Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

29 Pages·2007·0.47 MB·English

PhytochemRev(2007)6:277–305
DOI10.1007/s11101-006-9047-8
Catharanthus terpenoid indole alkaloids: biosynthesis
and regulation
Magdi El-Sayed Æ Rob Verpoorte
Received:29August2005/Accepted:23October2006/Publishedonline:10March2007
(cid:1)SpringerScience+BusinessMediaB.V.2007
Abstract Catharanthus roseus is still the only localization and regulation are discussed. Much
source for the powerful antitumour drugs vin- progress has been made at alkaloid regulatory
blastine and vincristine. Some other pharmaceu- level.Feedingprecursors,growthregulatorstreat-
tical compounds from this plant, ajmalicine and ments and metabolic engineering are good tools
serpentine are also of economical importance. to increase productivity of terpenoid indole alka-
Although C. roseus has been studied extensively loids. But still our knowledge of the late steps in
and was subject of numerous publications, a full the Catharanthus alkaloid pathway and the genes
characterizationof its alkaloidpathway is not yet involved is limited.
achieved. Here we review some of the recent
work done on this plant. Most of the work Keywords Indole alkaloids (cid:1) Biosynthesis (cid:1)
focussed on early steps of the pathway, particu- Catharanthus (cid:1) Indole pathway (cid:1) MEP pathway (cid:1)
larly the discovery of the 2-C-methyl-D-erythritol Regulation (cid:1) Terpenoids
4-phosphate (MEP)-pathway leading to terpe-
noids. Both mevalonate and MEP pathways are Abbreviations
utilized by plants with apparent cross-talk be- AACT Acetoacetyl-CoA thiolase
tweenthemacrossdifferentcompartments.Many ABA Abscisic acid
genes of the early steps in Catharanthus alkaloid AS Anthranilate synthase
pathway have been cloned and overexpressed to AVLB Anhydrovinblastine
improve the biosynthesis. Research on the late CMS 4-Cytidyl diphospho-2 C-methyl-D-
stepsinthepathwayresultedincloningofseveral erythritol synthase
genes. Enzymes and genes involved in indole CPR Cytochrome P450 reductase
alkaloid biosynthesis and various aspects of their CR Cathenamine reductase
DAT Acetyl CoA:deacetylvindoline 17-
O-acetyltransferease
M.El-Sayed(cid:1)R.Verpoorte(&)
D4H Desacetoxyvindoline 4-hydroxylase
DepartmentofPharmacognosy,Sectionof
DMAPP Dimethylallyl diphosphate
Metabolomics,InstituteofBiologyLeiden,Leiden
University,Leiden,TheNetherlands DXP 1-Deoxy-D-xylulose-5-phosphate
e-mail:[email protected] DXR 1-Deoxy-D-xylulose-5-phosphate
reducto isomerase
M.El-Sayed
DepartmentofBotany,AswanFacultyofScience, DXS 1-Deoxy-D-xylulose-5-phosphate
SouthValleyUniversity,Aswan,Egypt synthase
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278 PhytochemRev(2007)6:277–305
GAP Glyceraldehyde-3-phosphate predators, as messengers, attractants, repellents
G10H Geraniol 10-hydroxylase or as camouflage (Verpoorte 1998).
GPP Geranyl diphosphate Alkaloids are one of the largest classes of
HMG-CoA 3-Hydroxy-3-methylglutaryl-CoA secondary metabolites. They contain a heterocy-
HMGS 3-Hydroxy-3-methylglutaryl-CoA clic nitrogen usually with basic properties that
synthase makes them particularly pharmacologically
HMGR 3-Hydroxy-3-methylglutaryl-CoA active. Among them are the indole alkaloids
reductase whicharefoundmainlyinplantsbelongingtothe
IPP Isopentenyl diphosphate families: Apocynaceae, Loganiaceae, Rubiaceae
K Michaelis–Menten constant and Nyssaceae (Verpoorte et al. 1997).
