v 1 allergen
AAE belongs to the GDSL lipase family. Rau- protein families. However, homologous PR10
caffricine, a glucosylated derivative of vom- proteins from opium poppy are not catalyt-
ilenine, is found in R. serpentina cultures. Al- ically active. Biochemical characterization of
TYDC NCS
L-Dopa Dopamine
(S)-Norcoclaurine
4-Hydroxyphenylacetaldehyde 6OMT
Papaverine
CoOMT
CNMT
Cyp719A1
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
Cyp80A1 Bisbenzylisoquinoline
alkaloids
Cyp80B3
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DRS DRR
BBE
SalR
Cyp719A2
(S)-Stylopine (S)-Cheilanthifoline Laudanine
Salutaridinol Salutaridine
TNMT SalAT
MSH
(S)-cis-N-Methylstylopine Protopine Thebaine
Salutaridinol-7-O-acetate
P6H
Spontaneous
Dihydrosanguinarine 6-Hydroxyprotopine Neopinone Oripavine Morphinone
COR
recombinant T. avum NCS has been ac- cludes catechols and phenylpropanoids in
complished using a continuous enzyme assay addition to various BIA derivatives (42).
based on circular dichroism spectroscopy Coclaurine N-methyltransferases (CNMT)
that follows the generation of the enzymes have been cloned from opium poppy and
chiral product. These studies revealed a C. japonica and are more closely related to
reaction mechanism that involves a two- S-adenosyl-L-methionine (SAM)-dependent
step cyclization with a direct electrophilic cyclopropane fatty acid synthases than other
aromatic substitution (91). Recently, a N-methyltransferases (19, 35). The hydroxy-
second enzyme capable of producing only lation of N-methylcoclaurine is catalyzed by
the (S )-norcoclaurine enantiomer was iso- a P450 monooxygenase classied in the Cyp
lated from C. japonica; this enzyme displays subfamily 80B (65, 68, 125, 138).
sequence similarity to 2-oxoglutarate de- (S )-Reticuline is the central pathway in-
pendent dioxygenases (102). However, this termediate from which most BIA structural
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
enzyme does not possess a 2-oxoglutarate- types are derived. Only the dimeric bisben-
binding domain and requires ferrous ions, zylisoquinoline alkaloids are not produced
rather than 2-oxoglutarate or oxygen, for via (S )-reticuline. In this pathway, the cy-
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activity. Remarkably, two proteins in two tochrome P450 enzyme Cyp80A1 catalyzes
different protein families can catalyze the the regio- and stereoselective oxidative C-
same reaction in vitro. Nevertheless, the O phenol coupling of the reticuline pre-
relative participation of each enzyme in BIA cursor N-methylcoclaurine to produce the
biosynthesis in vivo must still be determined. bisbenzylisoquinoline alkaloid berbamunine
The conversion of (S )-norcoclaurine to (78). Another exception where reticuline
(S )-reticuline involves O-methylation at posi- does not represent an intermediate com-
tion 6, N-methylation, 3 -hydroxylation, and pound might be the biosynthesis of the sim-
a second 4 -O-methylation (Figure 2). Nor- ple N-demethylated BIA papaverine. The re-
coclaurine 6-O-methyltransferase (6OMT) cent discovery of a 7-O-methyltransferase
and 3 -hydroxy-N-methylcoclaurine 4 -O- specic for norreticuline (N7OMT, nor-
methyltransferase (4 OMT) are both class II reticuline 7-O-methyltransferase), but not
O-methyltransferases that display strict re- N-methylated analogs, suggests that (S )-
giospecicity. Cognate cDNAs have been ob- reticuline precursors might enter the path-
tained for each enzyme from opium poppy way to papaverine (S. Pienkny, J. Ziegler
and C. japonica (107, 121, 182). Additional & W. Brandt, manuscript in prepara-
6OMT homologs from Thalictrum tubero- tion). A second O-methyltransferase that
sum were functionally characterized and ex- acts on reticuline and catalyzes the forma-
hibit a broader substrate specicity that in- tion of laudanine was previously isolated
Figure 2
Biosynthesis of the benzylisoquinoline alkaloids berberine, morphine, and sanguinarine. Enzymes for
which corresponding molecular clones have been isolated are shown in bold. Abbreviations: 4 OMT,
3 -hydroxy-N-methylcoclaurine 4 -O-methyltransferase; 6OMT, norcoclaurine 6-O-methyltransferase;
7OMT, reticuline 7-O-methyltransferase; BBE, berberine bridge enzyme; CFS, cheilanthifoline
synthase; CNMT, coclaurine N-methyltransferase; CoOMT, columbamine O-methyltransferase; COR,
codeinone reductase; Cyp719A1, canadine synthase; Cyp719A2, stylopine synthase; Cyp719B1,
salutaridine synthase; Cyp80A1, berbamunine synthase; Cyp80B3, N-methylcoclaurine 3 -hydroxylase;
DBOX, dihydrobenzophenanthridine oxidase; DRR, 1,2-dehydroreticuline reductase; DRS,
1,2-dehydroreticuline synthase; MSH, N-methylstylopine 14-hydroxylase; NCS, norcoclaurine synthase;
P6H, protopine 6-hydroxylase; SalAT, salutaridinol 7-O-acetyltransferase; SalR, salutaridine:NADPH
7-oxidoreductase; SOMT, scoulerine 9-O-methyltransferase; STOX, (S )-tetrahydroxyprotoberberine
oxidase; TNMT, tetrahydroprotoberberine cis-N-methyltransferase; TYDC, tyrosine decarboxylase.
