6. Terpenes: C5 to C20 RA Macahig FM Dayrit HO CH3 3 _ 1 CO2 5 OH 3-(R)-MVA Introduction • Terpenes make up very prominent and characteristic group of plant secondary metabolites. Terpene metabolites range from volatile compounds with 10 carbons to colored polyenes with 40 carbons. • The word “terpene” comes from turpentine, the yellow to brown thick oleoresin which is obtained as an exudate from the terebinth tree (Pistacia terebinthus). • Terpenes are historically, culturally and economically important: • oleoresins, such as pine and eucalyptus oils; rubber (gutta percha) • distillates of the resin yield solvents and thinners • “essential oils” and perfumes, which are extracted from flowers and leaves by pressing, alcohol extraction or steam distillation • drugs and steroids 6. Terpenes: C5 to C20 (Dayrit) 2 Introduction Some characteristic terpenes: 10 AcO O OH CH3 H C CH3 1 O Ph O 3 2 O CH 9 7 8 3 Ph NH O CH3 CH3 CH3 H OH H 4 O Camphor: monoterpene from HO AcO PhCO Cinamomum camphora. 2 Taxol: antitumor diterpene from 21 24 20 23 26 Pacific yew, Taxus species 18 25 CH3 27 19 11 13 17 CH 3 14 1 9 10 8 3 5 6 HO Cholesterol: steroid originally Carotene: C40 terpene which is synthesized in isolated from gallstones; component of the chloroplast; important plant pigment; believed all cell membranes to be one of the important natural anti-oxidants. 6. Terpenes: C5 to C20 (Dayrit) 3 Introduction • In the late 19th century, Otto Wallach noted that upon chemical degradation, many of the products obtained had chemical formulas which were in multiples of 5 carbons. In the 1860s, these C5 units were called “isoprene” units. This is the basis of the “isoprene rule” which was formulated by Leopold Ruzicka. Isoprene represents the basic skeletal structure of the C5 unit. 2 n isoprene n natural rubber limonene The most prolific producer of isoprene-type polymers is the rubber tree, Hevea brasiliensis. 6. Terpenes: C5 to C20 (Dayrit) 4 Overview of terpene biogenesis • Isopentenyl diphosphate (IPP) is the C5 precursor of all isoprenoids. In plants, IPP is formed via two distinct OPP biosynthetic pathways: HO CH • The mevalonic acid (MVA) pathway 3 operates in the cytoplasm and is responsible for the smaller terpenes _ CO2 and the phytosterols OH • The methyl erythritol phosphate HO CH (MEP) pathway is responsible for the 3 OH chloroplast isoprenoids (-carotene, lutein, prenyl chains of chlorophylls HO and plastoquinone-9). OP 6. Terpenes: C5 to C20 (Dayrit) 5 The Mevalonic Acid (MVA) pathway • 3R-Mevalonic acid (MVA) is biosynthesized from three acetates. O + H HOCH3 HOCH3 O H3CSCoAOO 3 3 _ 1 H3C SCoA H3C SCoA CO25 1 5 OH O OO CoAS SCoA 3-(R)-MVA - H2C SCoA Note: 3S-MVA is an unnatural stereoisomer. There is no evidence that it is incorporated into terpenes. • MVA is converted to isopentenyl diphosphate (IPP) which is converted to its isomer, dimethylallyl diphosphate (DMAPP). HOCH3 HOCH 3 POCH3 3 -CO 2 ATP3 ATP 3 2 _ IPP _ 4.1.1.33 1CO5 1 1 OPP 2 CO2 5 5 OH OPP _ O O OPP 5.3.3.2 3-(R)-MVA5-diphospho-(R)-MVA DMAPP 4.1.1.336. Terpenes: C5: to C20Diphosphomevalonate (Dayrit) OPP 6 decarboxylase 5.3.3.2 : Isopentyl-diphosphate--isomerase The Mevalonic Acid (MVA) pathway MVA pathway for isoprenoid biosynthesis with labeling pattern from [1-13C]glucose metabolized via