m
LAMT Loganic acid methyltransferase Catharanthus roseus (L.) G. Don (Madagascar
MCS 2-C-Methyl-D-erythritol Periwinkle) is one of the most extensively inves-
2,4-cyclodiphosphate synthase tigated medicinal plants. The importance of this
MEP 2-C-methyl-D-erythritol plant is due to the presence of two antitumour
4-phosphate alkaloids, vinblastine and vincristine found in the
MVAK Mevalonate kinase leaves, and ajmalicine, an alkaloid found in the
MJ Methyljasmonates roots. All parts of this plant contain a variety of
M Relative molecular weight alkaloids, even seeds that were thought to have
r
MVA Mevalonic acid no alkaloids until Jossang et al. (1998) isolated
MVAPK 5-Diphosphomevalonate kinase two binsidole alkaloides from the seeds, vingr-
NMT-SAM Methoxy 2,16-dihydro-16- amine and methylvingramine. Cell suspension
hydroxytabersonine cultures of C. roseus are an alternative means
N-methyltransferase for the production of economically important
OMT O-Methyltransferase terpenoid indole alkaloids (TIAs). However, the
ORCA Octadecanoid-responsive yields are too low to allow commercial applica-
Catharanthus AP2/ERF-domain tion. The more than 100 C. roseus alkaloids that
SAM S-Adenosyl-L-methionine have been identified share many biosynthetic
SGD Strictosidine b-D-glucosidase steps. The early stages of alkaloid biosynthesis
SLS Secologanin synthase in C. roseus involve the formation of secologanin
STR Strictosidine synthase derived from the terpenoid (isoprenoid) biosyn-
T16H Tabersonine 16-hydroxylase thesis and its condensation with tryptamine to
THAS Tetrahydroalstonine synthase producethecentralintermediatestrictosidine,the
TIA Terpenoid indole alkaloids; common precursor for the monoterpenoid indole
TDC Tryptophan decarboxylase alkaloids (Fig. 1).
The terpenoid pathway
Terpenoids are the largest family of natural
Introduction products with over 30,000 compounds. They are
known to have many biological and physiological
Plant cells are considered to be excellent produc- functions. Formation of terpenoids proceeds via
ers of a broad variety of chemical compounds. two different pathways, the classical mevalonate
Many of these compounds are of high economic andthenewlydiscovered2-C-methyl-D-erythritol
value such as various drugs, flavours, dyes, 4-phosphate (MEP) pathway leading to isopente-
fragrances and insecticides. These compounds nyl diphosphate (IPP). In higher plants, the
usually play a role in the interaction of the plant mevalonate pathway operates mainly in the cyto-
with its environment, e.g. as toxins to defend plasm and mitochondria. The MEP pathway
the plant against micro-organisms or various operates in the plastids with a cross-talk between
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PhytochemRev(2007)6:277–305 279
H O
tryptamine N NH2 H OGlc
H H O
H3COOC secologanin
N+
N H
HN N+ H CH3 NHH NH OGlc H3HCOOHC OCH3
H O H O alstonine
H3COOC serpentine H3COOC
strictosidine
HNH N H CH3 HNH N CH3 HNH N+ NHH N CH3 HNH N H CH3
H3COOHC O H3COOHC O H3COOHC OH H3COOHC O H3COOHC O
ajmalicine cathenamine 4,21-dehydrogeissoschizine epicathenamine tetrahydroalstonine
N
H
N
H3COOC CH2OH N
N
N H H H H
NH H N N stemmadHe3CnOinOeC CH2OH COtOaCbHe3rsonine
H
akuammidine
secodine
N N H
N
H3COOC N H catharanthiHne COOCH3CH2CH3 CH3O N H OH OCOCH3
NH CH3COOCH3
NHO NH CH3 +N vindoline
perivine COOCH3
vindolinine H
N COOCH3
H N
OH
H3CO N H OCOCH3
H3C COOCH3
OH iminium
N
N
O
H
H N COOCH3 N
NH COOCH3 N H OH N
H3CO N H OH OCOCH3 H3CO H3CN vHiCnObOlaCsHOt3inCeOCH3 NH CHOOCH3 N
H3C COOCH3
leurosine OH
N OH H3CO H3NC H COOCHO3COCH3
3',4'-anhydrovinblastine
H
N COOCH3 N
H
OH
H3CO N H OCOCH3
OHC COOCH3
vincristine
Fig.1 DifferentCatharanthusindolealkaloidsbiosyntheticpathways
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280 PhytochemRev(2007)6:277–305
the two pathways where at least one metabolite Lange and Croteau 1999). The second pathway
can be exchanged. is mevalonate-independent (MEP pathway) and
leads to the formation of monoterpenes, diterp-
enes, tetraterpenes (carotenoids) and the prenyl
Formation of IPP side chains of chlorophyll (Eisenreich et al. 1996,
1997; Arigoni et al. 1997; Rohmer 1999).
