Review An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants † VINOD SHANKER DUBEY , RITU BHALLA* and RAJESH LUTHRA** †Neurobiotechnology Center, The Ohio State University, Columbus, Ohio 43210, USA *Plant Microbes Interaction Lab, Department of Biological Sciences, National University of Singapore, Singapore 117604 **CSIR Complex, Dr K S Krishnan Marg, Pusa, New Delhi 110 012, India †Corresponding author (Fax, 407-823-0956; Email, [email protected]) Terpenoids are known to have many important biological and physiological functions. Some of them are also known for their pharmaceutical significance. In the late nineties after the discovery of a novel non-mevalonate (non-MVA) pathway, the whole concept of terpenoid biosynthesis has changed. In higher plants, the conven- tional acetate-mevalonate (Ac-MVA) pathway operates mainly in the cytoplasm and mitochondria and synthesizes sterols, sesquiterpenes and ubiquinones predominantly. The plastidic non-MVA pathway however synthesizes hemi-, mono-, sesqui- and di-terpenes, along with carotenoids and phytol chain of chlorophyll. In this paper, recent developments on terpenoids biosynthesis are reviewed with respect to the non-MVA pathway. [Dubey V S, Bhalla R and Luthra R 2003 An overview of the non-mevalonate pathway for terpenoid biosynthesis in plants; J. Biosci. 28 637–646] 1. Introduction Terpenoid biosynthesis involves mostly head to tail addition of isopentenyl diphosphate (IPP, the active C5 Terpenoids constitute the largest family of natural plant isoprene unit), to its isomer dimethylallyl diphosphate products with over 30,000 members (Sacchettini and (DMAPP) synthesizing geranyl diphosphate (GPP, C10). Poulter 1997; Dewick 2002). Terpenoids are classified by Further, condensation of enzyme-bound geranyl diphos- the homologous series of number of five carbon isoprene phate with additional IPP units forms successively larger units in their structure: hemiterpenes C5 (1 isoprene unit), prenyl diphosphates e.g. farnesyl diphosphate (FPP, C15), monoterpenes C10 (2 isoprene units), sesquiterpenes C15 geranylgeranyl diphosphate (GGPP, C20), that might undergo (3 isoprene units), diterpenes C20 (4 isoprene units), tri- cyclization, coupling and/or rearrangement to produce terpenes C30 (6 isoprene units), tetraterpenes C40 (8 isoprene the parent carbon skeleton of sesquiterpenes and diter- units), polyterpenes (C5)n where ‘n’ may be 9–30,000 penes (figure 1; Singh et al 1989; McGarvey and Croteau (McGarvey and Croteau 1995). 1995; Luthra et al 1999b). GPP and FPP yield monoter- Keywords. Acetate-mevalonate; DOXP; non-MVA pathway; plants; terpenoids ________________ Abbreviations used: ATP, adenosine triphosphate; CDP, cytidine diphosphate; CDP-ME, 4-diphosphocytidyl-2-C-methyl-D- erythritol; CDP-ME2P, 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate; CTP, cytidine triphosphate; DMAPP, dimethylal- lyl diphosphate; DOXP, 1-deoxy-D-xylulose-5-phosphate; DXS, DOXP synthase; DXR, DOXP reductoisomerase; FPP, farnesyl diphosphate; GAP, glyceraldehyde-3-phosphate; GGPP, geranylgeranyl diphosphate; GPP, geranyl diphosphate; HMBPP, 1-hydroxy-2- methyl-2-(E)-butenyl-4-diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; MECP, 2C-methyl-D-erythritol 2, 4-cyclodiphosphate; MEP, 2-C-methyl-D-erythritol-4- phosphate; MVA, mevalonate; P, phosphate; PP, diphosphate; TPP, thiamin diphosphate. J. Biosci. | Vol. 28 | No. 5 | September 2003 | 637–646 | © Indian Academy of Sciences 637 638 Vinod Shanker Dubey, Ritu Bhalla and Rajesh Luthra pene and sesquiterpene skeletons, respectively. Further- In plants, terpenoids are synthesized via two IPP gen- more FPP and GGPP dimerize to produce parental pre- erating pathways i.e. acetate-mevalonate (Ac-MVA) and cursors to synthesize triterpenes and tetraterpenes, non-mevalonate (non-MVA) pathways (figure 2; Lichten- respectively. These parental precursors are subjected thaler 1999; Rohmer 1999). The present review is an up- to structural modification through oxidation, reduction, date in the area of plant terpenoid biosynthesis with isomerization, hydration, conjugation and/or other trans- reference to recent developments reported on the non- formations to give rise to a variety of terpenoids MVA pathway. (McGarvey and Croteau 1995). Terpenoids play multi- functional roles in plants, human health and commerce. Monoterpenes (C10) and sesquiterpenes (C15), the con- 2. Cytoplasmic Ac-MVA pathway for IPP stituents of essential oils, are important flavouring and synthesis and its restricted role in the fragrance agents in foods, beverages, cosmetics, per- biosynthesis of terpenoids fumes, soaps and exhibit specific biological, pharmaceu- tical and therapeutical activities as well (Singh