Natural Products (Secondary Metabolites)

CHAPTER 24 Natural Products (Secondary Metabolites)

Rodney Croteau Toni M. Kutchan Norman G. Lewis

CHAPTER OUTLINE Introduction

Introduction Natural products have primary ecological functions. 24.1 Terpenoids 24.2 Synthesis of IPP Plants produce a vast and diverse assortment of organic compounds, 24.3 Prenyltransferase and terpene the great majority of which do not appear to participate directly in synthase reactions growth and development. These substances, traditionally referred to 24.4 Modification of terpenoid as secondary metabolites, often are differentially distributed among skeletons limited taxonomic groups within the plant kingdom. Their functions, 24.5 Toward transgenic terpenoid many of which remain unknown, are being elucidated with increas- production ing frequency. The primary metabolites, in contrast, such as phyto- 24.6 Alkaloids sterols, acyl lipids, nucleotides, amino acids, and organic acids, are 24.7 Alkaloid biosynthesis found in all plants and perform metabolic roles that are essential 24.8 Biotechnological application and usually evident. of alkaloid biosynthesis Although noted for the complexity of their chemical structures research and biosynthetic pathways, natural products have been widely per- 24.9 Phenylpropanoid and ceived as biologically insignificant and have historically received lit- phenylpropanoid-acetate tle attention from most plant biologists. Organic chemists, however, pathway metabolites have long been interested in these novel phytochemicals and have 24.10 Phenylpropanoid and investigated their chemical properties extensively since the 1850s. phenylpropanoid-acetate Studies of natural products stimulated development of the separa- biosynthesis tion techniques, spectroscopic approaches to structure elucidation, and synthetic methodologies that now constitute the foundation of 24.11 Biosynthesis of lignans, lignins, contemporary organic chemistry. Interest in natural products was and suberization not purely academic but rather was prompted by their great utility 24.12 Flavonoids as dyes, polymers, fibers, glues, oils, waxes, flavoring agents, per- 24.13 Coumarins, stilbenes, fumes, and drugs. Recognition of the biological properties of myriad styrylpyrones, and arylpyrones natural products has fueled the current focus of this field, namely, 24.14 Metabolic engineering of the search for new drugs, antibiotics, insecticides, and herbicides. phenylpropanoid production: Importantly, this growing appreciation of the highly diverse biologi- a possible source of enhanced cal effects produced by natural products has prompted a reevalua- fibers, pigments, pharmaceuti- tion of the possible roles these compounds play in plants, especially cals, and flavoring agents in the context of ecological interactions. As illustrated in this chapter, many of these compounds now have been shown to have important

1250 Chapter 24 Natural Products (Secondary Metabolites) adaptive significance in protection against thus a natural product (Fig. 24.1). Even herbivory and microbial infection, as attrac- lignin, the essential structural polymer of tants for pollinators and seed-dispersing ani- wood and second only to cellulose as the mals, and as allelopathic agents (allelochem- most abundant organic substance in plants, icals that influence competition among plant is considered a natural product rather than species). These ecological functions affect a primary metabolite. plant survival profoundly, and we think it In the absence of a valid distinction reasonable to adopt the less pejorative term based on either structure or biochemistry, we “plant natural products” to describe sec- return to a functional definition, with prima- ondary plant metabolites that act primarily ry products participating in nutrition and es- on other species. sential metabolic processes inside the plant, and natural (secondary) products influenc- ing ecological interactions between the plant The boundary between primary and and its environment. In this chapter, we secondary metabolism is blurred. provide an overview of the biosynthesis of the major classes of plant natural products, Based on their biosynthetic origins, plant emphasizing the origins of their structural natural products can be divided into three diversity, as well as their physiological func- major groups: the terpenoids, the alkaloids, tions, human uses, and potential biotechno- and the phenylpropanoids and allied pheno- logical applications. lic compounds. All terpenoids, including both primary metabolites and more than 25,000 secondary compounds, are derived 24.1 Terpenoids from the five-carbon precursor isopentenyl diphosphate (IPP). The 12,000 or so known Terpenoids perhaps are the most structurally alkaloids, which contain one or more nitro- varied class of plant natural products. The gen atoms, are biosynthesized principally name terpenoid, or terpene, derives from the from amino acids. The 8000 or so phenolic fact that the first members of the class were compounds are formed by way of either the shikimic acid pathway or the malonate/ acetate pathway. Primary metabolite Secondary metabolite Primary and secondary metabolites cannot readily be distinguished on the basis of precursor molecules, chemical structures, or biosynthetic origins. For example, both primary and secondary metabolites are found among the diterpenes (C ) and triterpenes 20 COOH COOH (C30). In the diterpene series, both kaurenoic Kaurenoic acid Abietic acid acid and abietic acid are formed by a very similar sequence of related enzymatic reactions (Fig. 24.1); the former is an essential intermediate in the synthesis of gibberellins, N COOH N COOH i.e., growth hormones found in all plants H H (see Chapter 17), whereas the latter is a resin Proline Pipecolic acid component largely restricted to members of the Fabaceae and Pinaceae. Similarly, the es- Figure 24.1 sential amino acid proline is classified as a Kaurenoic acid and proline are primary metabolites, whereas the closely related compounds abietic primary metabolite, whereas the C6 analog acid and pipecolic acid are considered secondary pipecolic acid is considered an alkaloid and metabolites.

24.1–Terpenoids 1251 isolated from turpentine (“terpentin” in Ger- known hemiterpene is isoprene itself, a vol- man). All terpenoids are derived by repeti- atile product released from photosynthe- tive fusion of branched five-carbon units tically active tissues. The enzyme isoprene based on isopentane skeleton. These mono- synthase is present in the leaf plastids of nu-

mers generally are referred to as isoprene merous C3 plant species, but the metabolic units because thermal decomposition of rationale for the light-dependent production many terpenoid substances yields the alkene of isoprene is unknown (acclimation to high gas isoprene as a product (Fig. 24.2, upper temperatures has been suggested). Estimated panel) and because suitable chemical condi- annual foliar emissions of isoprene are quite tions can induce isoprene to polymerize in substantial (5 × 108 metric tons of carbon), multiples of five carbons, generating numer- and the gas is a principal reactant in the ous terpenoid skeletons. For these reasons, NOx radical–induced formation of tropo- the terpenoids are often called isoprenoids, spheric ozone (see Chapter 22, Fig. 22.37).