The mevalonate pathway The discovery of the MEP pathway for iso-
prenoid biosynthesis was reviewed by Rohmer
The early steps in the isoprenoid pathway com- (1999), Lichtenthaler (1999), Rohdich et al.
prise the enzymatic conversions involved in the (2001), Rodriguez-Concepcion and Boronat
synthesis of IPP. The mevalonate pathway starts (2002) and Dubey et al. (2003). The pathway
withthecouplingoftwomoleculesofacetyl-CoA was first discovered in studies of the biosynthesis
toformacetoacetyl-CoA.Thisstepiscatalysedby of bacterial hopanoids that are similar to sterols
the enzyme acetoacetyl-CoA thiolase (AACT). produced by eukaryotes and act as membrane
Condensation of acetoacetyl-CoA with another stabilizers. As hopanoids are chemically stable
molecule of acetyl-CoA to form 3-hydroxy-3- and easily isolated, they are very suited for
methylglutaryl-CoA (HMG-COA), is catalysed labelling experiments using stable isotopes fol-
by HMG-CoA synthase (HMGS). Reduction of lowed by NMR to determine the sites of incor-
HMG-CoA by HMG-CoA reductase (HMGR) poration in the molecules. Through the labelling
leads to the formation of mevalonate. Phosphor- experiments, it was thought to be trivial to
ylation of mevalonate to 5-diphosphomevalonate identify the isoprenoid units resulting from the
is catalysed by mevalonate kinase (MVAK) and mevalonic acid (MVA) route but, the pattern of
5-diphosphomevalonate kinase (MVAPK), then thelabelswascompletelydifferentanddidnotfit
5-diphosphomevalonate is decarboxylated by 5- the classical MVA pathway.
diphosphomevalonate decarboxylase to IPP. Sprenger (1996), in his labelling experiments
Isopentenyl diphosphate is considered as a on bacteria that utilize only hexoses, and espe-
building block of isoprenoids. The isomerization cially glucose as a carbon source, determined the
of IPP to form dimethylallyl diphosphate origin of isoprenic units of hopanoids as derived
(DMAPP) is a key step in the biosynthesis of from glucose. The labelling pattern was in accor-
isoprenoids. This step is catalysed by IPP isom- dancewithpyruvateasaprecursorofaC subunit
2
erase (E.C. 5.3.3.2; Ramos-Valdivia et al. 1997; and a triose phosphate derivative as precursor of
Verpoorte et al. 1997). DMAPP is condensed a C subunit.
3
with one IPP in a head-to-tail fashion generating For the MEP pathway, the biosynthetic
geranyl diphosphate(GPP), theprecursorforthe sequence leading to the formation of IPP in
monoterpenes including iridoids such as secolog- plants is still not completely identified (Fig. 2).
anin (Verpoorte et al. 1997; Contin 1999). The The complete pathway has been elucidated,
coupling reaction is catalysed by a prenyltrans- including the late steps in bacteria (for review
ferase while the enzymatic cyclization of GPP is see Rodriguez-Concepcion and Boronat 2002).
catalysed by a monoterpene synthase, GPP syn- The initial step of the pathway involves a
thase (Chappell 1995). condensation of pyruvate (C2 and C3) with D-
glyceraldehyde 3-phosphate to yield 1-deoxy-D-
Themevalonate-independentpathwayleading xylulose-5-phosphate (DXP). Chahed et al.