et al Earlier studies suggested mevalonate (MVA), a well- 1989; Dubey 1999; Mahmoud and Croteau 2002). known intermediate of Ac-MVA pathway, to be the key Figure 1. Synthesis of various classes of terpenoids in plants. The question mark (?) indicates the con- troversial role of isomerase via non-MVA route in which both IPP and DMAPP are reported to be synthe- sized independently. J. Biosci. | Vol. 28 | No. 5 | September 2003 Terpenoids biosynthesis in plants 639 Dimethylallyl diphosphate (DMAPP) Figure 2. Two independent pathways for biosynthesis of IPP and DMAPP in plants. The terminal steps [from MECP to IPP/DMAPP (marked by ‘?’)] are not well characterized in plants. However, the steps are shown on the basis of related studies done in E. coli and demonstration of orthologous genes in various plants, encoding such enzymes that catalyze the same reaction. The role of DOXP in biosynthesis of thiamin (vitamin B1) and pyridoxal (vitamin B6) and the known inhibitors (mevinolin and fosmidomycin) for each pathway are also shown. P, Phosphate; PP, diphosphate, and CMP, CDP, CTP and ATP are mono-, di- and triphosphates of cytidine and adenosine, respectively. J. Biosci. | Vol. 28 | No. 5 | September 2003 640 Vinod Shanker Dubey, Ritu Bhalla and Rajesh Luthra precursor of plant terpenoids (Croteau 1987; Lalitha and Earliest fore-runner data on plants include [13C]-labelled Ramasarma 1987; Bach 1995; Chappell 1995). The glucose incorporation into ginkgolides in Ginkgo biloba classical Ac-MVA pathway involves condensation of as a labelling pattern inconsistent with the Ac-MVA three units of acetyl CoA to form 3-hydroxy-3-methyl- route (Schwarz 1994). The [13C]-NMR data interpretation glutaryl coenzyme A (HMG-CoA), which after reduction of labelling patterns in various terpenoids, later suggested yields MVA. MVA is subsequently transformed to IPP that C-3, C-2 and C-1 of a triose could supply C-1, C-2 via three sequential steps involving phosphorylation and and C-4 of IPP, and that C-2 and C-3 of pyruvate could decarboxylation (figure 2A). The reduction of HMG-CoA supply C-3 and C-5 of IPP via the non-MVA pathway to MVA is catalyzed by HMG-CoA reductase (HMGR), a (Lichtenthaler 1999; Rohmer 1999). Accordingly, a bio- key regulatory enzyme of this pathway that has been chemical scheme was proposed with a head-to-head con- extensively studied (Rodwell et al 2000). densation of D-glyceraldehyde 3-phosphate (GAP, a triose) HMGR, a highly conserved enzyme occurring com- and ‘activated acetaldehyde’ derived from pyruvate, monly in eukaryotes, catalyzes the rate-limiting step of resulting in the formation of 1-deoxy-D-xylulose-5- IPP biosynthesis in animals and possibly also in the cyto- phosphate (DOXP) as the first precursor of this novel solic terpene biosynthesis in plants. In higher plants, biosynthetic pathway (step 1 in figure 2B). DOXP was HMGR is encoded in the nuclear genome by a multigene already known as a precursor for the biosynthesis of family. The characterization of HMGR from different thiamin (vitamin B1) and pyridoxal (vitamin B6) in plant species revealed developmental and organ-specific plants (Julliard and Douce 1991; Julliard 1992). expression of HMGR isoforms (Rodwell et al 2000). Fur- In the non-MVA pathway, addition of a C2 unit (derived thermore, HMGR activity responds to a variety of envi- from pyruvate decarboxylation) to a C3 unit (a triose ronmental and physiological signals including light, plant phosphate or its derivative) involved a transketolase type growth regulators, inhibitors, phosphorylation, metabolic reaction, as well as a rearrangement similar to that feed back, wounding and plant pathogens (Chappell et al observed in the biosynthesis of branched-chain amino 1995; Luthra et al 1999a). However, correlation between acids such as valine (cited in Luthra et al 1999b). cytosolic HMGR activity and the biosynthesis of plas- Glyceraldehyde-3-phosphate (GAP) and pyruvate were tid-bound prenyllipids like chlorophyll or carotenoids is later identified as the direct precursors of IPP (Rohmer not fully understood, and HMGR isoforms with typical 1999). The subsequent steps of this pathway in plants leader peptides to target these into the chloroplasts have have been demonstrated by various radio-tracer studies not been reported (Rodwell et al 2000). There are some (Fellermeier et al 1998; Lichtenthaler 1999; McCaskill indications of existence of a plastidic HMGR, however, and Croteau 1999), and various enzymes and the genes whether HMGR plays any role in the biosynthesis of involved have also been identified (Lichtenthaler 2000; plastidic terpenoids is a moot point (McCaskill and Cro- Eisenreich et al 2001; Rohdich et al 2001). The existence teau 1998).