although researchers have known for well C10 terpenoids, although they consist of over 100 years that isoprene itself is not two isoprene units, are called monoterpenes; the biological precursor of this family of as the first terpenoids isolated from turpen- metabolites. tine in the 1850s, they were considered to be the base unit from which the subsequent nomenclature is derived. The monoterpenes 24.1.1 Terpenoids are classified by the are best known as components of the vol- number of five-carbon units they contain. atile essences of flowers and of the essential oils of herbs and spices, in which they make The five-carbon (isoprene) units that make up as much as 5% of plant dry weight. Mono- up the terpenoids are often joined in a “head- terpenes are isolated by either distillation or to-tail” fashion, but head-to-head fusions are extraction and find considerable industrial also common, and some products are formed use in flavors and perfumes. by head-to-middle fusions (Fig. 24.2, lower The terpenoids that derive from three panel). Accordingly, and because extensive isoprene units contain 15 carbon atoms and structural modifications with carbon–carbon are known as sesquiterpenes (i.e., one and bond rearrangements can occur, tracing the one-half terpenes). Like monoterpenes, original pattern of isoprene units is some- many sesquiterpenes are found in essential times difficult. oils. In addition, numerous sesquiterpenoids The smallest terpenes contain a single act as phytoalexins, antibiotic compounds isoprene unit; as a group, they are named produced by plants in response to microbial hemiterpenes (half-terpenes). The best challenge, and as antifeedants that discour- age opportunistic herbivory. Although the plant hormone abscisic acid is structurally a

sesquiterpene, its C15 precursor, xanthoxin, is not synthesized directly from three isoprene Isopentane Isoprene units but rather is produced by asymmetric cleavage of a C40 carotenoid (see Chapter 17). The diterpenes, which contain 20 car-

bons (four C5 units), include phytol (the hydrophobic side chain of chlorophyll), the gibberellin hormones, the resin acids of conifer and legume species, phytoalexins, Head-to-tail Head-to-head Head-to-middle and a host of pharmacologically important metabolites, including taxol, an anticancer Figure 24.2 agent found at very low concentrations Terpenes are synthesized from C5 units. The upper panel shows the structures of the isopentane skeleton and isoprene gas. The lower panel shows how different (0.01% dry weight) in yew bark, and for- patterns of isoprene unit assembly yield a variety of different structures. For ex- skolin, a compound used to treat glaucoma. ample, the triterpene squalene is formed by head-to-head fusion of two molecules Some gibberellins have only 19 carbon atoms of farnesyl diphosphate (FPP), which itself is the product of the head-to-tail fu- and are considered norditerpenoids since sion of isopentenyl diphosphate (IPP) and geranyl diphosphate (GPP) (see Fig. 24.7). The monoterpene pyrethrin I (see Fig. 24.10) results from a head-to-middle they have lost 1 carbon through a metabolic fusion of two C5 units. cleavage reaction (see Chapter 17).

1252 Chapter 24 Natural Products (Secondary Metabolites) The triterpenes, which contain 30 car- rule,” emphasizing mechanistic considera- bon atoms, are generated by the head-to- tions of terpenoid synthesis in terms of

head joining of two C15 chains, each of electrophilic elongations, cyclizations, and which constitutes three isoprene units joined rearrangements. This hypothesis ignores the head-to-tail. This large class of molecules in- precise character of the biological precursors cludes the brassinosteroids (see Chapter 17), and assumes only that they are “isoprenoid” the phytosterol membrane components (see in structure. As a working model for ter- Chapter 1), certain phytoalexins, various penoid biosynthesis, the biogenetic isoprene toxins and feeding deterrents, and compo- rule has proved essentially correct. nents of surface waxes, such as oleanolic Despite great diversity in form and acid of grapes. function, the terpenoids are unified in their The most prevalent tetraterpenes (40 common biosynthetic origin. The biosynthe- carbons, eight isoprene units) are the carote- sis of all terpenoids from simple, primary noid accessory pigments which perform metabolites can be divided into four overall essential functions in photosynthesis (see steps: (a) synthesis of the fundamental pre- Chapter 12). The polyterpenes, those con- cursor IPP; (b) repetitive additions of IPP to taining more than eight isoprene units, in- form a series of prenyl diphosphate ho- clude the prenylated quinone electron carri- mologs, which serve as the immediate pre- ers (plastoquinone and ubiquinone; see cursors of the different classes of terpenoids; Chapters 12 and 14), long-chain polyprenols (c) elaboration of these allylic prenyl diphos- involved in sugar transfer reactions (e.g., phates by specific terpenoid synthases to dolichol; see Chapters 1 and 4), and enor- yield terpenoid skeletons; and (d) secondary mously long polymers such as rubber (aver- enzymatic modifications to the skeletons age molecular mass greater than 106 Da), (largely redox reactions) to give rise to the often found in latex. functional properties and great chemical di- Natural products of mixed biosynthetic versity of this family of natural products. origins that are partially derived from terpenoids are often called meroterpenes. For example, both cytokinins (see Chapter 17) 24.2 Synthesis of IPP and numerous phenylpropanoid compounds contain C5 isoprenoid side chains. Certain al- 24.2.1 Biosynthesis of terpenoids is kaloids, including the anticancer drugs vin- compartmentalized, as is production of the cristine and vinblastine, contain terpenoid terpenoid precursor IPP. fragments in their structures (see Fig. 24.34). Additionally, some modified proteins in- Although terpenoid biosynthesis in plants, clude a 15- or 20-carbon terpenoid side chain animals, and microorganisms involves simi- that anchors the protein in a membrane (see lar classes of enzymes, important differences Chapter 1). exist among these processes. In particular, plants produce a much wider variety of terpenoids than do either animals or microbes, 24.1.2 A diverse array of terpenoid a difference reflected in the complex organi- compounds is synthesized by various zation of plant terpenoid biosynthesis at the conserved reaction mechanisms. tissue, cellular, subcellular, and genetic lev- els. The production of large quantities of ter- At the turn of the 20th century, structural in- penoid natural products as well as their sub- vestigations of many terpenoids led Otto sequent accumulation, emission, or secretion Wallach to formulate the “isoprene rule,” is almost always associated with the pres- which postulated that most terpenoids could ence of anatomically highly specialized be constructed hypothetically by repetitively structures. The glandular trichomes (Fig. joining isoprene units. This principle provid- 24.3A, B) and secretory cavities of leaves ed the first conceptual framework for a com- (Fig. 24.3C) and the glandular epiderms of mon structural relationship among terpenoid flower petals generate and store or emit ter- natural products (Box 24.1). Wallach’s idea penoid essential oils that are important be- was refined in the 1930s, when Leopold cause they encourage pollination by insects. Ruzicka formulated the “biogenetic isoprene The resin ducts and blisters of conifer species

24.2–Synthesis of IPP 1253 Early investigators formulated rules for identifying and naming Box 24.1 isoprenoid structures.