to the formation of IPP (MEP pathway) (2000) isolated and characterized the cDNA
(crdxs) encoding for 1-deoxy-D-xylulose-5-phos-
The biosynthesis of IPP, the central precursor of phate synthase (DXS) from C. roseus. The
all isoprenoids, proceeds via two separate path- enzyme that catalyses this reaction belongs to a
ways in plants. The mevalonate pathway leads to familyoftransketolases.Inthesecondstepofthis
the formation of triterpenes (sterols) and certain pathway,rearrangementandreductionofDXPto
sesquiterpenes (Newman and Chappell 1999; MEPtakesplace.Theenzymecatalysingthisstep
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PhytochemRev(2007)6:277–305 281
I. Mevalonate pathway II. MEP pathway
O
O
O COOH
SCoA + H OP
OH
acetyl CoA pyruvate glyceraldehyde 3-phsophate
O
acetoacetyl-CoA DXS
CO
thiolase SCoA 2
OH
O O O
OP
SCoA OH
acetoacetyl-CoA 1-deoxy-D-xylulose 5-phosphate
O NADPH
HMG-CoA DXR
synthase SCoA NADP+
HO O HO
SCoA OP
COOH OH OH
hydroxymethyl-glutaryl-CoA 2-C-methyl-D-erythritol 4-phosphate
CTP
HMG-CoA NADPH Mg2+ CMS NH2
reductase PPi
N
HO O HO O O
N O
O P O P O O
OH
OH OH OH OH
COOH
OHOH
mevalonate 4-cytidyl diphospho-2C-methyl-D-erythritol
ATP
MVA kinase ATP CMK NH
2
ADP
O N
HO O HO P O O O N O
OH O P O P O O
OP
OH OH OH OH
COOH
OHOH
mevalonate phosphate 4-cytidyl diphospho-2C-methyl-D-erythritol 2-phosphate
MVAP kinase ATP MCS
CMP
O OH
HO O
O O
P O
OP P P
O OH
COOH
OH OH
mevalonate diphosphate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate
MVAPP
ATP O O
decarboxylase
O P O P OH
OH OH OH
1-hydroxy-2-methyl 2(E) butenyl 4 diphosphate
OP P IPP isomerase OP P IPP isomerase OP
IPP DMAPP IPP
Fig.2 ThebiosynthesisofIPPviamevalonatepathwayandMEPpathwaywithinvolvedenzymes
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282 PhytochemRev(2007)6:277–305
is DXP reducto isomerase (DXR). Grolle et al. ring of loganin forms the secologanin (Fig. 3).
(2000) cloned the gene encoding this enzyme Conversion of loganin to secologanin is of partic-
from the bacterium Zymomonas mobilis. Veau ular interest, the enzyme catalysing this reaction
et al. (2000) reported the cloning and expression is secologanin synthase (SLS). In C. roseus,
of cDNAs encoding crdxr and crmecs (reducto Contin (1999) attempted to identify the enzyme
isomerase and 2-C-methyl-D-erythritol-2,4-cyc- involved in bioconversion of loganin to secolog-
lodiphosphate synthase) from C. roseus. The anin with no success. She reported that the
IPPisformedfinallyinlowratefromisopentenyl conversion probably involves a cytochrome P450
monophosphate via a step catalysed by the enzyme. This enzyme was finally detected and
enzymeisopentenylmonophosphatekinase.Con- characterized in a cell suspension culture of
tin et al. (1998) proved that the terpenoid moiety Lonicera japonica (Yamamoto et al. 2000). It is
of the TIAs, secologanin is not derived from the a membrane-associated enzyme belonging to the
mevalonate pathway but instead from the MEP group of cytochrome P450 monooxygenases and
pathway using a cell suspension culture of its reaction requires NADPH and oxygen. Irmler
C. roseus. The late steps are not characterized et al. (2000) reported that the activities of
yetinC.roseus.4-Cytidyldiphospho-2C-methyl- CYP72A1 from C. roseus expressed in E. coli
D-erythritol synthase was first cloned from Ara- converts loganin into secologanin and confirmed
bidopsis thaliana and expressed in Escherichia it as SLS. This enzyme was previously purified
coli(Rohdichetal.2000).Althoughtherearefew from C. roseus by Mangold et al. (1994) and
radiotracer studies in plants which demonstrated thought to have G10H activity but did not show
the possible phophorylation role of 4-diphospho- any hydroxylase activity with 11 substrates for
cytidyl-2-C-methyl-D-erythritol kinase (CMK) in cytochrome P450 reactions.