from opium poppy (121). (R,S )-Reticuline nium compounds. Subsequent hydroxylation
7-O-methyltransferase (7OMT) does not ac- by (S )-cis-N-methylstylopine 14-hydroxylase
cept N-demethylated BIA substrates, but is ac- yields protopine, which is further hydrox-
tive toward phenolic compounds. ylated by protopine 6-hydroxylase to dihy-
A major branch pathway that gives rise drosanguinarine. Both enzymes have been de-
to many BIA classes begins with the forma- tected in protopine alkaloid-containing cell
tion of (S )-scoulerine by the berberine bridge cultures, and their characterization suggests
enzyme (BBE) (Figure 2). This enzyme has that they are P450 monooxygenases (132,
been cloned from several sources (28, 36, 138) 156). Dihydrobenzophenanthridine oxidase,
and was recently characterized thoroughly which converts dihydrosanguinarine to san-
(174, 175). BBE belongs to a novel family guinarine, has been puried from Sanguia-
of avoproteins that possess two covalent at- naria canadensis (7).
tachment sites for avin adenine dinucleotide An alternative branch for the metabolism
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
(FAD); one is histidine and the other is cys- of (S )-scoulerine in some species involves the
teine. The cysteinylation of the cofactor in- formation of (S )-tetrahydrocolumbamine by
creases the midpoint redox potential to a value scoulerine 9-O-methyltransferase (SOMT)
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higher than that observed for other avo- (Figure 2) (155). The cDNA encoding
proteins, thereby facilitating hydride abstrac- SOMT from C. japonica was the rst re-
tion of (S )-reticuline. This step represents the ported clone for an O-methyltransferase
rst half reaction toward the conversion to specically implicated in BIA metabolism.
(S )-scoulerine. The biosynthesis of ben- All O-methyltransferases in the BIA pathway
zophenanthridine alkaloids is initiated by the exhibit considerable homology. Tetrahydro-
formation of two methylenedioxy bridges columbamine is converted to columbamine,
resulting in (S )-cheilanthifoline and (S )- which is methylated by columbamine
stylopine (Figure 2). Both reactions are cat- O-methyltransferase (CoOMT) to yield
alyzed by P450-dependent monooxygenases palmatine (106). Recombinant CoOMT is
and two cDNAs coding for stylopine syn- active only against protoberberine alkaloid
thase have been cloned from E. californica and substrates, such as scoulerine or tetrahy-
classied as Cyp719A2 and Cyp719A3 (67). drocolumbamine, but not against other
Both recombinant proteins show the same re- BIA derivatives. The subsequent formation
giospecicity for methylendioxy bridge for- of a methylenedioxy bridge is catalyzed
mation, but Cyp719A2 only converts (S )- by canadine synthase, a P450 enzyme
cheilanthifoline to (S )-stylopine. In contrast, that belongs to the Cyp719A family (68).
Cyp719A3 also accepts compounds without Cyp719A1 displays high substrate specicity
a pre-existing methylenedioxy bridge. (S )- for tetrahydrocolumbamine and does not
Stylopine is subsequently N-methylated to accept columbamine. As such, a parallel path
(S )-cis-N-methylstylopine by tetrahydropro- to berberine via this columbamine is unlikely.
toberberine N-methyltransferase (TNMT). (S )-Canadine, or (S )-tetrahydroberberine
On the basis of homology to CNMT, a as it is also called, is oxidized by either (S )-
cDNA encoding TNMT has been isolated canadine oxidase or (S )-tetrahydroberberine
and functionally characterized from opium oxidase (STOX), which catalyze the same
poppy (86). The enzyme shows a narrow sub- reaction but show substantially different
strate range in that it converts only tetrahy- biochemical properties.
droprotoberberine alkaloids with dimethoxy Whereas all pathways downstream of reti-
or methylenedioxy functional groups at C2/3 culine begin with the (S )-epimer, conversion
and C9/10, respectively. TNMT is also one to the (R )-epimer of reticuline is a required
of only a few plant enzymes able to cat- entry step into the morphinan alkaloid biosyn-
alyze the formation of quaternary ammo- thetic pathway (Figure 2). The epimerization
of reticuline is a two-step process that in- morphine consist of two demethylations and
volves the oxidation of (S )-reticuline by 1,2- one reduction. Both the demethylation from
dehydroreticuline synthase and the reduction thebaine to neopinone, which isomerises to
SDR: short chain
of 1,2-dehydroreticuline to (R )-reticuline. codeinone, and from codeine to morphine are dehydrogenases/
Both steps have been characterized biochem- not yet understood and no enzymes capable of reductases
ically and the enzymes have been partially pu- catalyzing either reaction have been detected. Calystegines:
ried (23, 63). Intramolecular carbon-carbon Codeinone reductase has been puried and nortropane alkaloids
phenol coupling between C2 of the benzyl cloned from opium poppy, and, in contrast thought to serve as
and C4a of the isochinoline moiety leads to SalR, belongs to the aldo-keto reductase nutritional sources
for soil
to the formation of salutaridine. This en- (AKR) family (83, 160).