In the late 1800s, chemists struggled concept, known as the isoprene rule, ophile to yield the observed terpenoid to define the structures of the monoter- earned Wallach the Nobel Prize in Chem- products. This proposal, which Ruzicka penes. The mixed results achieved by istry in 1910. called the biogenetic isoprene rule, can these efforts are illustrated by the numer- By the 1930s, faced with a bewilder- be stated simply: A compound is “iso- ous structures proposed for camphor ing array of terpenoid substances, prenoid” if it is derived biologically from

(C10H16O; see structures at left of figure, Leopold Ruzicka and his contemporaries an “isoprenoid” precursor, with or with- which include the names of the proposers sought to develop a unifying principle out rearrangements. Ruzicka’s concept and the dates proposed). Chromatograph- that could rationalize the natural occur- differs from Wallach’s in its emphasis on ic purification techniques and spectro- rence of all of the known terpenoids, biochemical origin rather than structure. scopic methods for structure elucidation even those that did not strictly fit Wal- The great strength of the biogenetic iso- were not available to these early lach’s isoprene rule. Ruzicka’s ingenious prene rule lay in its use of mechanistic chemists, who relied on the preparation solution to the problem was to focus on considerations to classify the bulk of of crystalline derivatives to assess purity reaction mechanisms and ignore the pre- known terpenoids, including structures and on chemical degradation studies to cise character of the biological precursor, that did not strictly follow Wallach’s iso- determine structures. Systematic study of assuming only that it had a terpenoid prene rule. Application of the biogenetic the monoterpenes led the German structure during reaction. He hypothe- isoprene rule to the origin of several of chemist Otto Wallach to recognize that sized the involvement of electrophilic re- the common monoterpene skeletons is il- many terpenoid compounds might be actions that generated carbocationic inter- lustrated in the right panel of the figure constructed by joining isoprene units, mediates, which underwent subsequent (note the bornane skeleton from which

generally in a repetitive head-to-tail C5 addition, cyclization, and in some cas- camphor is derived). Ruzicka was award- fashion, as in Bredt’s correct proposed es skeletal rearrangement before elimina- ed the Nobel Prize in Chemistry in 1939. structure for camphor (see figure). This tion of a proton or capture by a nucle-

+ O

(Meyer, 1870) (Hlasiwetz, 1870) +

+ O O + Pinane skeleton α-Terpinyl cation Terpinen-4-yl cation

(Bredt, 1893) (Tiemann, 1895)

+ O + +

(Bouveault, 1897) (Perkin, 1898) Fenchane skeleton Bornane skeleton Thujane skeleton

(Fig. 24.3D) produce and accumulate a defen- polyterpenes such as rubber. These special- sive resin consisting of turpentine (monoter- ized structures sequester natural products pene olefins) and rosin (diterpenoid resin away from sensitive metabolic processes and acids). Triterpenoid surface waxes are formed thereby prevent autotoxicity. Most structures and excreted from specialized epidermis, of this type are nonphotosynthetic and must and laticifers produce certain triterpenes and therefore rely on adjacent cells to supply

1254 Chapter 24 Natural Products (Secondary Metabolites) the carbon and energy needed to drive ter- IPP to the cytosol for use in biosynthesis, penoid biosynthesis. and vice versa. Mitochondria, a third com- A more fundamental, and perhaps uni- partment, may generate the ubiquinone versal, feature of the organization of ter- prenyl group by the acetate/mevalonate penoid metabolism exists at the subcellular pathway, although little is known about the

level. The sesquiterpenes (C15), triterpenes capability of these organelles for terpenoid (C30), and polyterpenes appear to be pro- biosynthesis. duced in the cytosolic and endoplasmic reticulum (ER) compartments, whereas iso- 24.2.2 Hydroxymethylglutaryl-CoA

prene, the monoterpenes (C10), diterpenes reductase, an enzyme in the (C20), tetraterpenes (C40), and certain preny- acetate/mevalonate pathway, lated quinones originate largely, if not exclu- is highly regulated. sively, in the plastids. The evidence now indicates that the biosynthetic pathways for The basic enzymology of IPP biosynthesis by the formation of the fundamental precursor way of the acetate/mevalonate pathway is IPP differ markedly in these compartments, widely accepted (Fig. 24.4). This cytosolic with the classical acetate/mevalonate path- IPP pathway involves the two-step conden- way being active in the cytosol and ER and sation of three molecules of acetyl-CoA the glyceraldehyde phosphate/pyruvate catalyzed by thiolase and hydroxymethyl- pathway operating in the plastids. Regula- glutaryl-CoA synthase. The resulting tion of these dual pathways may be difficult product, 3-hydroxy-3-methylglutaryl-CoA to assess, given that plastids may supply (HMG-CoA), is subsequently reduced by

(A) (B)

St B E 100 µ

X Sh

P S L L S Sh

Figure 24.3 (A) Scanning electron micrograph of the leaf surface of thyme. The round structures are peltate glandular trichomes, in which monoterpenes and sesquiterpenes are synthesized. (B) Light micrograph of a glandular tri- chome from spearmint, shown in longitudinal section. C, subcuticular space; S, secretory cells; St, stalk; B, basal cell; E, epidermal cell. (C) Light micrograph of a secretory cavity in a lemon leaf, shown in cross-section. L, lumen; Sh, sheath cells; P, parenchyma cell. (D) Light micrograph of a resin duct in wood of Jeffrey pine, shown in cross-section. X, secondary xylem.

24.2–Synthesis of IPP 1255 HMG-CoA reductase in two coupled reac- HMG-CoA reductase is one of the most tions that form mevalonic acid. Two sequen- highly regulated enzymes in animals, being tial ATP-dependent phosphorylations of largely responsible for the control of choles- mevalonic acid and a subsequent phospho- terol biosynthesis. Accumulated evidence rylation/elimination-assisted decarboxylation indicates that the plant enzyme, which is yield IPP. located in the ER membrane, is also highly regulated. In many cases, small gene families, each containing multiple members, en- O Acetyl-CoA code this reductase. These gene families are CH3 C S CoA expressed in complex patterns, with individ- O ual genes exhibiting constitutive, tissue- or Acetyl-CoA development-specific, or hormone-inducible CH3 C S CoA expression. Specific HMG-CoA reductase Thiolase genes can be induced by wounding or path- CoASH ogen infection. The activity of HMG-CoA re- O O ductase may be subject to posttranslational Acetoacetyl- CH3 C CH2 C S CoA CoA regulation, for example, by a protein kinase O cascade that phosphorylates and thereby in- Acetyl-CoA activates the enzyme. Allosteric modulation HMG-CoA CH3 C S CoA probably also plays a regulatory role. Prote- synthase olytic degradation of HMG-CoA reductase CoASH protein and the rate of turnover of the corre- HO O 3-Hydroxy- sponding mRNA transcripts may also influ- 3-methyl- ence enzyme activity. Researchers have not CH3 C CH2 C S CoA glutaryl-CoA (HMG-CoA) arrived at a unified scheme that explains CH2 COOH how the various mechanisms that regulate 2 NADPH HMG-CoA reductase facilitate the produc- HMG-CoA + tion of different terpenoid families. The 2 NADP reductase precise biochemical controls that influence CoASH activity have been difficult to assess in vitro HO because the enzyme is associated with the Mevalonic acid ER membrane. A model proposed to ration- CH3 C CH2 CH2OH (MVA) alize the selective participation of HMG-CoA