the pathway, the complete enzymology and its
molecular analysis is not available in a plant Characterized enzymes involved in the
system. The gene encoding 2-C-methyl-D-erythr- biosyntheticpathwayleadingtotheformation
itol 2,4-cyclodiphosphate synthase has been dem- of secologanin
onstrated in Arabidopsis but the enzyme was not
fully characterized. AACT (E.C. 2.3.1.9) and HMGS (E.C. 4.1.3.5)
The iridoid pathway Both AACT and HMGS activities were found to
be present in C. roseus by using an HPLC
The first steps in the pathway leading to the method specially developed to determine HMG-
formation of secologanin are the formation of CoA metabolizing enzyme activities. Using this
geraniol followed by hydroxylation into 10- method, three HMG-CoA catabolizing activities
hydroxylgeraniol, catalysed by the cytochrome were discovered in C. roseus suspension cultured
P450enzymegeraniol10-hydroxylase(G10H).In cells in addition to HMGR (Van der Heijden
the presence of NAD+ or NADP+, 10-hydroxy- et al. 1994). These enzymes are instable and
geraniol is oxidized into 10-oxogeranial. The sensitive to high salt concentrations. AACT and
enzyme responsible for this step is an oxido HMGS were partially purified from a cell
reductase (Madyastha and Coscia 1979). 10- suspension culture of C. roseus (Van der Heijden
Oxogeraniol is converted to iridodial by and Verpoorte 1995).
cyclization. NADPH:cytochrome P450 reductase
(CPR) is essential for the G10H catalysed reac- 3-Hydroxy-3-methylglutaryl-CoA reductase
tion.Intheformationof7-deoxyloganicacidfrom (E.C. 1.1.1.34)
iridodial, so far, no enzymes have been described
(Contin 1999). Methylation of loganic acid to The HMG reductase has been purified from a
form loganin is catalysed by S-adenosyl-L- number of species besides C. roseus and its
methionine:loganic acid methyltransferase characteristics and regulation mechanisms have
(LAMT).Finallythecleavageofthecyclopentane been the subject of extensive reviews (Chappell
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PhytochemRev(2007)6:277–305 283
OH OH OH CHO
CHO
10--hydroxy geranial
G10H
OH
OHC CHO
OH
geraniol 10-hydroxy geraniol 10-oxogeraniol 10-oxogeranial
cyclase
CH3
HO CH3 CH3
H H
H OGlc
OGlc
H
H
LAMT H O HOOC O CHO
HOOC H
loganic acid 7-deoxyloganic acid
HO CH3 iridodial
H
OGlc
CHO
H O SLS H
OGlc
CH3OOC
H
O
loganin CH3OOC
secoloanin
Fig.3 The biosynthesis of secologanin from geraniol. G10H geraniol 10-hydroxylase, LAMT loganic acid methyltrans-
ferase,SLSsecologaninsynthase
1995; Bach 1995; Stermer et al. 1994; Verpoorte quite stable. It is strongly inhibited by farnesyl
et al. 1997; Schulte 1998). Overexpression of the diphosphate. MVAK activity depends on the
hmgr gene in C. roseus hairy roots resulted in an presence of the divalent ions, Mg2+ and Mn2+
increase in alkaloid levels. A clone with high which are effective in sustaining the activity. It
hybridization signal produced more ajmalicine also has a broad pH optimum between 7 and
and catharanthine than the control, whereas the 10withamaximumactivityaroundpH9(Schulte
clone with low hybridization signal increased the et al. 2000). MVAPK from C. roseus was also
productionofserpentineuptosevenfold(Ayora- purified and characterized (Schulte et al. 1999).
Talavera et al. 2002).
5-Diphosphomevalonate decarboxylase (MVAPP
MVAK(E.C.2.7.1.36)andMVAPK(E.C.2.7.4.2) decarboxylase, E.C. 4.1.1.33)
The phosphorylation of MVA to the mono- and The MVAPP decarboxylase that forms IPP from
di-phosphate ester (MVAP and MVAPP) has MVAPP has not yet been given much attention
been extensively studied in C. roseus plants and and has only been characterized in a few plant
cell cultures (Schulte 1998). MVAK from C. species (Verpoorte et al. 1997; Contin 1999). The
roseus cell cultures was purified to homogeneity formation of IPP was reviewed by Ramos-Valdi-
and characterized. The enzyme showed to be via et al. (1997).
