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provided by Springer - Publisher Connector Phytochem Rev (2007) 6:277–305 DOI 10.1007/s11101-006-9047-8

Catharanthus terpenoid indole alkaloids: biosynthesis and regulation

Magdi El-Sayed Æ Rob Verpoorte

Received: 29 August 2005 / Accepted: 23 October 2006 / Published online: 10 March 2007 Springer Science+Business Media B.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. Feeding precursors, growth regulators treat- 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 characterization of its alkaloid pathway 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 Á Biosynthesis Á focussed on early steps of the pathway, particu- Catharanthus Á Indole pathway Á MEP pathway Á larly the discovery of the 2-C-methyl-D-erythritol Regulation Á 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 tween them across different compartments. 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- steps in the pathway resulted in cloning of several 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 Á R. Verpoorte ( ) D4H Desacetoxyvindoline 4-hydroxylase Department of Pharmacognosy, Section of Metabolomics, Institute of Biology Leiden, Leiden DMAPP Dimethylallyl diphosphate University, Leiden, The Netherlands DXP 1-Deoxy-D-xylulose-5-phosphate e-mail: [email protected] DXR 1-Deoxy-D-xylulose-5-phosphate reducto isomerase M. El-Sayed Department of Botany, Aswan Faculty of Science, DXS 1-Deoxy-D-xylulose-5-phosphate South Valley University, Aswan, Egypt synthase

123 278 Phytochem Rev (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 which are found mainly in plants belonging to the IPP Isopentenyl diphosphate families: Apocynaceae, Loganiaceae, Rubiaceae

Km Michaelis–Menten constant and Nyssaceae (Verpoorte et al. 1997). 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

Mr Relative molecular weight alkaloids, even seeds that were thought to have 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 produce the central intermediate strictosidine, 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 and the newly discovered 2-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

123 Phytochem Rev (2007) 6:277–305 279

HO NH H N 2 OGlc tryptamine H H O H3COOC secologanin N+ N H + H CH3 N NH N H H N O CH H H 3 H OGlc H3COOC H H O O H3COOC H3COOC alstonine serpentine strictosidine

+ N N N N N N N N N H H N H H H H CH H H H CH H CH3 H 3 H CH3 3 H H H H O H O O O H COOC H3COOC H COOC H3COOC H3COOC 3 3 OH ajmalicine cathenamine 4,21-dehydrogeissoschizine epicathenamine tetrahydroalstonine

N H N H COOC 3 CH2OH N N H H N H H N H COOC CH2OH COOCH3 N 3 H N H stemmadenine tabersonine H akuammidine secodine

N N H N H COOC CH2CH3 OH 3 N H H COOCH3 CH O N OCOCH3 catharanthine 3 H CH3 NH COOCH3 N O N CH3 + H N vindoline H COOCH perivine 3 vindolinine H N COOCH3 H N

OH H3CO N OCOCH H 3 H3C COOCH3 OH N iminium N O H N H COOCH3 N N H N COOCH3 H N OH H H3CO N OCOCH3 OH H N OCOCH H3C COOCH3 COOCH3 H3CO N 3 H N H H3C COOCH3 vinblastine leurosine OH H3CO N OCOCH OH H 3 N H3C COOCH3

H 3',4'-anhydrovinblastine N COOCH 3 N H

OH OCOCH H3CO N 3 H OHC COOCH3 vincristine

Fig. 1 Different Catharanthus indole alkaloids biosynthetic pathways 123 280 Phytochem Rev (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 with the coupling of two molecules of acetyl-CoA was first discovered in studies of the biosynthesis to form acetoacetyl-CoA. This step is catalysed by 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 the labels was completely different and did not fit 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- dance with pyruvate as a precursor of a C2 subunit 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 aC3 subunit. with one IPP in a head-to-tail fashion generating For the MEP pathway, the biosynthetic geranyl diphosphate (GPP), the precursor for the 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- The mevalonate-independent pathway leading 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 family of transketolases. In the second step of this the formation of triterpenes (sterols) and certain pathway, rearrangement and reduction of DXP to sesquiterpenes (Newman and Chappell 1999; MEP takes place. The enzyme catalysing this step

