Introduction Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol150.Issue1.2000:

Plant Science 150 2000 59 – 69 Mevalonate kinase activity in Catharanthus roseus plants and suspension cultured cells Anna E. Schulte 1 , Eva M. Llamas Dura´n, Robert van der Heijden , Robert Verpoorte Di6ision of Pharmacognosy, LeidenAmsterdam Center for Drug Research, Gorlaeus Laboratories, P.O. Box 9502 , 2300 RA Leiden, The Netherlands Received 17 May 1999; received in revised form 10 August 1999; accepted 27 August 1999 Abstract Mevalonate kinase is an early enzyme in plant isoprenoid biosynthesis. Its activity was studied in different parts of Catharanthus roseus L. G. Don Apocynaceae plants and in C. roseus suspension cultured cells. In the plant specific mevalonate kinase activities were found to be relatively high in the fruits, stem, roots, flowers and buds, and relatively low in young and completely elongated leaves. In suspension cultured cells, the specific mevalonate kinase activity increased during the exponential phase of growth, reaching a maximum of 0.5 nkatmg protein at 4 days after subculturing. After 6 days, which corresponded to the beginning of the stationary phase, the specific activity decreased. After transferring 14-day-old C. roseus cells to a medium known to induce the production of terpenoid indole alkaloids, an increase in the specific mevalonate kinase activity was observed, reaching a maximum of 1.7 nkatmg protein at 8 days after transfer. The alkaloid accumulation in the cultures was monitored, and ajmalicine was found to be the major product. Under standard conditions, mevalonate kinase activity showed a diurnal rhythm, with highest activities at 12:00 and 24:00. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords : Catharanthus roseus; Apocynaceae; Terpenoid biosynthesis; Mevalonate kinase; Mevalonate www.elsevier.comlocateplantsci