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284 PhytochemRev(2007)6:277–305
IPP isomerase (E.C. 5.3.3.2) fiedfromC.roseusplants(MadyasthaandCoscia
1979)andfromcellcultures(Meijeretal.1993b).
Isopentenyl diphosphate isomerase was partially The protein showed a M of 79,000 and the
r
purified from C. roseus cultures (Ramos-Valdivia activityisdependentonNADPH,FADandFMN
et al. 1998). IPP-isomerase activity was also ascofactors.InC.roseustheCPRmRNAlevelis
determined in 5-day-old C. roseus suspension enhanced by fungal elicitor treatments (Meijer
cultured cells, treated with Pythium aphanider- et al. 1993a, b, Lopes Cardoso et al.,1997). Like
matum elicitor preparation. A slight inhibition of the yeast and animal CPRs, C. roseus protein
the enzyme was observed during the first 120 h contains a hydrophobic domain close to the
after elicitor treatment (Moreno et al. 1996). N-terminus which serves as a membrane anchor.
Steady-state mRNA levels observed in C. roseus
Geranyl diphosphate synthase (E.C. 5.1.1.1) plants were higher in flowers and much lower in
leaves and stems while intermediate in the roots.
The enzyme GPP synthase is not yetinvestigated
in C. roseus (Contin 1999). Cyclase
Geraniol 10-hydroxylase The dialdehyde (10-oxogeranial/10-oxoneral) is
cyclized to iridodial. The enzyme responsible for
This enzyme is a cytochrome P450 monooxygen- the cyclization has not yet been purified from C.
ase dependent on NADPH. G10H is regarded as roseus but was obtained from Rauwolfia serpen-
a potential site for regulatory control in the tina (Uesato et al. 1986, 1987; Verpoorte et al.
biosynthesis of secologanin. Studies by Schiel 1997).
etal.(1987)showedthatG10Hactivityisinduced
when C. roseus cell cultures are transferred to an Loganic acid methyltransferase (E.C. 2.1.1.50)
induction medium known to enhance alkaloid
accumulationandthatthereisacloserelationship In the formation of 7-deoxyloganic acid from
between G10H activity and alkaloid accumula- iridodial, so far no enzymes have been described
tion. Also McFarlane et al. (1975) demonstrated (Contin 1999). The 7-hydroxylation to afford
that G10H is feedback inhibited by the TIAs loganic acid must precede its methylation, as
catharanthine, vinblastine and vindoline. The K suggested byenzymatic studies with the S-adeno-
i
ofcatharanthineinhibition(1 mM)isintherange syl-L-methionine:LAMT partially purified from
of alkaloid concentration in C. roseus (0.3– C. roseus seedlings (Madyastha et al. 1973). This
1 mM). Madyastha et al. (1976) partially purified enzyme catalyses the transfer of a methyl group
G10H from C. roseus seedlings. Meijer et al. to loganic acid to form loganin. Contin (1999)
(1993a)purifiedG10H from C.roseus suspension measured the activity of LAMT in C.roseus cells
cultured cells in a four-step procedure after cultured on three different media and found that
solublization with cholate. The protein showed a the activity is restricted to the early period of
M of 56,000 and a K of 5.5 lM geraniol and growth similarly to the results obtained by Guar-
r m
11 lMnerol.Also,Colluetal.(2001)purifiedthis naccia et al. (1974) and Madyastha and Coscia
enzymefrom C.roseus cell cultures followingthe (1979) in C. roseus seedlings where maximum
method developed by Meijer et al. (1993a) with activity of the enzyme was recorded just after
some modifications. germination.