CH2 COOH reductase in the biosynthesis of different ATP mevalonate-derived terpenoids is shown MVA kinase in Figure 24.5. ADP HO 24.2.3 In plastids, IPP is synthesized from Mevalonic acid pyruvate and glyceraldehyde 3-phosphate. CH3 C CH2 CH2O P 5-phosphate (MVAP)

CH2 COOH The plastid-localized route to IPP involves ATP a different pathway, demonstrated in green MVAP kinase algae and many eubacteria as well as plants. ADP In this pathway, pyruvate reacts with thia- HO mine pyrophosphate (TPP) to yield a Mevalonic acid two-carbon fragment, hydroxyethyl-TPP, CH3 C CH2 CH2O P P 5-diphosphate (MVAPP) which condenses with glyceraldehyde 3-

CH2 COOH phosphate (see Chapter 12, Fig. 12.41, for Figure 24.4 similar TPP-mediated C transfers catalyzed The acetate/mevalonate ATP 2 pathway for the forma- MVAPP by transketolase). TPP is released to form a tion of IPP, the basic decarboxylase five-carbon intermediate, 1-deoxy-D-xylulose ADP + P i five-carbon unit of ter- 5-phosphate, which is rearranged and re- penoid biosynthesis. CO2 + H2O duced to form 2-C-methyl-D-erythritol 4- Synthesis of each IPP CH3 Isopentenyl phosphate and subsequently transformed unit requires three C CH2 CH2O P P diphosphate molecules of acetyl-CoA. CH2 (IPP) to yield IPP (Fig. 24.6, upper pathway). The

1256 Chapter 24 Natural Products (Secondary Metabolites) HMG-CoA MVA Phytoalexins Sterols MVA HMG-CoA

Cytoplasm Glycosylation Lumen site Lumen

Transmembrane domains

N C N-terminal Catalytic domain sequence Linker Linker

Figure 24.5 Model for the membrane topology of HMG-CoA reductase sis of sesquiterpenoid phytoalexins contain an N-linked glycosyla- (HMGR). The protein includes a highly variable hydrophilic N-ter- tion site exposed to the ER lumen. Differences in N-terminal se- minal sequence (blue), a conserved membrane anchor (orange), a quences and extent of glycosylation may affect targeting of HMGR highly variable linker sequence (green and purple), and a highly to various ER domains and to other organelles of the endomem- conserved, cytosol-exposed, C-terminal catalytic domain (yellow). brane system (see Chapters 1 and 4). ER, endoplasmic reticulum; Isoforms of HMGR that are associated with elicitor-induced synthe- MVA, mevalonic acid.

E CH2O P CH2O P HO TPP CH3 OH COO– C H C OH TPP E H C OH NADPH C CH2

C O CH3 HO C H H O C H H2C C O P

CO CH O P H OH CH3 2 2 HO C TPP E O C + OH NADP Pyruvate + H C OH H TPP E CH3 CH3

C 1-Deoxy-D-xylulose- 2-C-Methyl-D-erythritol- H O 5-phosphate 4-phosphate

GAP

2 Acetyl-CoA OS–CoA 5

C CH2OH 1 O S–CoA 3 C 4 O P P CH2 CH2 2 IPP COOH CH3 HO C CH HO C CH 3 2 NADPH 3 CH3 5 C O CH2 CH2 3 1 C 4 O P P CH3 O S–CoA COOH COOH 2 2 CoASH + IPP Pyruvate Acetyl-CoA HMG-CoA 2 NADP Mevalonic + acid CoASH

Figure 24.6 Feeding studies distinguish two pathways of isoprenoid biosynthe- from labeled pyruvate and GAP by the plastid-localized pathway sis. When glucose isotopically labeled at C-1 is transformed by gly- will be labeled at C-1 and C-5 (upper panel), whereas IPP formed colytic enzymes and pyruvate dehydrogenase, the label subse- from labeled acetyl-CoA by way of the cytosolic acetate/meval- quently appears in the methyl groups of pyruvate and acetyl-CoA onate pathway will be labeled at C-2, C-4, and C-5 (lower panel). and in C-3 of glyceraldehyde 3-phosphate (GAP). IPP synthezised

1257 discovery of this new pathway for IPP for- can be used to determine the 13C-labeling mation in plastids suggests that these organ- pattern of each isoprene unit in a terpenoid elles, presumed to have originated as pro- compound, allowing researchers to infer the karyotic endosymbionts, have retained the labeling pattern of the corresponding IPP bacterial machinery for the production of this units (Fig. 24.6). key intermediate of terpenoid biosynthesis. The details of the glyceraldehyde 3- phosphate/pyruvate pathway and the en- 24.3 Prenyltransferase and terpene zymes responsible have not yet been fully synthase reactions defined. However, products of the two IPP biosynthesis pathways can be easily dis- Prenyltransferase enzymes generate the al- tinguished in experiments that utilize lylic diphosphate esters geranyl diphosphate [1-13C]glucose as a precursor for terpenoid (GPP), farnesyl diphosphate (FPP), and ger- biosynthesis. Nuclear magnetic resonance anylgeranyl diphosphate (GGPP). Reactions (NMR) spectroscopy (see Chapter 2, Box 2.2) that these compounds undergo (often cyclizations), which are catalyzed by terpene synthases, yield a wide variety of terpenoid compounds. Both prenyltransferases and ter- C5 CH2OCHP P 2O P P pene synthases utilize electrophilic reaction Isopentenyl Dimethylallyl Hemiterpenes mechanisms involving carbocationic inter- diphosphate diphosphate IPP isomerase mediates, a feature of terpenoid biochemistry. Enzymes in both groups share similar P P i properties and contain conserved sequence elements, such as an aspartate-rich DDxxD C10 CH2O P P motif involved in substrate binding, which Geranyl Monoterpenes may participate in initiating divalent metal diphosphate ion–dependent ionizations. CH2O P P (IPP)

P P i 24.3.1 Repetitive addition of C units is C CH O P P 5 15 2 carried out by prenyltransferases. Farnesyl Sesquiterpenes 2 diphosphate IPP is utilized in a sequence of elongation CH2O P P (IPP) reactions to produce a series of prenyl P P i diphosphate homologs, which serve as the

C20 CH2O P P immediate precursors of the different families of terpenoids (Fig. 24.7). Isomerization of 2 Geranylgeranyl Diterpenes IPP by IPP isomerase produces the allylic diphosphate isomer dimethylallyl diphosphate (DMAPP), 2 P P i