microorganisms
zyme belongs to the P450 monooxygenase
family (48). The gene encoding salutaridine Tropane alkaloids and nicotine. Tropane
synthase (SalSyn) was recently cloned from alkaloids are an important class of plant-
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
opium poppy on the basis of its higher expres- derived anticholinergic compounds, such as
sion in morphine-containing Papaver species hyoscyamine and scopolamine, that occur
and the enzyme was then functionally char- in several Hyoscyamus, Atropa, and Datura
by Washington State University on 11/01/11. For personal use only.
acterized (A. Gesell, F.C. Huang, J. Ziegler species. Calystegines, which function as se-
& T.M. Kutchan, manuscript in preparation). lective glucosidase inhibitors, are more widely
The SalSyn protein shows high homology spread than hyoscyamine and scopolamine in
to the methylenedioxybridge-forming P450- the plant kingdom and occur mainly in the
dependent enzymes, and was classied as Solanaceae and Convolvulaceae (30). Nico-
Cyp719B1. The next step in the pathway is tine, the active principle in Nicotiana species,
catalyzed by salutaridine reductase (SalR); a acts on nicotinic acetylcholine receptors to
cognate cDNA was obtained via the same cause a variety of physiological effects, in-
approach used for SalSyn (183). Functional cluding addiction. Tropane alkaloid and nico-
characterization of the recombinant enzyme tine biosynthesis begin with the methyla-
showed the stereospecic reduction of the tion of putrescine to N-methylputrescine
keto group to 7(S )-salutaridinol, which is also by putrescine N-methyltransferase (PMT)
reported for the puried enzyme from opium (Figure 3). The isolation of the cDNA en-
poppy (49). The enzyme belongs to the fam- coding PMT from tobacco was one of the
ily of short chain dehydrogenases/reductases rst examples of the successful integration
(SDR), but unlike many other enzymes in of metabolite and gene expression proles
this family it exhibits a higher molecular as a strategy to isolate cDNAs implicated
weight and is monomeric. The stereospecic in plant secondary metabolite biosynthe-
reduction of salutaridine is required for the sis (61). Homologous cDNAs from other
next step, which is catalyzed by salutaridi- plants that produce tropane alkaloids, such
nol 7-O-acetyltransferase (SalAT). This en- as Hyoscyamus niger, Atropa belladonna, and
zyme specically acetylates the 7(S )-epimer Solanum tuberosum, have also been isolated
of salutaridinol to salutaridinol-7-O-acetate (147, 153, 157). The second step in the
(82). With considerable sequence homology pathway is the oxidative deamination of N-
to the acetylating enzymes from the MIA methylputrescine to 4-methylaminobutanal
pathway, SalAT also belongs to the BAHD by N-methylputrescine oxidase (MPO). This
family of acetyltransferases (52). The in- enzyme belongs to a class of amine ox-
troduced acetyl group is eliminated sponta- idases that require copper as a cofactor.
neously, leading to the formation of an oxide This property was exploited in a homology-
bridge between C-4 and C-5 to yield thebaine, based cloning strategy to isolate the MPO
the rst pentacyclic alkaloid of the pathway cDNA from tobacco (59). The central inter-
(82). The nal steps toward the biosynthesis of mediate N-methyl-1 -pyrrolium cation for
ODC
Putrescine
NAD
biosynthesis
intermediate 3,6-Dihydronicotinone
N-Methylputrescine
MPO
Spontaneous
4-Methylaminobutanal N-Methyl-1-pyrrolium
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
cation Nicotine
Figure 3
Cyp82E4
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Biosynthesis of the
tropane alkaloids
hyoscyamine and TR-II
scopolamine, the
calystegines, and
nicotine. Hygrine Tropinone Pseudotropine Nornicotine
Molecular clones TR-I
have been isolated
for all enzymes
shown.
Abbreviations:
Cyp80F1, littorine
mutase/
monooxygenase; Tropine Littorine
Cyp82E4, nicotine
N-demethylase; Cyp80F1
H6H, hyoscyamine
6-hydroxylase;
MPO,
Calystegine A3
methylputrescine
oxidase; ODC,
ornithine Hyoscyamine Hyoscyamine aldehyde
decarboxylase;
PMT, putrescine H6H
N-
methyltransferase;
TR-I, tropinone Calystegine B1 Calystegine B2
reductase I; TR-II,
tropinone
reductase II. Scopolamine
tropane alkaloid and nicotine biosynthesis Nicotine can be N-demethylated to form nor-
results from the spontaneous cyclization of nicotine, which is an undesirable derivative
4-methylaminobutanal. For nicotine, the owing to its role as the precursor of the car-
cation is condensed with nicotinic acid to form cinogen N -nitrosonornicotine. On the basis
3,6-dihydronicotine, which subsequently un- of the differential abundance of transcripts
dergoes dehydrogenation to nicotine by en- corresponding to several P450-encoding
zymes that remain poorly characterized. cDNAs in tobacco varieties acccumulating
Tropinone reductase I (TR-I) catalyzes the cDNA for which has been cloned from
reduction of tropinone to tropine, which several tropane alkaloid-containing plants
possesses a 3 conguration. In contrast, (88, 95, 154).