NADPH:cytochrome P450 reductase Secologanin synthase (E.C. 1.3.3.9)
(E.C. 1.6.2.4)
Secologanin synthase belongs also to the cyto-
Cytochrome P450 reductase functions in electron chrome P450 family. This gene was cloned 12
transfer from NADPH and is essential for all years ago from C. roseus and was first thought to
cytochrome P450 monooxygenases. It was puri- encode G10H (Vetter et al. 1992) but recently it
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PhytochemRev(2007)6:277–305 285
was shown that it encodes the enzyme that 2-C-methyl-D-erythrose and the reduction of this
converts loganin to secologanin (Irmler et al. aldose to MEP. The free aldose phosphate was
2000).ThiscytochromeP450enzymeacceptsonly notdetectedandthepresenceofthe5-phosphate
loganin as substrate with an optimum catalysing group on DXP was required for the enzymatic
reaction at a pH of 7.5 (Yamamoto et al. 2000). conversion.
Enzymes involved in the MEP pathway
Isopentenyl monophosphate kinase (E.C. 2.7.1.)
leading to the formation of IPP
LangeandCroteau(1999)reportedthecloningof
1-Deoxy-D-xylulose 5-phosphate synthase (E.C. the gene encoding IPK from peppermint and E.
4.1.3.37)
coli. This kinase catalyses the phosphorylation of
isopentenylmonophosphateasthelaststepofthe
Bacteria, fungi, yeasts and plants are capable of
biosynthetic sequence to IPP. This enzyme
synthesizing 1-deoxy-D-xylulose (DX) or its belongstoaconservedclassoftheGHMPfamily
5-phosphate (DXP) from pyruvate and from
of kinases that includes galactokinase, homoser-
D-glyceraldehyde or from its phosphate (GAP). ine kinase, MVAK and phosphomevalonate
The enzymatic activity in this step is thiamine
kinase. This enzyme was thought to catalyse the
diphosphate dependent and probably related to
last step in the MEP pathway, however Rohdich
pyruvate dehydrogenase. As the reaction is not
etal.(2000)showedthattheoverexpressedCMK
specific,thesystemcanacceptacyloinsinplaceof
protein from tomato does not have any IPK
pyruvate as acetyl donor and also different
activity, even if very high concentrations of
aldoses, yielding 1-deoxyketoses with C , C or
5 6 recombinant enzyme were used. The detected
C skeletons.
7 IPK activity could not be metabolically relevant
The enzyme catalyses the concomitant decar-
and cannot confirm the final steps leading to IPP
boxylation of pyruvate and the condensation of
synthesis.
the resulting (hydroxyethyl) thiamine on free
GAP, yielding deoxyxylulose or its phosphate
(DXP). As free glyceraldehyde is not a usual
cellularmetabolite,GAPandDXParethoughtto Localization of the enzymes involved in the
be the normal substrate and product of the pathwaysleadingto theformation ofsecologanin
synthase.
In the mevalonate pathway, the enzymes are
1-Deoxy-D-xylulose 5-phosphate reducto localizedinthecytosolandproducetheprecursor
isomerase (E.C. 1.1.1.267) of triterpenes and sesquiterpenes. Nothing has
been reported on the localization of AACT/
The rearrangement of DX or DXP yields 2-C- HMGS enzymes in C. roseus although they were
methyl-D-erythrose or its 4-phosphate and the purified from a cell suspension cultures (Van der
reduction of these products yields 2-C-methyl-D- Heijden et al. 1994; Van der Heijden and
erythritol(ME)orits4-phosphate(MEP;Duvold Verpoorte 1995). The radish AACT and HMGS
et al. 1997a,b, Sagner et al. 1998). have been reported as membrane-associated
UsingE.colimutantsthatwereauxotrophicto enzymes (Weber and Bach 1994; Bach et al.
ME, a gene that complemented in these mutants 1994). It has been suggested that HMGR might
theregioncodingforIPPbiosynthesiswascloned be located in mitochondria and plastids of plants
and led to the identification of the enzyme (Gary 1987; Stermer et al. 1994). The regulated
responsible for the conversion of DXP into degradation of HMGR has been indicated to be
MEP (Takahashi et al. 1998). This reducto- completely localized in the endoplasmic reticu-
isomerase enzyme is NADPH-dependent and luminyeast(HamptonandRine1994).Evidence
requires Mn2+ as cofactor. It catalyses two con- that HMGR is located within endoplasmic retic-
secutive steps: the rearrangement of DXP into ulum as well as in spherical, vesicular structures
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286 PhytochemRev(2007)6:277–305
derived from endoplasmic reticulum has been Localization of the non-mevalonate pathway
recently given (Leivar et al. 2005). leading to the formation of IPP
The plant enzymesMVAK andMVAPK were
presumedtobepredominantlycytosolicbuthave Thebiosynthesisofmono-,di-andtetraterpenoids
been proven to be present in plastids as well in plants seems to occur in plastids, where the
(McKaskill and Croteau 1995; Albrecht and MEP pathway is localized (Rohmer 1999; Lange
Sandmann 1994). The subcellular localization of and Croteau 1999).In plastids, theDXPpathway
MVAK and MVAPK was studied in suspension operates to supply IPP for the synthesis of mon-
cultured cells of C. roseus and it was shown that oterpenes, diterpenes and carotenoids. Table 1
most of the activity of both enzymes was located summarizes the information about the known
in thecytosolic fraction. MVAK activity was also enzymesinvolvedinTIAbiosynthesisinC.roseus.