123 Phytochem Rev (2007) 6:277–305 281

I. Mevalonate pathway II. MEP pathway O O O COOH SCoA + HOP OH acetyl CoA pyruvate glyceraldehyde 3-phsophate O acetoacetyl-CoA DXS CO2 thiolase SCoA OH OO O O P SCoA OH acetoacetyl-CoA 1-deoxy-D-xylulose 5-phosphate O NADPH HMG-CoA DXR synthase + SCoA NADP

HO O HO SCoA O P COOH OH OH hydroxymethyl-glutaryl-CoA 2-C-methyl-D-erythritol 4-phosphate CTP HMG-CoA NADPH Mg2+ CMS NH2 reductase PPi N O HO O O HO N O O PO P O O OH OH OH OH OH COOH OH OH mevalonate 4-cytidyl diphospho-2C-methyl-D-erythritol

ATP MVA kinase ATP CMK NH2 ADP O N O HO P O O O HO N O OH O P OP O O O P 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 O PP P O OH COOH OH OH mevalonate diphosphate 2-C-methyl-D-erythritol 2,4-cyclodiphosphate MVAPP O decarboxylase ATP O O P OP OH OH OH OH 1-hydroxy-2-methyl 2(E) butenyl 4 diphosphate

O P P O P P O P IPP isomerase IPP isomerase IPP DMAPP IPP Fig. 2 The biosynthesis of IPP via mevalonate pathway and MEP pathway with involved enzymes

123 282 Phytochem Rev (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 IPP is formed finally in low rate from isopentenyl conversion probably involves a cytochrome P450 monophosphate via a step catalysed by the enzyme. This enzyme was finally detected and enzyme isopentenyl monophosphate kinase. 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 yet in C. roseus. 4-Cytidyl diphospho-2 C-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 (Rohdich et al. 2000). Although there are few 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- biosynthetic pathway leading to the formation 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 P450 enzyme geraniol 10-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. In the formation of 7-deoxyloganic acid from (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). Finally the cleavage of the cyclopentane been the subject of extensive reviews (Chappell

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OH OH OH CHO CHO 10--hydroxy geranial

G10H

OH OHC CHO OH geraniol 10-hydroxy geraniol 10-oxogeraniol 10-oxogeranial

cyclase CH3 CH 3 HO CH 3 H H H OGlc OGlc H H H O O CHO LAMT HOOC HOOC H loganic acid 7-deoxyloganic acid iridodial HO CH 3

H OGlc CHO H O SLS H OGlc CH 3OOC H O loganin CH 3OOC secoloanin

Fig. 3 The biosynthesis of secologanin from geraniol. G10H geraniol 10-hydroxylase, LAMT loganic acid methyltrans- ferase, SLS secologanin synthase

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 10 with a maximum activity around pH 9 (Schulte clone with low hybridization signal increased the et al. 2000). MVAPK from C. roseus was also production of serpentine up to sevenfold (Ayora- purified and characterized (Schulte et al. 1999). Talavera et al. 2002). 5-Diphosphomevalonate decarboxylase (MVAPP MVAK (E.C. 2.7.1.36) and MVAPK (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).

123 284 Phytochem Rev (2007) 6:277–305

IPP isomerase (E.C. 5.3.3.2) fied from C. roseus plants (Madyastha and Coscia 1979) and from cell cultures (Meijer et al. 1993b).

Isopentenyl diphosphate isomerase was partially The protein showed a Mr of 79,000 and the purified from C. roseus cultures (Ramos-Valdivia activity is dependent on NADPH, FAD and FMN et al. 1998). IPP-isomerase activity was also as cofactors. In C. roseus the CPR mRNA level is 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 yet investigated 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). et al. (1987) showed that G10H activity is induced when C. roseus cell cultures are transferred to an Loganic acid methyltransferase (E.C. 2.1.1.50) induction medium known to enhance alkaloid accumulation and that there is a close relationship 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 Ki suggested by enzymatic studies with the S-adeno- of catharanthine inhibition (1 mM) is in the range 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) purified G10H 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

Mr of 56,000 and a Km of 5.5 lM geraniol and growth similarly to the results obtained by Guar- 11 lM nerol. Also, Collu et al. (2001) purified this naccia et al. (1974) and Madyastha and Coscia enzyme from C. roseus cell cultures following the (1979)inC. 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