tropinone reductase II (TR-II) catalyzes the
formation of -tropine, which has a 3 con- Purine alkaloids. Purine alkaloids are
guration. Cognate cDNAs show that both derived from purine nucleotides. The
NADPH-dependent enzymes belong to the most prominent members of this class of
SDR family and exhibit considerable amino compounds are theobromine and caffeine.
acid sequence similarity (76, 110, 111, 113). Purine alkaloid biosynthesis begins with
Domain-swapping experiments and site- the N-methylation of xanthosine at position
directed mutagenesis led to the identication 7 by 7-methylxanthosine synthase (XRS),
of the substrate-binding domain responsible which is also known as xanthosine 7-N-
for the opposite stereospecicity of each methyltanferase (XMT) (Figure 4). Cognate
reductase. Tropine then condenses with the cDNAs for XMT have been isolated from
Figure 4
Biosynthesis of the
purine alkaloids
caffeine and
XMT theobromine.
Molecular clones
H2O Ribose have been isolated
7-Methylxanthine for all enzymes
shown.
Abbreviations: CS,
caffeine synthase;
Xanthosine 7-Methylxanthosine MXMT CS/TS
DXMT, 1,7-
dimethylxanthosine
methyltransferase;
MXMT,
DXMT 7-methylxanthine
CS methyltransferase;
TS, theobromine
synthase, XMT,
Caffeine Theobromine xanthosine 7-N-
methyltanferase.
Coffea arabica (103, 158). The second step in lation at position N-1 (117, 158). These
the purine alkaloid pathway is the hydrolysis N-methyltransferases possess more then
of xanthosine to 7-methylxanthine, although 80% amino acid similarity and phylogenetic
the responsible enzyme has not yet been analysis suggests that they are more closely
puried or characterized. However, detailed related to carboxyl-methyltransferases than
structural investigations of XMT suggest a to other N-methyltransferases.
coupled reaction for 7-N-methylation and
nucleoside cleavage catalyzed by a single Pyrrolizidine alkaloids. Pyrrolizidine alka-
enzyme (97). Several cDNAs with different loids are produced constitutively in various
substrate specicities have been obtained plants as a defense against hebivores and as
from tea, coffee, or cacao that catalyze the next a component of the complex chemical ecol-
two N-methylations. Caffeine synthase (CS) ogy between plants and animals (55). These
performs dual methylations, rst at position alkaloids are composed of a necine base and
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
3 to form theobromine, and then at position one or more necic acids; the latter are re-
1 to yield caffeine (74, 104). Several cDNAs sponsible for their structural diversity. Necine
encoding related enzymes that catalyze only biosynthesis begins with the condensation of
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single N-methylations have been isolated, in- spermidine and putrescine to form homosper-
cluding 7-methylxanthine methyltransferase midine by homospermidine synthase (HSS)
(MXMT1 and MXMT2) and theobromine (116) (Figure 5). The reaction mechanism
synthase (TS), which methylate position N-3, is identical to that of deoxyhyposine syn-
and 3,7-dimethylxanthine methyltransferase thase (DHS), which transfers the aminobutyl
(DXMT), which catalyzes the nal methy- moiety to a lysine residue of the eukaryotic
Spermidine
HSS
Homospermidine
Putrescine
Necine base
Figure 5
Biosynthesis of
pyrrolizidine
alkaloids. A
molecular clone
has been isolated
for only one
biosynthetic
enzyme.
Abbreviation: HSS,
homospermidine Senecionine Heliotrine
synthase. Clivorine
initiation factor 5A precursor protein. Ad- ajmaline biosynthesis. The structure of STR
ditionally, both enzymes share extensive se- from R. serpentina revealed a novel six-bladed
quence homology, suggesting the recruitment -propeller protein and site-directed muta-
Homology-based
of HSS from DHS, with subsequent opti- genesis showed the importance of a key gluta- model: an
mization to facilitate the role of HSS in sec- mate residue in catalysis (93). The structure of atomic-resolution
ondary metabolism. Phylogenetic analysis of the enzyme complexed with the natural sub- protein structure
HSS and DHS homologs from angiosperms strates tryptamine and secologanine provided based on amino acid
sequence
that represent several unrelated plant lineages insight into the architecture of the substrate
suggests at least four independent recruitment binding sites, which could then be engineered Substrate docking:
a mathematical
events for HSS (128). The remaining compo- to accommodate several different tryptamine
procedure used to
nents of pyrrolizidine alkaloid metabolism are and secologanin analogs (18, 90). The three- identify ligand-
not well understood. dimensional structure of SGD revealed the binding sites in
expected (/)8 barrel fold typical for family proteins
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
The biomimetic exploitation of enzymes, es- architecture. Ruppert and coworkers (133)
pecially those that display strict stereospeci- reported preliminary X-ray crystallographic
city, is an appealing strategy for the commer- data for the homologous enzyme RG. A com-
cial production of pharmaceutical alkaloids. parision of the structures of SGD and RG will
Equally intriguing is the synthesis of novel al- be interesting because both enzymes occur in
kaloids via protein engineering aimed at al- the same organism and catalyze the same type
tering the substrate specicity of biosynthetic of reaction, but differ in substrate specicity.