1. Introduction

Isoprenoid biosynthesis is one of the major pathways in plants leading to a huge number of compounds. Many of these play important roles in growth and development, such as abscisic acid, chlorophyll, ubiquinone, sterols and phytoalexins [1,2]. The common precursor of all isoprenoids is isopentenyl diphosphate IPP. The demand and competition for this precursor must be extremely high, as a consequence of its unique position in this physiologically important pathway. At present, two pathways are known that result in the biosynthesis of IPP, i.e., the mevalonate MVA pathway [1 – 3], and the 2-C-methyl- D -erythritol 4-phosphate MEP pathway at the 4th European Symposium on Plant Isoprenoids Barcelona, April 21 – 23 1999 it was agreed to use only the names ‘‘Rohmer pathway’’ after its discoverer or ‘‘MEP pathway’’ after 2-C-methyl- D -erythritol 4- phosphate, the first committed precursor for the recently discovered non-mevalonate pathway of IPP biosynthesis. [4,5]. The MVA pathway is supposed to be exclusively cytosolic and in plants the MEP pathway has been shown to be localized in the chloroplasts. As to the mevalonate pathway, it consists of a sequence of six enzyme reactions Fig. 1. First, three molecules of acetyl-CoA are condensed to form 3-hydroxy-3-methylglutaryl-CoA HMG- CoA, which is reduced to MVA by HMG-CoA reductase HMGR; EC 1.1.1.34. MVA is subse- Corresponding author. Fax: + 31-71-527-4511. E-mail addresses : a.e.schultestm.tudelft.nl A.E. Schulte, heij- denlacdr.leidenuniv.nl R. van der Heijden 1 Present address: Section Enzymology, Department of Biotechnol- ogy, Delft University of Technology, Julianalaan 67, 2628 BC, Delft, The Netherlands. 0168-945200 - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 6 4 - 8 quently phosphorylated to the diphosphate before it is decarboxylated to form IPP. In this sequence of enzyme reactions HMGR has been recognised as an important enzyme for the regulation of substrate flux into isoprenoid biosynthesis [1,2,6]. In addition, IPP isomerase EC 5.3.3.2 [7], and MVA kinase MK; EC 2.7.1.36 have been indi- cated to be regulators of the metabolic flux to- wards isoprenoids. Considering plant MK, its regulatory role has been proposed due to the fact that in vitro the enzyme has been found to be inhibited by geranyl diphosphate GPP and farnesyl diphosphate FPP, two later intermediates of isoprenoid biosynthesis, in cotyledons of Phaseolus 6ulgaris and Cucumis melo, and in He6ea latex [8]. This characteristic has also been shown in yeast, hu- man, and rat MK [9 – 11]. In addition, the rat liver MK has been found to be regulated by sterols on the protein [10] and mRNA [12] levels. Indeed, the presence of a sterol regulatory element, known to control gene expression of HMGR and HMG- CoA synthase, has been recognised in the pro- moter region of the human MK gene [13]. At present, the genes for MK have been cloned from yeast [9], rat [12], human [14] and Arabidop- sis thaliana [15], and the sequences showed to be highly homologous.Therefore, regulatory charac- teristics as found for mammalian MK might also hold for MK from a plant source. Consequently, MK is expected to be an important enzyme in the regulation of isoprenoid biosynthesis in plants. Indeed, MK activity has been correlated with the accumulation of phytoalexins in potato tuber after wounding and elicitation [16], and with increased rubber production in Guayule plants after treat- ment with a bioregulator [17]. However, the importance of the MVA pathway in the biosynthesis of isoprenoids of plastidial origin has been questioned since the discovery of the presence of the MEP pathway in plant chloro- plasts. Certainly, the MVA pathway is predomi- nantly localized in the cytosol. Nevertheless, MK activity was detected in transforming maize etio- plasts [18], and labeling studies showed the pres- ence of the MVA pathway in the leucoplasts of peppermint glandular cells [19], and immature chloroplasts of young spinach plants [20]. In addition, numerous labeling studies have been performed on seedlings showing incorpora- tion of MVA into alkaloids reviewed in [21]. On the other hand, labeling studies with photo- mixotrophic suspension cultures of Catharanthas roseus have indicated that the plastidial iso- prenoids were derived from the MEP pathway [22]. In addition, the ultimate precursor of the terpenoid moiety of terpenoid indole alkaloids, i.e. secologanin, has been shown to be derived from the MEP pathway in a specially selected, secolo- ganin-accumulating green cell-line of C. roseus in Fig. 1. Biosynthesis of isopentenyl diphosphate according to the mevalonate pathway. AACT, acetoacetyl-CoA thiolase; HMGS, HMG-CoA synthase; HMGR, HMG-CoA reductase; MK, mevalonate kinase; PMK, 5-phosphomevalonate kinase; PMDC, 5-diphosphomevalonate decarboxylase; and IPP, isopentenyl diphosphate. our laboratory [23]. More studies will be necessary to determine the possibility of cross-talk and ex- change of intermediates between the MVA and MEP pathways in the formation of different ter- penoid classes, in particular in relation to the differentational and physiological state of the cell. Imbault et al. [24] showed that the inhibition of endogenous MVA production by pravastatin treatment blocked the alkaloid biosynthesis; sup- plying such cells with exogenous MVA allowed accumulation of alkaloids, but did not result in incorporation of MVA. Recently, it was suggested that isoprenylated proteins are involved in the regulation of secologanin biosynthesis. These proteins may thus form the link between the MVA and the MEP pathway in alkaloid biosynthesis [25]. The MVA pathway thus seems to play an essential role in the biosynthesis of terpenoid-in- dole alkaloids, either in a direct or an indirect way. Therefore, in continuation of our studies on the early steps of isoprenoid biosynthesis [26] atten- tion was focused on MK. MK was studied in C. roseus plants and suspension cultured cells grown under standard conditions and on a medium known to induce the accumulation of terpenoid- indole alkaloids [27].

2. Materials and methods