glycolysis. (Rohmer, Pure Appl Chem 2003) 6. Terpenes: C5 to C20 (Dayrit) 7 The Methyl Erythritol Phosphate (MEP) pathway OH HOCH • Glyceraldehyde-3- 2 Glyceraldehyde- O PO 3-phosphate) (C phosphate (GAP) and CHO 3 OH OH O phosphoenolpyruvate (PEP) HO OH Phosphoenol are formed from glucose. Glucose H3C CO2H pyruvate3) (C • GAP condenses with PEP to form MEP. MEP is converted to IPP which forms its isomer DMAPP. OH PO CHO HOCH3 Glyceraldehyde-3-phosphate3) (C OH O IPP HO OPP OP H3C CO2H Methylerythritol phosphate Phosphoenol3) pyruvate 5) (MEP) (C (C DMAPP OPP 6. Terpenes: C5 to C20 (Dayrit) 8 The Methyl Erythritol Phosphate (MEP) pathway MEP pathway for the biosynthesis of isoprenoids with labeling pattern from [1-13C]glucose metabolized via glycolysis. (Rohmer, Pure Appl Chem 2003) 6. Terpenes: C5 to C20 (Dayrit) 9 Evolution of the MVA and MEP pathways • The MVA pathway was originally thought to be the obligatory intermediate for all terpenes. (This is the pathway assumed in pre-2000 literature.) • The MEP pathway was first found in eubacteria and green algae, and was later shown to operate in the plant’s chloroplast. It is hypothesized that the MEP evolved first, and was incorporated into plants from cyanobacteria. • Some fungi and yeasts have been shown to use the MVA pathway. Because the plant cytosol uses the MVA pathway, it is believed that the higher evolved organisms (fungi and yeast) may be the source of the plant’s nuclear DNA. • The co-occurrence of two distinct major metabolic pathways in plant cells is unique for isoprenoid formation in plant cells. 6. Terpenes: C5 to C20 (Dayrit) 10 “Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes” (Lange et al., PNAS, 97(24): 13172–13177, Nov 21, 2000) (p 1) • IPP is “the central intermediate in the biosynthesis of isoprenoids, the most ancient* and diverse class of natural products. Two distinct routes of IPP biosynthesis occur in nature: the MVA pathway and the recently discovered DXP** pathway.” “The evolutionary history of the enzymes involved in both routes and the phylogenetic distribution of their genes across genomes suggest that: the MVA pathway is germane to archaebacteria, that the DXP pathway is germane to eubacteria, and that eukaryotes have inherited MVA their genes for IPP biosynthesis from DXP (MEP) prokaryotes.” * In evolutionary terms, the fats are6. Terpenes:probably C5 the to older C20 (Dayrit) group! 11 ** DXP (deoxyxylulose 5-phosphate) pathway = MEP pathway “Isoprenoid biosynthesis: The evolution of two ancient and distinct pathways across genomes” (Lange et al., PNAS, 97(24): 13172–13177, Nov 21, 2000) (p 2) “The occurrence of genes specific to the DXP pathway is restricted to plastid-bearing eukaryotes, indicating that these genes were acquired from the cyanobacterial ancestor of plastids. “However, the individual phylogenies of these genes, with only one exception, do not provide evidence for a specific affinity between the plant genes and their cyanobacterial homologues. The results suggest that: lateral gene transfer between eubacteria subsequent to the origin of plastids has played a major role in the evolution of this MVA DXP (MEP) pathway.” 