enzymes. Recent attention has focused on elu- VS was the rst BAHD acyltransferase family
cidating the reaction and substrate binding enzyme for which a three-dimensional struc-
mechanisms of key alkaloid biosynthetic en- ture was obtained (92). The protein consists
zymes. The structures of several enzymes have of two major domains of similar size con-
been determined and substrate-binding pock- nected by a large loop. Whereas site-directed
ets have been characterized. mutagenesis and structural analysis conrm
The rst detailed structures of enzymes in- the importance of a HXXXD motif for
volved in plant alkaloid metabolism were ob- catalysis, a second highly conserved motif in
tained by X-ray crystallography for TR-I and members of the BAHD family is distant from
TR-II, which show strict product specici- the active site and is considered essential for
ties and catalyze either tropine or -tropine proper folding rather than catalysis. Molec-
formation. Site-directed mutagenesis of se- ular modeling and site-directed mutagenesis
lected amino acids in the substrate-binding established the classication of PNAE as a
domains resulted in a mutual conversion of member of the / hydrolase superfamily
the product specicities for TR-I and TR-II and identied the amino acid residues that
(114). A homology-based model of the three- participate in the catalytic triad (96).
dimensional structure of PMT was generated The structure of SalR (involved in BIA
on the basis of the crystal structure of the pu- biosynthesis) was analyzed by homology mod-
tative ancestral enzyme spermidine synthase, elling (47). An additional helix near the cat-
which suggested the identity of amino acids alytic site not found in multimeric SDR-type
responsible for the distinction between these enzymes appears to contribute to substrate
two enzymes (157). specicity. Substrate docking studies and site-
Most X-ray crystallographic data have directed mutagenesis revealed several amino
been obtained for enzymes implicated in acids implicated in the binding of the morphi-
MIA metabolism, especially those involved in nan alkaloid precursor salutaridine. It will be
interesting to compare the substrate-binding tures (182, 183, 184). A recent oral genome
site of SalR with that of COR, which specif- project contributed 11,000 ESTs from the
ically reduces codeinone, an alkaloid with a BIA-accumulating plant E. californica (17), and
Laticifer: a plant
cell or vessel that similar structure, to salutaridine. The po- the release of a C. japonica database has also
contains latex tential of homology modelling and substrate been announced (69, 75). NapGen, a consor-
docking is well demonstrated in studies in- tium of Canadian investigators, has sequenced
volving the O-methyltransferases implicated more than 400,000 ESTs from a wide vari-
in BIA metabolism (S. Pienkny, J. Ziegler & ety of plants that produce health-related sec-
W. Brandt, manuscript in preparation). Al- ondary compounds. In this project, 46,000
though highly similar to 6OMT, the mod- ESTs were sequenced from cell cultures
elling and substrate docking results for a novel of eight BIA-accumulating species including
O-methyltransferase precluded norcoclaurine E. californica, T. avum, Nandina domestica,
as a likely substrate, and rather suggested and Papaver bracteatum (D.K. Liscombe, J.
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
the O-methylation of norreticuline at posi- Ziegler & P.J. Facchini, manuscript in prepa-
tion 7. Experimental evidence has conrmed ration). More than 56,000 ESTs have been ob-
this prediction and led to the identication of tained from leaf, leaf epidermis, and roots of
by Washington State University on 11/01/11. For personal use only.
aromatic amino acid metabolism were also in- Two different signal transduction pathways
duced in opium poppy and C. roseus in re- were identied. One cascade is jasmonate-
sponse to the increased demand for the pre- dependent and responds to high elicitor
Metabolomics: the
cursors of BIA and MIA biosynthesis (129, concentrations. The other is jasmonate- systematic study of
184). Surprisingly, the expression of genes as- independent and is triggered by low elic- the chemical
sociated with nitrogen metabolism was not af- itor concentrations (38). The jasmonate- ngerprints left
fected in opium poppy cultures, suggesting independent pathway involves G proteins behind by specic
cellular processes
that the cells have a constitutive and suf- that interact and activate phospholipase A2,
cient capacity for nitrogen assimilation even which leads to a transient proton efux from RNAi: RNA
interference
during periods of increased demand (184). the vacuole and a subsequent activation of
However, substantial changes occurred in the other cytoplasmic components (60, 164).
levels of primary metabolites involved in the
production of energy molecules potentially
Transgenic Approaches
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
of correlation networks that integrate tran- tended to increase the synthetic capacity of
scriptomic and metabolomics data revealed desired product via the overexpression of cer-
a correlation between the expression of al- tain genes, or to divert pathways to previ-
kaloid biosynthetic genes and corresponding ously under-represented or novel compounds
metabolic products (129). through posttranscriptional gene silencing.