123 Phytochem Rev (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). This cytochrome P450 enzyme accepts only not detected and the presence of the 5-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 Lange and Croteau (1999) reported the cloning of 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 isopentenyl monophosphate as the last step of the Bacteria, fungi, yeasts and plants are capable of biosynthetic sequence to IPP. This enzyme synthesizing 1-deoxy-D-xylulose (DX) or its belongs to a conserved class of the GHMP family 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 et al. (2000) showed that the overexpressed CMK specific, the system can accept acyloins in place of 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 cellular metabolite, GAP and DXP are thought to Localization of the enzymes involved in the be the normal substrate and product of the pathways leading to the formation of secologanin synthase. In the mevalonate pathway, the enzymes are 1-Deoxy-D-xylulose 5-phosphate reducto localized in the cytosol and produce the precursor 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) or its 4-phosphate (MEP; Duvold Verpoorte 1995). The radish AACT and HMGS et al. 1997a,b, Sagner et al. 1998). have been reported as membrane-associated Using E. coli mutants that were auxotrophic to enzymes (Weber and Bach 1994; Bach et al. ME, a gene that complemented in these mutants 1994). It has been suggested that HMGR might the region coding for IPP biosynthesis was cloned 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 lum in yeast (Hampton and Rine 1994). 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

123 286 Phytochem Rev (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 enzymes MVAK and MVAPK were presumed to be predominantly cytosolic but have The biosynthesis of mono-, di- and tetraterpenoids 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, the DXP pathway 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 the cytosolic fraction. MVAK activity was also enzymes involved in TIA biosynthesis in C. 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 but in Castor beans, a mitochondrial and proplastidial IPP isomerase A cDNA clone for radish AACT has been cloned 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 screen an A. thaliana cDNA library, four positive 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 sively in the plastids. A connection of the pathway mRNA could indicate a regulatory mechanism of 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 expressed in E. coli (Diez et al. 1997). In addition 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- The enzyme G10H is associated with (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), Solanaceae species. Four HMGR genes have been 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- CPR catalysing cytochrome P450 monooxygenase thesis and cell growth. The activation pattern of reactions is a membrane-bound flavoprotein, the second HMGR gene hmg2 by wounding and closely linked to the P450 protein. The expression 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. internal phloem parenchyma, i.e. in cells different 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

123 Phytochem Rev (2007) 6:277–305 287

Table 1 Enzymes involved in biosynthesis of indole alkaloids of C. roseus Enzyme Abbreviation Cofactors Product Localization

Anthranilate synthase AS Mg2+ Anthranilate Plastid Tryptophan decarboxylase TDC PP, PQQ Tryptamine Cytosol Isopentenyl diphosphate IPP Mg2+,Mn2+ 3,3-Dimethylallyl Plastid isomerase isomerase diphosphate Geraniol 10-hydroxylase G10H Haeme 10-Hydroxygeraniol Provacuolar membrane NAPDH:cytochrome CPR NADPH, – Provacuolar P450 reductase FAD, membrane FMN SAM:loganic acid LAMT SAM Loganin – methyltransferease Secologanin synthase SLS NADPH Secologanin – Strictosidine synthase STR – Strictosidine Vacuole Strictosidine b-glucosidase SGD – [Cathenamine] Endoplasmic reticulum Cathenamine reductase CR NADPH Ajmalicine – Geissoschizine dehydrogenase – NADP+ 4,21-Dehydrogeis- – soschizine Tetrahydroalstonine synthase THAS NADPH Tetrahydroalstonine – Tabersonine 16-hydroxylase T16H Haeme, 16-Hydroxytabersonine Endoplasmic NADPH reticulum 11-Hydroxytabersonine OMT – 16-Methoxytabersonine – O-methyltransferase SAM:methoxy 2,16-dihydro-16- NMT SAM Desacetoxyvindoline Thylakoid hydroxytabersonine membranes N-methyltransferase Desacetoxyvindoline D4H 2- Oxoglutarate, Desacetylvindoline 17-hydroxylase Fe2+, ascorbate Cytoplasm Acetyl CoA:deacetylvindoline DAT Acetyl CoA Vindoline Cytoplasm 17-O-acetyltransferase Acetyl CoA:minovincinine-O- MAT Acetyl CoA Echitovenine Cytoplasm acetyltransferase Anhydrovinblastine synthase AVLB – Anhydrovinblastine Vacuole synthase shown to be able to increase HMGR mRNA cytochrome P450 cDNAs and their corresponding levels after a transient suppression (Maldonada- mRNAs were induced in alkaloid production when Mendosa et al. 1994). compared to normal growth medium (Vetter et al. The gene of deoxyxylulose 5-phosphate syn- 1992). Measuring the expression of the gene in thase has been isolated from E. coli (Sprenger different parts of the plant showed that mRNA et al. 1997; Lois et al. 1998) and cloned from levels were highest in the flowers. Attempts to higher plants, Mentha · piperita (Lange et al. clone the G10H gene by immunoscreening of a C. 1998) and C. roseus (Chahed et al. 2000; Veau roseus cDNA library were not successful because et al. 2000). The gene encoding the DXR enzyme the antibodies raised against the purified G10H was cloned from higher plants such as peppermint protein were not sufficiently specific (Meijer et al. (Lange and Croteau 1999), A. thaliana (Lange 1993a). Multiple cDNAs encoding P450s from A. and Croteau 1999; Schwender et al. 1999) and C. thaliana were isolated by employing a PCR strat- roseus (Veau et al. 2000). egy with degenerate oligonucleotide primers de- Molecular cloning of G10H has been previously signed on amino acid sequences conserved attempted using several different approaches. between plant cytochrome P450s (Mizutani et al. Differential screening of a C. roseus cDNA library 1998). The gene was cloned by Collu et al. (2001) resulted in the isolation of two highly homologous and expressed in C. roseus and yeast cells. It is