C30

Squalene Triterpenes

2 P P i

C40

Phytoene Tetraterpenes

Figure 24.7 The major subclasses of terpenoids are biosynthesized from the derived from the corresponding intermediates by sequential head- basic five-carbon unit, IPP, and from the initial prenyl (allylic) to-tail addition of C5 units. Triterpenes (C30) are formed from two diphosphate, dimethylallyl diphosphate, which is formed by iso- C15 (farnesyl) units joined head-to-head, and tetraterpenes (C40) are merization of IPP. In reactions catalyzed by prenyltransferases, formed from two C20 (geranylgeranyl) units joined head-to-head. monoterpenes (C10), sesquiterpenes (C15), and diterpenes (C20) are

1258 Chapter 24 Natural Products (Secondary Metabolites) which is considered the first prenyl diphos- wide range of nonterpenoid compounds, phate. Because DMAPP and related prenyl including proteins. diphosphates contain an allylic double bond, The most extensively studied prenyl- these compounds can be ionized to generate transferase, farnesyl diphosphate synthase, resonance-stabilized carbocations. Once plays an important role in cholesterol bio- formed, a carbocation intermediate of n car- synthesis in humans. Farnesyl diphosphate bons can react with IPP to yield a prenyl synthases from microbes, plants, and ani- diphosphate homolog containing n + 5 car- mals exhibit high sequence conservation. The bons. Thus, the reactive primer DMAPP un- first enzyme of the terpenoid pathway to be dergoes condensation with IPP to yield the structurally defined is recombinant avian

C10 intermediate GPP. Repetition of the reac- farnesyl diphosphate synthase, the crystal tion cycle by addition of one or two structure of which has been determined. molecules of IPP provides FPP (C15) or GGPP (C20), respectively. Each prenyl homolog in the series arises as an allylic diphos- 24.3.2 The enzyme limonene synthase is a phate ester that can ionize to form a reso- model for monoterpene synthase action. nance-stabilized carbocation and condense with IPP in another round of elongation The families of enzymes responsible for the (Fig. 24.8). formation of terpenoids from GPP, FPP, and The electrophilic elongation reactions GGPP are known as monoterpene, sesquiter-

that yield C10, C15, and C20 prenyl diphos- pene, and diterpene synthases, respectively. phates are catalyzed by enzymes known col- These synthases use the corresponding lectively as prenyltransferases. GPP, FPP, and prenyl diphosphates as substrates to form GGPP are each formed by specific prenyl- the enormous diversity of carbon skeletons transferases named for their products (e.g., characteristic of terpenoids. Most terpenoids farnesyl diphosphate synthase). The new al- are cyclic, and many contain multiple ring lylic double bond introduced in the course systems, the basic structures of which are of the prenyltransferase reaction is common- determined by the highly specific synthases. ly in the trans geometry, although this is not Terpenoid synthases that produce cyclic always the case: The transferase responsible products are also referred to as “cyclases,” for rubber biosynthesis introduces cis-double although examples of synthases producing bonds, which are responsible for the elastici- acyclic products are also known. ty of that polymer. Prenylation reactions are A diverse array of monoterpene syn- not limited to elongations involving IPP; the thases has been isolated from essential oil- same basic carbocationic mechanism permits producing angiosperm species and resin- the attachment of prenyl side chains to atoms producing gymnosperms. These enzymes of carbon, oxygen, nitrogen, or sulfur in a use a common mechanism in which ionization

IPP + O P P OPP O P P + H H 2+ H M + R R 1 2 3 R O P P R P P i P P Allylic diphosphate ester (n carbons)i Allylic diphosphate ester (n + 5 carbons) 1 A divalent metal cation promotes 2 The cation is added to IPP, generating 3 Deprotonation of the enzyme-bound the ionization of an allylic diphosphate a tertiary carbocation that corresponds intermediate yields an allylic (prenyl) substrate, yielding a charge-delocalized to the next C5 isoprenolog. diphosphate five carbons longer than cation that probably remains paired with the starting substrate. the pyrophosphate anion.

Figure 24.8 The prenyltransferase reaction.

24.3–Prenyltransferase and Terpene Synthase Reactions 1259 O P P of GPP leads initially to the tertiary allylic pines, spruces, and firs. The compounds are isomer linalyl diphosphate (LPP; Fig. 24.9). toxic to bark beetles and their pathogenic This isomerization step is required because fungal symbionts, which cause serious dam- GPP cannot cyclize directly, given the pres- age to conifer species worldwide. Many con- ence of the trans-double bond. Ionization ifers respond to bark beetle infestation by of the enzyme-bound LPP intermediate pro- up-regulating synthesis of monoterpenes, Geranyl diphosphate motes cyclization to a six-membered ring a process analogous to the production of 2+ M carbocation (the α-terpinyl cation), which antimicrobial phytoalexins, when under P P i may undergo additional electrophilic cycli- pathogen attack (Fig. 24.11). Other monoter- + zations, hydride shifts, or other rearrange- penes have quite different functions. Thus, ments before the reaction is terminated by linalool (see Fig. 24.10) and 1,8-cineole emit- deprotonation of the carbocation or capture ted by flowers serve as attractants for polli- by a nucleophile (e.g., water). Variations on nators, including bees, moths, and bats. this simple mechanistic scheme, involving 1,8-Cineole and camphor act as foliar feed- subsequent reactions of the α-terpinyl carbo- ing deterrents to large herbivores such as O P P cation, are responsible for the enzymatic for- hares and deer and also may provide a mation of most monoterpene skeletons (see competitive advantage to several angio- Box 24.1). sperm species as allelopathic agents that The simplest monoterpene synthase re- inhibit germination of the seeds of other action is catalyzed by limonene synthase, a species. (3S)-Linalyl diphosphate useful model for all terpenoid cyclizations Exceptions to the general pattern of (transoid rotamer) (Fig. 24.9). The electrophilic mechanism of head-to-tail joining of isoprene units seen action used by limonene synthase can be in limonene, the pinenes, and most other O P P viewed as an intramolecular equivalent of monoterpenes derived from GPP are the the prenyltransferase reaction (see Fig. 24.8). “irregular” monoterpenes. An example of Synthases that produce acyclic olefin prod- this type is the family of insecticidal mono- ucts (e.g., myrcene) and bicyclic products (α- terpene esters called pyrethrins, found in and β-pinene) from GPP are also known, as Chrysanthemum and Tanacetum species. are enzymes that transform GPP to oxy- These monoterpenoids, which exhibit a (3S)-Linalyl diphosphate (cisoid rotamer) genated derivatives such as 1,8-cineole and head-to-middle joining of C5 units, have bornyl diphosphate (Fig. 24.10), the precur- gained wide use as commercial insecticides 2+ M sor of camphor (see Box 24.1). because of their negligible toxicity to mam- An interesting feature of the monoter- mals and their limited persistence in the pene synthases is the ability of these en- environment (see Fig. 24.10). P P i + zymes to produce more than one product; for example, pinene synthase from several plant sources produces both α- and β-pinene. 24.3.3 Sesquiterpene synthases generate The pinenes are among the most common several compounds that function in monoterpenes produced by plants and are plant defense. principal components of turpentine of the The electrophilic mechanisms for the forma-