However, such experiments also provide in-
sights into the regulatory architecture of alka-
Gene Regulation loid pathways, especially if the predicted out-
and Signal Transduction come is not observed. Unexpected metabolic
Progress has continued on the identication consequences resulting from single-enzyme
of promoter elements and transcription fac- perturbations of alkaloid pathways suggest the
tors involved in the regulation of several MIA existence of key rate-limiting steps, potential
biosynthetic genes (165). The recent applica- multienzyme complexes, or unsuspected com-
tion of a genomics approach based on DNA partmentalization.
macroarrays constructed from ESTs has led to Metabolic engineering of low-scopola-
the isolation of the rst transcription factor mine A. belladonna plants via the intro-
putatively involved in the regulation of BIA duction of a constitutively expressed H6H
metabolism. Transcripts that encode a subset transgene led to an increase in scopolamine
of transcriptional regulators showed coordi- accumulation (179). Similarly, a shift in the
nate expression with respect to BIA biosyn- accumulation of hyoscyamine in favor of
thetic genes in berberine-producing C. japon- scopolamine occurred when the H6H trans-
ica cell cultures. One of these regulators, a gene was introduced into A. beatica, suggest-
WRKY transcription factor, was shown by ing that the enzyme is a rate-limiting step
RNAi and overexpression analysis to specif- in scopolamine biosynthesis (180). However,
ically regulate the expression of BIA biosyn- the unpredictability of metabolic engineering
thetic genes (75). With respect to early signal in tropane alkaloid biosynthesis was demon-
transduction events, considerable effort has strated by the constitutive coexpression of
been focused on events associated with the in- PMT and H6H. The transgenes caused only
duction of alkaloid metabolism in E. californica modest increases in alkaloid accumulation
cell cultures, which accumulate benzophenan- when expressed alone, but exhibited a syner-
thridine alkaloids such as sanguinarine in re- gistic effect on alkaloid levels when expressed
sponse to treatment with a fungal elicitor. together (181).
In MIA biosynthesis, the rst attempts of BBE expression leads to undetectable lev-
at metabolic engineering focused on the els of downstream metabolites and increased
expression of constitutive TDC and STR cellular pools of amino acids, revealing the im-
Latex: the milky
cytoplasm of transgenes in C. roseus cell cultures (31). pact of perturbations in alkaloid metabolism
specialized cells However, only unsustained increases in al- on primary metabolism (123). Interestingly,
called laticifers kaloid accumulation were reported, which antisense suppression of BBE does not re-
raises concerns about the actual relation- sult in the accumulation of its substrate (S )-
ship between the transgenes and the initial reticuline or any other upstream alkaloid, sug-
phenotype (16). Overexpression of enzymes gesting the occurrence of metabolic channels
responsible for production of the indole moi- that are abolished in the absence of BBE.
ety, such as anthranilate synthase (AS), re- In contrast, RNAi lines targeting BBE in E.
sulted in higher levels of tryptophan, but californica cell cultures result in high (S )-
not alkaloids (64, 66). Monoterpenoid path- reticuline content (43). Furthermore, lauda-
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
way reactions have long been considered nine is detected, implying that the pathway is
the rate-limiting steps in MIA production. redirected toward the O-methylation of reti-
In this context, overexpression of a modi- culine by 7OMT. Transgenic opium poppy
by Washington State University on 11/01/11. For personal use only.
from the pathway is unaffected (4). However, to increase pest resistance. Overexpression
the plants accumulate the far upstream inter- of all three N-methyltransferases in trans-
mediate (S )-reticuline rather than codeinone, genic Nicotiana tabacum leads to a substantial
Idioblast: an
the substrate of COR. Whether this is based (5 ug g1 fresh weight) accumulation of caf- individual cell that
on a feedback mechanism that inhibits the en- feine in leaves (77, 159). This is sufcient to differs greatly from
tire morphinan branch pathway or the im- reduce by 50% pest damage caused by feeding its neighbors
pairment of a required metabolic channel due of the tobacco cutworm Spodoptera litura.
to the absence of COR is not known. The
presence of multienzyme complexes in mor- ALKALOID TRAFFICKING
phine biosynthesis is supported by experi-
The cell biology of alkaloid metabolism in
ments that involve the suppression of SalAT.
plants has recently emerged as an exciting
RNAi-silenced SalAT plants show an accumu-
eld of research. Although the biosynthetic
lation of salutaridine, which is normally not
pathways leading to various alkaloid types
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
a Pericycle b
Phloem Idioblast Epidermis
Xylem
Endodermis Idioblast Laticifer
Cortex Epidermis
Epidermis
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
c d
by Washington State University on 11/01/11. For personal use only.
Phloem
Phloem
Xylem Xylem
Endodermis Endodermis
Cortex Cortex
Immature
e f endodermis
Pericycle
Xylem
Vascular
cambium
Companion Sieve
cell element
Laticifer Stele
Cortex
Protoderm
Parenchyma
Figure 6
Alkaloid biosynthetic pathways are associated with a diversity of cell types. The tissue-specic
localization (red ) of enzymes and/or gene transcripts are shown for the biosynthesis of tropane alkaloids
in (a) Atropa belladonna and Hyoscyamus niger roots, (b) terpenoid indole alkaloids in Catharanthus roseus
leaves, (c) pyrrolizidine alkaloids in Senecio vernalis roots, (d ) pyrrolizidine alkaloids in Eupatorium
cannabinum roots, (e) benzylisoquinoline alkaloids in Papaver somniferum vascular bundles, and
( f ) benzylisoquinoline alkaloids in Thalictrum avum roots.