6. Terpenes: C5 to C20 (Dayrit) 12 The MVA and MEP pathways: taxonomic distribution Organism Pathways Bacteria MVA or MEP Archaea MVA Green Algae MEP Fungi MVA Plants MVA and MEP Animals MVA 6. Terpenes: C5 to C20 (Dayrit) 13 The MVA and MEP pathways: practical implications • The mevalonate-independent methylerythritol phosphate (MEP) pathway is present in many bacteria and in the chloroplasts of all phototrophic organisms. It represents an alternative to the well-known MVA pathway, which is present in animals, fungi, plant cytoplasm, archaebacteria, and some eubacteria. • The MEP pathway in these bacteria represents a novel selective target for antibacterial and antiparasitic drugs. • The MEP pathway is also present in nonphototrophic eukaryotes, but belonging to phyla related to phototrophic unicellular eukaryotes, such as the parasite responsible for malaria, Plasmodium falciparum. This presents a potential target for a new class of antibacterial and antiparasitic drugs. 6. Terpenes: C5 to C20 (Dayrit) 14 The MVA and MEP pathways:DXS, 1-deoxy- dpractical-xylulose-5-phosphate implications synthase DXR, 1-deoxy-d-xylulose-5-phosphate reductoisomerase HMGR, 3-hydroxy-3-methylglutaryl coenzyme A HDS, hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase reductase IDS, isopentenyl diphosphate dimethylallyl diphosphate synthase IDI, isopentenyl diphosphate isomerase IDI, isopentenyl diphosphate isomerase Roberts, Nature Chemical Biology, 2007. Biology, Chemical Nature Roberts, Compartmentalized biosynthesis of IPP and DMAPP via the cytosolic MVA and the plastidic MEP pathways. 6. Terpenes: C5 to C20 (Dayrit) 15 OPP Hemiterpenes (rare) DMAPP, C5 OPP The terpene family is formed by Monoterpenes condensation of C5 OPP (IPP) units: geranyl pyrophosphate, C10 • C10, monoterpenes OPP • C15, sesquiterpenes • C20, diterpenes. OPP Sesquiterpenes farnesyl pyrophosphate, C15 OPP OPP Diterpenes geranylgeranyl pyrophosphate, C20 6. Terpenes: C5 to C20 (Dayrit) 16 Terpene chains are produced by condensation of DMAPP with IPP in “head-to-tail” manner. DMAPP is the “starter unit” while IPP is the nucleophile which lengthens the terpene chain. :Base H OPP OPP -OPP _ OPP IPP X DMAPP X Enz Enz _ -Enz-X OPP OPP farnesylpyrophosphate geranylpyrophosphate OPP OPP _ IPP X etc. Enz 6. Terpenes: C5 to C20 (Dayrit) 17 C30 terpenes are formed by head-to-head dimerization of C15 sesquiterpenes. This leads to the triterpenes, steroids, and carotenes. OPP + PPO (farnesyl pyrophosphate, C15) x 2 squalene, C30 Triterpenes, C30 Steroids OPP + PPO (geranylgeranyl pyrophosphate, C20) x 2 C40 18 Carotenes, C40 HOCH2 Plastids Cytosol O Overview of OH OH HO OH OP Terpene CoA-SCOCH OH 3 Glucose PO CHO CO2H Biosynthesis GAP PEP in Plants H3C OH HO CH3 OH MVA HOC MEP 2 HO OPP OP R OPP OPP OPP OPP DMAPP, C5 Prenyl IPP, C5 DMAPP, C5 IPP, C5 side-chain Monoterpenes OPP Monoterpenes OPP geranyl pyrophosphate, C10 geranyl pyrophosphate, C10 IPP, C5 IPP, C5 Sesquiterpenes Sesquiterpenes OPP OPP farnesyl pyrophosphate, C15 farnesyl pyrophosphate, C15 IPP, C5 H Polyprenyl R side-chain Head-to-head n Diterpenes dimerization OPP geranylgeranyl pyrophosphate, C20 OPP Diterpenes geranylgeranyl pyrophosphate, C20 Head-to-head dimerization Triterpenes & Steroids squalene,6. Terpenes: C30 C5 to C20 (Dayrit) 19 Carotenoids, C40 Estimates of number of structural groups and compounds known for each of the major types of terpenes.