tion of the C15 sesquiterpenes from FPP Figure 24.9 closely resemble those used by monoterpene (–)-Limonene synthase catalyzes the simplest of all synthases, although the increased flexibility P P i terpenoid cyclizations and serves as a model for this of the 15-carbon farnesyl chain eliminates + reaction type. Ionization of GPP, assisted by divalent α-Terpinyl cation metal ions, provides the delocalized carbocation– the need for the preliminary isomerization diphosphate anion pair, which collapses to form the step except in forming cyclohexanoid-type P P i enzyme-bound tertiary allylic intermediate linalyl compounds. The additional C5 unit and dou- diphosphate. This required isomerization step, fol- ble bond of FPP also permit formation of a lowed by rotation about the C-2–C-3 single bond, overcomes the original stereochemical impediment to greater number of skeletal structures than direct cyclization of the geranyl precursor. A subse- in the monoterpene series. The best known quent assisted ionization of the linalyl diphosphate sesquiterpene synthase of plant origin is epi- α ester promotes an anti-endo-cyclization to the -ter- aristolochene synthase from tobacco, the pinyl cation, which undergoes deprotonation to form limonene, a compound now thought to be an impor- crystal structure of which has been deter- (–)-Limonene tant cancer preventive in humans. mined (Fig. 24.12). This enzyme cyclizes FPP

1260 Chapter 24 Natural Products (Secondary Metabolites) and catalyzes a methyl migration to yield the olefin precursor of the phytoalexin capsidiol, which is elicited by pathogen attack. Vetispiradiene synthase from potato provides the olefin precursor of the phytoalexin lubimin in this species, whereas α Myrcene -Pinene δ-cadinene synthase from cotton yields the OH olefin precursor of the important defense compound gossypol, the latter being currently studied as a possible male contraceptive (Fig. 24.13). Some sesquiterpene synthases involved in the production of conifer resin are capable of individually producing more Limonene Linalool than 25 different olefins.

O 24.3.4 Diterpene synthases catalyze two distinct types of cyclization reactions.

Two fundamentally different types of enzy- β-Pinene 1,8-Cineole matic cyclization reactions occur in the COOR transformation of GGPP to diterpenes (Fig. O P P 24.14). The first resembles the reactions catalyzed by monoterpene and sesquiterpene synthases, in which the cyclization involves ionization of the diphosphate ester and at- Bornyl diphosphate Pyrethrin I tack of the resulting carbocation on an interi- Figure 24.10 or double bond of the geranylgeranyl sub- Structures of monoterpenes, including insecticidal strate. An example of this type is casbene α β compounds ( - and -pinene, pyrethrin), pollinator synthase, which is responsible for produc- attractants (linalool and 1,8-cineole), and antiher- bivory agents (1,8-cineole). tion of the phytoalexin casbene in castor bean. Taxadiene synthase from yew species uses a mechanistically similar, but more complex, cyclization to produce the tricyclic olefin precursor of taxol. Abietadiene synthase from grand fir ex- emplifies the second type of cyclization, in which protonation of the terminal double bond to generate a carbonium ion initiates the first cyclization to a bicyclic intermediate (labdadienyl diphosphate, also known as copalyl diphosphate). Ionization of the diphosphate ester promotes the second cyclization step to give the tricyclic olefin product, abietadiene; a single enzyme catalyzes both

Figure 24.11 Mass attack by mountain pine beetles on a lodgepole pine (Pinus contorta) bole. Each white spot on the trunk represents a beetle entry point at which resin has been secreted. This tree has survived the attack because turpentine production was sufficient to kill all of the bark beetles, which have been “pitched out” by resin outflow. On evaporation of the turpentine and exposure to air, the diterpenoid resin acids form a solid plug that seals the wound.

24.3–Prenyltransferase and Terpene Synthase Reactions 1261 cyclization steps. Oxidation of a methyl 24.3.5 Triterpene synthesis proceeds group yields abietic acid (see Fig. 24.1), one from squalene, tetraterpene synthesis of the most common diterpenoid resin acids from phytoene. of conifers and important for wound sealing in these species. Fossilization of this resin Before cyclization can occur in the triterpene

produces amber. (C30) series, two molecules of FPP (C15) are first joined in a head-to-head condensation to produce squalene (see Fig. 24.7). The cata- lyst, squalene synthase, is a prenyltransferase that catalyzes a complex series of cationic C terminus (548) rearrangements to accomplish the chemically difficult chore of joining the C-1 carbons of 2 3 1 two farnesyl residues. Squalene is usually oxidized to form the 2,3-epoxide, oxidosqua- 4 8 lene, and then cyclized in a protonation- 7 5 6 initiated reaction to produce, for example, 36 534 A K J 521 I D J/K loop OH C H1 W273 G2 FHP R266 H2 E HO

R264 H3 α -1 Capsidiol 255 D1 F Mg2+ G1 D2 A/C loop N terminus (17)

Figure 24.12 epi-Aristolochene Schematic view of epi-aristolochene synthase com- plexed with the substrate analog farnesyl hydroxy- epi-Aristolochene phosphonate (FHP). Blue rods represent α-helices in synthase the N-terminal domain; red rods represent α-helices in the C-terminal domain. Loop regions shown in purple are disordered in the native enzyme. Three Mg2+ ions and arginines 264 and 266 are involved in the initial steps of the reaction and are labeled near δ-Cadinene Vetispiradiene the entrance to the active site. Tryptophan 273, which synthase O P P synthase serves as the general base in the final deprotonation FPP step, is shown within the hydrophobic active-site pocket. The substrate analog FHP is shown in ball- and-stick representation, highlighted with yellow carbon–carbon bonds. Naming of helices in the C-terminal domain corresponds to the convention used for FPP synthase.

δ-Cadinene Vetispiradiene

CHO OH OH CHO CHO HO OH

HO HO OH Figure 24.13 Structures of sesquiterpenes biosynthetical- ly derived from FPP. The end products Gossypol Lubimin function in plant defense. (Sesquiterpene dimer)

1262 Chapter 24 Natural Products (Secondary Metabolites) Casbene synthase O P P

Taxadiene Geranylgeranyl synthase Casbene diphosphate

Abietadiene synthase

Taxadiene

O P P

Abietadiene synthase

Labdadienyl Abietadiene (copalyl) diphosphate

Figure 24.14 Cyclization of GGPP to form the diterpenes casbene, taxadiene, and abietadiene. Cyclization can proceed by one of two distinct mechanims, only one of which yields the intermediate labdadienyl (copalyl) diphosphate.