Scopolamine
Pericycle Ornithine Arginine
cell types. TDC and STR are abundant in
roots (151), but also occur in photosynthetic
organs, whereas T16H (149), D4H (162), and
DAT (150) are restricted to young leaves and Ornithine
Arginine
other shoot organs where vindoline biosyn-
thesis occurs. In situ hybridization and im-
Phloem Xylem
munocytochemical localization studies have
shown that Cyp72A1, TDC, and STR lo-
calize to the epidermis of stems, leaves, and
ower buds (70, 151) (Figure 6b). In roots,
these enzymes occur in cells near the api-
cal meristem. In contrast, D4H and DAT
are associated with laticifers and idioblasts
of shoots, but are absent from roots. Lati-
cifers and idioblasts are distributed through- Figure 7
out the mesophyll in leaves, often several cell Putative trafcking of pathway intermediates and products in tropane
layers away from the epidermis; thus, vin- alkaloid metabolism. Red arrows show specic enzyme-catalyzed reactions.
doline biosynthesis involves at least two dis- Blue arrows represent the purported intercellular translocation of
unspecied compounds.
tinct cell types and requires the intercellu-
lar translocation of pathway intermediates.
Moreover, gene transcripts that encode en- sis in C. roseus are not known. Interestingly,
zymes involved in the MEP pathway, along the identication of gene transcripts encod-
with geraniol 10-hydroxylase, colocalize to ing vindoline biosynthetic enzymes in specic
the internal phloem parenchyma of young C. cell types isolated by laser-capture microdis-
roseus aerial organs (15, 120). These results section and enriched via carborundum abra-
suggest the translocation of vindoline biosyn- sion was interpreted to suggest that the leaf
thetic intermediates from the internal phloem epidermis is independently capable of produc-
to the epidermis and from the epidermis to ing 16-methoxytabersonine (109). Upper leaf
laticifers and idioblasts (Figure 8). As is the epidermis has been implicated in several sec-
case in tropane alkaloid metabolism, the spe- ondary metabolic pathways (94), complicating
cic pathway intermediates that undergo in- the assignment of common precursors to spe-
tercellular translocation in MIA biosynthe- cic cell types. The role of internal phloem
Deacetyl- vindoline
vindoline trast to the general monophyletic origin of
Vindoline
Vindoline BIA biosynthesis (87), pyrrolizidine alkaloid
Laticifer
pathways have evolved in at least four differ-
ent angiosperm lineages (128). The differen-
Deacetyl-
vindoline tial localization of a key enzyme was inter-
MEP pathway
? preted to support the polyphyletic origin for
Vindoline
Geraniol pyrrolizidine alkaloid biosynthesis (6). In con-
10-Hydroxy- Spongy
IPAP mesophyll trast, the monophyletic origin of BIA biosyn-
geraniol
idioblast
IPAP IPAP thesis (87) and the differential localization of
IPAP gene transcripts in T. avum and opium poppy
suggest the migration of pathway intermedi-
Vascular
tissues ates between cell types.
IPAP
IPAP In opium poppy, BIA accumulation
occurs in the articulated laticifers found
Figure 8 adjacent or proximal to sieve elements of the
Putative trafcking of pathway intermediates and products in phloem (32). The cytoplasm of laticifersor
monoterpenoid indole alkaloid metabolism. Red arrows show specic latexcontains a full complement of cellular
enzyme-catalyzed reactions. Blue arrows represent the purported
organelles and many large vesicles to which
intercellular translocation of unspecied compounds. IPAP, internal
phloem-associated parenchyma. alkaloids are sequestered. Although laticifers
were long considered to be the site of BIA
and epidermis in the supply of terpenoid pre- biosynthesis and accumulation, the cellular
cursors to alkaloid biosynthesis requires fur- localization of BIA biosynthetic enzymes
ther evaluation. and gene transcripts has shown that alkaloid
Pyrrolizidine alkaloids also share a com- synthesis involves other cell types (13, 34,
mon metabolic pathway in restricted yet di- 170). Initial in situ hybridization experiments
verse taxa (54). In Senecio species, pyrrolizidine demonstrated the localization of TYDC gene
alkaloids are produced in actively growing transcripts to the phloem, but not to laticifers
roots as senecionine N-oxides, which are (34). The morphinan pathway enzymes
transported via the phloem to above-ground salutaridine synthase (Cyp719B1) and salu-
organs (54). Senecionine N-oxides are sub- taridine:NADPH 7-oxidoreductase (SalR)
sequently modied by one or two species- are also not detected in isolated latex (48, 49).