recovered from an organellar and microsomal
fraction. MVAPK activity was detected in the
organelle fraction (Schulte et al. 1999, 2000). Genes-encoding enzymes involved in the
The localization of IPP isomerase in C. roseus biosynthesis of secologanin
has not been determined yet butin Castor beans,
a mitochondrial and proplastidial IPP isomerase AcDNAcloneforradishAACThasbeencloned
have been detected (Green et al. 1975). In by complementation in yeast (Vollack and Bach
glandular trichomes of peppermint, it was found 1995). Using the radish cDNA as a probe to
that the cytoplasmic MVA pathway was blocked screenanA.thalianacDNAlibrary,fourpositive
at the level of HMGR and that the IPP utilized clones have been isolated of which three were
for both plastidial monoterpene and cytosolic identical and the fourth shown to be an antisense
sesquiterpene biosynthesis is synthesized exclu- (Pin˜as et al. 1997). The presence of antisense
sivelyintheplastids.Aconnectionofthepathway mRNAcould indicate aregulatorymechanismof
was proposed at the level of IPP that requires controlling translation of AACT mRNA.
translocation of IPP to the different compart- A cDNA encoding the A. thaliana HMGS
ments and the presence of an isoform of IPP has been cloned (Montamat et al. 1995) and
isomerase in each compartment (McKaskill and expressedinE.coli(Diezetal.1997).Inaddition
Croteau 1995). Ramos-Valdivia et al. (1997) to the A. thaliana HMGS gene, also the HMGS
extensively reviewed the IPP isomerase. gene has been cloned from Schizosaccharomyces
In Lithospermum erythrorhizon, the enzyme pombe (Katayama et al. 1995).
GPP synthase involved in the biosynthesis of Plants have a small gene-family of HMGR
naphthoquinones was found to be present in the isoenzymes that are differentially expressed and
cytosol (Sommer et al. 1995). respond to a variety of developmental and envi-
TheenzymeG10Hisassociatedwith(pro)vac- ronmental signals (Stermer et al. 1994; Enjuto
uolar membranes (Madyastha et al. 1977) rather et al. 1994; Weissenborn et al. 1995; Korth et al.
than with endoplasmic reticulum where many 1997). HMGR genes have been well studied in
P450s are found (Nebert 1979). Collu (1999), Solanaceaespecies.FourHMGRgeneshavebeen
studied the localization of G10H in C. roseus cell isolated from tomato. One of them, the hmg1
suspension cultures and confirmed that this gene has been found to be involved in aspects
enzyme is localized in vacuolar membranes. The related to primary metabolism like sterol biosyn-
CPRcatalysingcytochromeP450monooxygenase thesis and cell growth. The activation pattern of
reactions is a membrane-bound flavoprotein, the second HMGR gene hmg2 by wounding and
closelylinkedtotheP450protein.Theexpression elicitation has suggested a role in the plant’s
of the three identified C. roseus MEP pathway defence (Weissenborn et al. 1995). Also potato,
genes and the G10H genes was found to be in has at least three HMGR genes (Stermer et al.
internalphloemparenchyma,i.e.incellsdifferent 1994). A C. roseus hmg cDNA has been cloned
than the other known alkaloid biosynthesis (Maldonada-Mendosa et al. 1992). In C. roseus
related genes (Burlat et al. 2004). cell suspensions, methyl jasmonate has been
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