the common sterol cycloartenol (Fig. 24.15), thase. A series of desaturation steps precedes a precursor of many other phytosterols and cyclization in the tetraterpene (carotenoid) brassinosteroids (see Chapter 17). Several al- series, usually involving formation of six- ternative modes of cyclization in the triter- membered (ionone) rings at the chain termi- pene series are also known, such as that ni to produce, for example, β-carotene from leading to the pentacyclic compound lycopene (see Chapter 12, Fig. 12.7). β-amyrin, the precursor of oleanolic acid found in the surface wax of several fruits (Fig. 24.15). Preliminary evidence suggests 24.4 Modification of terpenoid skeletons that sesquiterpene biosynthesis and triterpene biosynthesis (both of which utilize cy- Subsequent modifications of the basic parent tosolic FPP as a precursor) are reciprocally skeletons produced by the terpenoid syn- regulated during the induced defense re- thases are responsible for generating the

sponses, such that production of C15 defen- myriad different terpenoids produced by sive compounds is enhanced and C30 synthe- plants. These secondary transformations sis is repressed. most commonly involve oxidation, reduc-

The tetraterpenes (C40) are produced by tion, isomerization, and conjugation reac- joining two molecules of GGPP in head-to- tions, which impart functional properties to head fashion to produce phytoene, in a man- the terpenoid molecules. Several oxygenated ner analogous to the formation of squalene derivatives of parent terpenoids have al- (see Fig. 24.7). The reaction is catalyzed by ready been described in this chapter, includ- phytoene synthase, which deploys a mecha- ing capsidiol, lubimin, gossypol, abietic acid, nism very similar to that of squalene syn- and oleanolic acid.

24.4–Modification of Terpenoid Skeletons 1263 HO

OH HO

HO HO

O Cycloartenol O Brassinolide

COOH O

Oxidosqualene

HO HO Figure 24.15 β Structures of triterpenes. -Amyrin Oleanolic acid This class of squalene- derived products includes brassinosteroid regulators of plant Many of the hydroxylations or epoxida- reductase to produce (+)-cis-isopulegone. An growth and surface wax tions involved in introducing oxygen atoms isomerase next moves the remaining double components. into the terpenoid skeletons are performed bond into conjugation with the carbonyl by cytochrome P450 mixed-function oxidas- group, yielding (+)-pulegone. One regiospe- es. Because these reactions are not unique to cific, NADPH-dependent, stereoselective terpenoid biosynthesis, this section will not reductase converts (–)-pulegone to either focus on specific enzyme types but rather on (+)-isomenthone or the predominant species, the general role of secondary transforma- (–)-menthone. Similar reductases produce tions as the wellspring of diversity in ter- the menthol isomers from these ketones. penoid structure and function. (–)-Menthol greatly predominates among the menthol isomers (constituting as much as 40% of the essential oil) and is the compo- 24.4.1 The conversion of (–)-limonene to nent primarily responsible for the character- (–)-menthol in peppermint and carvone in istic flavor and cooling sensation of pepper- spearmint illustrates the biochemistry of mint. The menthol isomers are often found terpenoid modification. as acetate esters, formed by the action of an acetyl CoA-dependent acetyltransferase. The The principal and characteristic essential oil menthol and menthyl acetate content of pep- components of peppermint (Mentha piperita) permint oil glands increases with leaf matu- and spearmint (M. spicata) are produced by rity. Environmental factors greatly influence secondary enzymatic transformations of oil composition. Water stress and warm (–)-limonene (Fig. 24.16). In peppermint, night growth conditions both promote the a microsomal cytochrome P450 limonene accumulation of the more-oxidized pathway 3-hydroxylase introduces an oxygen atom intermediates such as (+)-pulegone. at an allylic position to produce (–)-trans- The pathway in spearmint is much isopiperitenol. A soluble NADP+-dependent shorter. In this instance, a cytochrome P450 dehydrogenase oxidizes the alcohol to a ke- limonene 6-hydroxylase specifically intro- tone, (–)-isopiperitenone, thereby activating duces oxygen at the alternative allylic posi- the adjacent double bond for reduction by a tion to produce (–)-trans-carveol, which soluble, NADPH-dependent, regiospecific is oxidized to (–)-carvone by the soluble

1264 Chapter 24 Natural Products (Secondary Metabolites) O P P 1 6 2

5 3 O 4 OH

Geranyl (–)-Limonene (–)-trans- (–)-Isopiperitenone diphosphate Isopiperitenol

(–)-trans-Carveol (+)-cis-Isopulegone

(–)-Carvone (+)-Pulegone

O O

(–)-Menthone (+)-Isomenthone

Figure 24.16 Essential oil synthesis in peppermint and spearmint. In peppermint, (–)-limonene is converted to (–)-isopiperitenone, OOCCH OH OH OH OH which is modified to form 3 (–)-menthol and related compounds. In spearmint, (–)-limonene is converted (–)-Menthyl (–)-Menthol (+)-Neomenthol (+)-Isomenthol (+)-Neoisomenthol to (–)-carvone by a acetate two-step pathway.

1265 NADP+-dependent dehydrogenase. Al- mals. Monoterpene lactones include nepeta- though most of the enzymatic machinery lactone (the active principle of catnip as well present in peppermint oil glands is also as an aphid pheromone), a member of the present in spearmint, the specificity of these iridoid family of monoterpenes, which are enzymes is such that (–)-carvone is a very formed by a cyclization reaction quite different poor substrate. Consequently, carvone, the from that of other monoterpenes (Fig. 24.17). characteristic component of spearmint flavor, The limonoids are a family of oxygenat- accumulates as the major essential oil com- ed nortriterpene antiherbivore compounds. ponent (about 70%). Similar reaction se- Like the sesquiterpene lactones, these sub- quences initiated by allylic hydroxylations stances taste very bitter to humans and prob- and subsequent redox metabolism and con- ably to other mammals as well. A powerful jugations are very common in the monoter- insect antifeedant compound is azadirachtin pene, sesquiterpene, and diterpene classes. A, a highly modified limonoid from the neem tree (Azadirachta indica). Other oxygenated triterpenoid natural products with unusual 24.4.2 Some terpenoid skeletons are biological properties include the phytoecdy- extensively decorated. sones, a family of plant steroids that act as hormones and stimulate insect molting; the Reactions similar to those responsible for saponins, so named because of their soap- essential oil production in mints generate like, detergent properties; and the cardeno- myriad terpenoid compounds of biological lides, which, like the saponins, are glyco- or pharmaceutical interest. Such reactions sides, in that they bear one or more attached convert sesquiterpene olefin precursors to sugar residues. Ingestion of α-ecdysone by phytoalexins (see Fig. 24.13), allelopathic insects disrupts the molting cycle, usually agents, and pollinator attractants. Additional with fatal consequences. The saponins and sesquiterpenes generated by modifying cardenolides are toxic to many vertebrate olefin precursors include juvabione (Fig. herbivores; this family of compounds in- 24.17), a compound from fir species that cludes well-known fish poisons and snail exhibits insect juvenile hormone activity; poisons of significance in the control of sirenin, a sperm attractant of the water mold schistosomiasis. Many of these products are allomyces; and artemisinin, a potent anti- also cardioactive and anticholesterolemic malarial drug from annual wormwood agents of pharmacological significance. Digi- (Artemisia annua, also known as Qinghaosu, toxin, the glycone (glycosylated form) of a plant used in traditional Chinese medicine digitoxigenin (Fig. 24.17) extracted from fox- since about 200 B.C.). A related enzymatic glove (Digitalis), is used widely in carefully reaction sequence converts the parent diter- prescribed doses for treatment of congestive pene olefin taxadiene to the anticancer drug heart disease. taxol in yew species, in which the basic ter- The broad range of insect and higher an- penoid nucleus is modified extensively by a imal toxins and deterrents among the modi- complex pattern of hydroxylations and acy- fied triterpenes leaves little doubt as to their lations. Esters of phorbol (another highly role in plant defense. Interestingly, some her- oxygenated diterpene) produced by species bivores have developed the means to cir- of the Euphorbiaceae are powerful irritants cumvent the toxic effects of these terpenoids and cocarcinogens. After introduction of a and adapt them to their own defense pur- hydroxyl group, subsequent oxidation can poses. The classical example of this phe- generate a carboxyl function such as that nomenon is the monarch butterfly, a special- found in abietic acid (see Fig. 24.1) and ist feeder on milkweeds (Asclepias) which oleanolic acid, and also provide the struc- contain cardenolides that are toxic to most tural elements for lactone ring formation. herbivores and are even associated with live- Sesquiterpenes bearing such lactone rings, stock poisoning. Monarch caterpillars, how- e.g., costunolide, are produced and accumu- ever, feed on milkweeds and accumulate the lated in the glandular hairs on the leaf sur- cardenolides without apparent ill effects. As faces of members of the Asteraceae, where a result, both caterpillars and the adult but- some of these compounds serve as feeding terflies contain enough cardenolides to be repellents to herbivorous insects and mam- toxic to their own predators such as birds.