specic reactions (i.e., hydroxylation, dehy- Seven other biosynthetic enzymes6OMT,
CNMT, (S )-N-methylcoclaurine 3 -
Parenchyma
hydroxylase (Cyp80B3), 4 OMT, BBE,
SalAT, and CORlocalize to sieve elements Companion
in opium poppy and their corresponding gene cell
Laticifer
transcripts localize to associated companion
cells (13, 135) (Figure 6e). The localization
of BIA metabolism components to phloem
cells distinct from laticifers predicts the in- Alkaloid
biosynthetic Alkaloids
tercellular transport of alkaloid biosynthetic enzymes
enzymes and pathway intermediates and/or
products. The implication of sieve elements Sieve
also breaks a long-standing paradigm in element
plant biology (Figure 9). Previously, sieve
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
detected. Removal of COR was suggested to able sequence information (21). However, the
disrupt a metabolic channel composed of mor- growing sequence database makes the appli-
phinan branch pathway enzymes, resulting in cation of this technique feasible in studying
the accumulation of an alkaloid intermediate mutations in alkaloid-containing plants. The
produced by enzymes that are not part of development of sequencing techniques that
the same complex. Interestingly, thebaine can generate large datasets within short times
and oripavine, intermediates upstream of will probably be applied to alkaloid produc-
substrates acted upon by COR, accumulate ing plants and will provide the basis for many
to high levels in some opium poppy cul- sequence-based approaches that are now lim-
tivars (101); thus, researchers expected an ited to plants with sequenced genomes such
accumulation of morphinan branch pathway as Arabidopsis and rice. Whether genome
intermediates. An alternative interpretation sequencing projects in alkaloid-containing
not considered by Allen and coworkers (4) plants will be feasible remains to be seen, be-
Annu. Rev. Plant Biol. 2008.59:735-769. Downloaded from www.annualreviews.org
of two enzymes involved in the epimerization 1 Gbp for rice). The predicted function of al-
of (S )-reticuline to (R )-reticuline. The po- kaloid biosynthetic enzymes and other com-
tential homology between the two reductases ponents based on the in vitro characterization
could lead to cosilencing. of gene products must be conrmed in vivo.
However, functional genomics remains prob-
lematic for most alkaloid-producing plants
PERSPECTIVE owing to general limitations in established
Major progress in the elucidation of alka- genetic transformation technologies. Virus-
loid biosynthetic pathways and their regula- induced gene silencing is a fast method for the
tion has been obtained by application of large generation of transiently transformed plants
scale genomic tools. However, there are sev- and works well in a model organism such
eral techniques, whose applications are still as N. benthamiana (130), and protocols for
more or less exclusively conned to model or- opium poppy and E. californica have recently
ganisms, that might further our understand- been developed (62, 169). Thus, the impact
ing of plant alkaloid biosynthesis. Proteome of suppression of candidate genes on alkaloid
analysis in alkaloid-accumulating plants has biosynthesis can be readily examined before
been undertaken (26, 71, 115), but is still lim- stable transformation is applied. Considering
ited by the lack of peptide mass databases for the progress in the investigation of alkaloid
the investigated species, which does not allow metabolism achieved in the last 5 years, a look
protein identication by simple peptide mass to the future promises further strong devel-
ngerprinting. Targeted induced local lesions opments in the discovery of regulatory net-
in genomes (TILLING) as a tool to discover works that lead to alkaloid accumulation in
point mutations is also dependent on avail- plants.
SUMMARY POINTS
1. The application of genomics technologies has expedited the discovery of new alkaloid
biosynthetic genes that encode enzymes and regulatory proteins with novel functions.
2. Large-scale, integrated transcriptomics and metabolomics analyses are providing ini-
tial hints about the regulation of alkaloid pathways.
though the endoplasmic reticulum is a favored site for alkaloid formation, biosynthetic
enzymes have been associated with chloroplast thylakoid membranes, mitochondria,
vacuoles, and the cytosol.
FUTURE ISSUES
1. Despite impressive advances facilitated by the application of genomics technologies to
the discovery of novel genes that encode alkaloid biosynthetic enzymes, a substantial
understanding of the regulatory components of alkaloid pathways has been achieved
only for terpenoid indole alkaloid metabolism. Genomics approaches should lead to
the identication of regulatory genes involved in benzylisoquinoline alkaloid synthesis
and other alkaloid pathways.
2. The impact of posttranslational modication of alkaloid biosynthetic enzymes on
product accumulation is a black box. Proteomic approaches might explain why the
expression levels of some biosynthetic genes do not correlate with alkaloid proles.
3. Although the involvement of multiple cell types in the biosynthesis and accumula-
tion of many alkaloids has been established, the identity of the pathway intermediates
and/or products that undergo intercellular transport is not known. Moreover, the
mechanisms of transportsymplastic involving plasmodesmata, or apoplastic involv-
ing specic transporters or channelsare poorly understood.
4. The existence of metabolic channels as a common feature of alkaloid biosynthetic
pathways has been widely purported, but only scant evidence is available to support
their existence. Establishing the interactions among biosynthetic enzymes and other
pathway components will facilitate the rational engineering of alkaloid metabolism in
plants and microorganisms.
DISCLOSURE STATEMENT
The authors are not aware of any biases that might be perceived as affecting the objectivity of
this review.
ACKNOWLEDGMENTS
J.Z. was funded through a Canada Research Chair in Plant Metabolic Processes Biotechnology
held by P.J.F. Research support from the Natural Sciences and Engineering Research Council
of Canada to P.J.F. is also gratefully acknowledged.
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