123 288 Phytochem Rev (2007) 6:277–305 strongly induced by methyljasmonate (MJ). Trans- from chorismate. This step is catalysed by the genic C. roseus cells showed that G10H activity is enzyme anthranilate synthase (AS, E.C. 4.1.3.27). correlated to the ability of the cells to accumulate The second step is formation of N-(5-phosphori- TIAs, although the increased G10H is not the only bosyl) anthranilate and this step is catalysed by requirement for increased alkaloid accumulation the enzyme phosphoribosyl diphosphate (PR)- (Collu et al. 2002). This gene was classified as a new anthranilate transferase (E.C. 4.1.1.48). The third member of the CYP76B plant P450 family. step is formation of 1-(O-carboxyphenylamino)- Cytochrome P450 reductase is cloned from C. 1-deoxyribulose phosphate. This step is catalysed roseus cell cultures by Meijer et al. (1993b). by the enzyme PR-anthranilate isomerase (E.C. Immunoscreening of a C. roseus cDNA expression 5.3.1.24). The following step is formation of library resulted in the isolation of a partial indole-3-glycerol phosphate. This step is catalysed NADPH:CPR clone. The clone was identified on by the enzyme indole-3-glycerol phosphate syn- the basis of sequence homology with CPRs from thase (E.C. 4.1.1.48). The next step is formation yeast and animals. The gene is encoded by a single of indole which is catalysed by the enzyme copy and the protein consists of 714 amino acids. tryptophan synthase a (E.C. 4.2.1.20). The last step is formation of L-tryptophan which is catal- The indole pathway ysed by the enzyme tryptophan synthase b (E.C. 4.2.1.20). This enzyme was detected in the prote- Biosynthesis of tryptophan omics approach as being induced when alkaloid production is induced (Jacobs et al. 2005). Tryptophan is an aromatic amino acid derived from chorismate via anthranilate. Chorismate Enzymes involved in the biosynthesis originates from the shikimate pathway (Fig. 4). of L-Tryptophan L-Tryptophan is formed through a biosynthetic pathway consisting of five enzymatically con- Anthranilate synthase (E.C. 4.13.27) trolled steps (Poulsen and Verpoorte 1991; Verpoorte et al. 1997; Bongaerts 1998; Whitmer Anthranilate synthase was first isolated and puri- 1999). The first step is formation of anthranilate fied to apparent homogeneity from C. roseus

- - COO COO- COO H OH 1 NH N 2 2 OH O COO- O

OH 3 OPi chorismate anthranilate N-(5-phosphoribosyl) anthranilate OH COO- O OH

N OPi 5 OPi 4 H N N OH OH H H indole indole-3-glycerol phosphate 1-(O-carboxyphenylamino)-1-deoxyribulose phosphate 6 COO-

NH2

N H tryptophan

Fig. 4 Biosynthesis of L-tryp