1266 Chapter 24 Natural Products (Secondary Metabolites) O O O OH O COOCH3 O HO O Juvabione Sirenin Artemisinin (insect juvenile (sperm attractant in (antimalarial drug) hormone analog) water molds)

OH OOCCH3 O OH OH NH O O HO

O O OH OH H OOCCH3 O O O HO OH O Taxol O Phorbol Costunolide (anticancer drug) (irritant and cocarcinogen) (insect repellent, mammal antifeedant)

OH COOCH O 3 HO O OH OH C O O O O

HO O H3CCOO OH OH H3COOC O O HO O Nepetalactone Azadirachtin A α-Ecdysone (active principle of catnip) (insect antifeedant) (disrupts insect molting cycle)

O O

O O Figure 24.17 Terpenoids formed by OH secondary transforma- HO tions of parent cyclic HO H compounds. The yellow highlighting delineates Hecogenin, Digitoxigenin, the terpenoid portion of the aglycone of a saponin the aglycone of digitoxin, a cardenolide the molecule taxol. (a detergent) (treatment of congestive heart disease)

1267 24.5 Toward transgenic an even greater challenge, given that little terpenoid production metabolic context exists in these cases. In such species, issues of subcellular sites of With recent success in the cloning of genes synthesis, requirements for sufficiency of that encode enzymes of terpenoid synthesis, precursor flux, and the fate of the desired the transgenic manipulation of plant ter- product might present additional difficulties. penoid metabolism may present a suitable Clearly, targeting a terpenoid synthase to the avenue for achieving a number of goals. Sev- cellular compartment containing the appro-

eral agriculturally important crop species priate C10, C15, C20, or C30 precursor will be have been bred selectively to produce rela- an important consideration. Sufficient flux of tively low amounts of unpalatable terpenoid IPP at the production site to drive the path- defense compounds; in the process, these way also will be essential. Because con- cultivars have lost not only defense capabili- straints in precursor flow ultimately will ties but also, in some cases, quality attributes limit the effectiveness of transgenes for sub- such as flavor and color. The selective rein- sequent pathway steps, information about troduction of terpenoid-based defense the flux controls on IPP biosynthesis in both chemistry is certainly conceivable, as is the cytosol and plastid, and about the interac- engineering of pathways into fruits and veg- tions of these controls, is sorely needed. etables to impart desirable flavor properties. Very few published examples of the ge- The aroma profiles of ornamental plant netic engineering of terpenoid metabolism species might be modified by similar ap- are currently available, although two notable proaches. Likewise, transgene expression successes have been achieved in the area of might accelerate the rate of slow biosynthetic terpenoid vitamins. The ratio of beneficial steps and thereby increase the yields of tocopherol (vitamin E) isomers in oilseeds essential oils used in flavors and perfumes, has been altered by this means, and an in- phytopharmaceuticals (e.g., artemisinin creased concentration of β-carotene (a vita- and taxol), insecticides (e.g., pyrethrins and min A precursor) in both rice kernels and azadirachtin), and a wide range of industrial rapeseed has been obtained by manipulating intermediates that are economically inacces- the carotenoid pathway. In another, caution- sible by traditional chemical synthesis. ary example, however, overexpression in a The genetic engineering of terpenoid- transgenic tomato of the enzyme that diverts based insect defenses is particularly appeal- GGPP to carotenoids resulted in a dwarf ing, given the array of available monoterpene, phenotype, an unintended consequence of sesquiterpene, diterpene, and triterpene depleting the precursor of the gibberellin compounds that are toxic to insects not plant hormones. adapted to them. Attracting predators and parasitoids of the target insect or modifying host attractants, oviposition stimulators, and 24.6 Alkaloids pheromone precursors offers even more so- phisticated strategies for pest control. For ef- 24.6.1 Alkaloids have a 3000-year history fective transgenic manipulation of such ter- of human use. penoid biosynthetic pathways, promoters for tissue-specific, developmentally controlled, For much of human history, plant extracts and inducible expression are required, as are have been used as ingredients in potions promoters for targeting production to secre- and poisons. In the eastern Mediterranean, tory structures of essential oil plants and use of the latex of the opium poppy (Papaver conifers. The latter are the most likely somniferum; Fig. 24.18) can be traced back at species for initial manipulation because they least to 1400 to 1200 B.C. The Sarpagandha already are adapted for terpenoid accumula- root (Rauwolfia serpentina) has been used in tion, and the antecedent and subsequent India since approximately 1000 B.C. Ancient metabolic steps are largely known. people used medicinal plant extracts as The engineering of terpenoid biosyn- purgatives, antitussives, sedatives, and treat- thetic pathways into plant species that do ments for a wide range of ailments, includ- not ordinarily accumulate these natural ing snakebite, fever, and insanity. As the use products presents a greater opportunity but of medicinal plants spread westward across

1268 Chapter 24 Natural Products (Secondary Metabolites)