Biosynthesis of IsoprenoidsDavid Wang’s Natural Products Class

Terpene• Mankind has used terpenes that are extracted from

plants for many different purposes — as fragrances and flavors, as pharmaceutical agents and as insecticides.

• Aside from their immense commercial value, terpene products have important biological functions in plants.– Plant growth and development (e.g. gibberellin phytohormones) – Represent important tools in the various interactions of plants

with the environment. • Volatile and non-volatile terpenes are implicated in the

attraction of both pollinators and predators of herbivores, in protection against photooxidative stress, in mediating thermotolerance, and in direct defense against microbes and insects.

Terpenoids

o Occurrence

n The softwood resin canals are filled with oleoresin.n Oleoresin: monoterpenoids and especially resin acids

(diterpenoids) are dominant and commercially important oleoresin constituent.

n The parenchyma resin of both softwoods and hardwoods contains triterpenoids and steroids, main occurring as fatty acid esters.

n Some tree produce rubber, gutta percha, and balata, which polyterpenes.

Classification and Biosynthesis of Terpenoids

n Terpenoids is formally derived from isoprene units and are sometimes called isoprenoids.

n The name terpene was given to hydrocarbons which were detected in terpentine oil.

n Terpenes are known as the large group of hydrocarbons made up of isoprene units (C5H8). Their respective derivatives with hydroxyl, carbonyl, and carboxyl functions are not hydrocarbons but strictly speaking terpenoids. → It has been proposed to call both the terpene hydrocarbons and their derivatives collectively terpenoids.

Myrcene

Greek Bay (Laurus nobilis)

Myrcene is found in the essential oil of bay leaves as well as hops. It is used as an intermediate in the manufacture of perfumes.

Hops (Humulus lupulus)

Geraniol and Linalool

Geraniol is isomeric with linalool, constitutes the major part of the oil of roses and is also found in essential oils of citronella, lemon grass, and others.

Geraniol Linalool

citronella

Menthol

Menthol is a well-known monoterpene which is found in the essential oil of peppermint and other members of the mint family.

Sesquiterpenoidsn More than 10000 sesquiterpenoids have been identified,

representing a wide variety compounds of different skeletal types from acyclic to tetra cyclic systems.

α-muurolene δ-cadinene α-cadinolα-cedrene

logifloene juniperol nootkatin chanootin

cadinenes

santonin

Wormwood (Artemisia maritima)

caryophyllene

Cloves (Syzygium aromaticum)

Dandelion (Taraxacum officinale )

tetrahydroridentin B

Classification of Terpenes (Terpenoids)

Classification and Biosynthesis of

Terpenoids

• Isoprene itself had been characterized as a decomposition product from various natural cyclic hydrocarbons, and was suggested as the fundamental building block for isoprenoids.

• Isoprene is produced naturally but is not involved in the formation of these compounds, and the biochemically active isoprene units were identified as the DMAPP (dimethylallyl diphosphate) and IPP (isopentenyl diphosphate).

Classification and Biosynthesis of Terpenoids

n Relative few of natural terpenoids conform exactly to the simply concept of a linear head-to-tail combination of isoprene units as seen with geraniol, farnesol, geranylgeraniol.

n Squalene and phytoene display a tail-to-tail linkage at the centre of the molecules.

n Most terpenoids are modified further by cyclization reactions, but the head-to-tail arrangement of the units can usually still be recognized such as menthol, bisabolene, and taxadiene.

Head-to-tail coupling mechanism of terpenoids and steroids

Biosynthesis of Terpenoids and Steroids

Biosynthesis of Terpenoids

n The prenylation reaction is stereospecific, mediated by a

type of prenyl transferase enzyme, which generates mainly

isoprenoids with trans configuration.

n In contrast, rubber has an all-cis configuration because its

synthesis is controlled by another type of prenyl transferase

enzyme.

Synthesis of IPP

n The glandular trichomes and secretory cavities of leaves and the glandular epidermis of flower petals generate and store or emit terpenoid essential oils that are important because they encourage pollination by insects.

n The resin ducts and blisters of conifer species the carbon and energy needed to drive terpenoids biosynthesis.

Synthesis of IPP

n A more fundamental, and perhaps universal, feature of the organization of terpenoids metabolism exists at the subcellular level.

n The sesquiterpenes (C15), triterpenes (C30), and polyterpenes appear to be produced in the cytosolic and endoplasmic reticulum (ER) compartments, whereas isoprene, the monoterpenes (C10), diterpenes (C20), tetraterpenes (C40), and certain prenylated quinones originate largely, if not exclusively, in the plastids.

Synthesis of IPP

n The evidence now indicates that the biosynthetic pathways for the formation of the fundamental precursor IPP differ markedly in these compartments, with the classical acetate/mevalonate pathway being active in the cytosol and ER and the glyceraldehyde phosphate/pyruvate pathway operating in the plastids.

n Regulation of these dual pathways may be difficult to assess, given that plastids may supply IPP to the cytosol for use in biosynthesis, and vice versa.

n Mitochondria, a third compartment, may generate the ubiquinone prenyl group by the acetate/mevalonate pathway, although little is known about the capability of these organelles for terpenoids biosynthesis.

Head-to-tail coupling mechanism of terpenoids and steroids

萜類化合物之生合成路徑

C30

Organization of terpene biosynthesis in plants

Hydroxymethylglutaryl-CoA reductase, an enzyme in the acetate/mevalonate pathway, is highly regulated

n The basic enzymology of IPP biosynthesis by way of the acetate/mevalonate pathway is widely accepted.

n This cytosolic IPP pathway involves the two-step condensation of three molecules of acetyl-CoA catalyzed by thiolase and hydroxymethylglutaryl-CoA synthase.

羥甲基戊二酸輔 A

Hydroxymethylglutaryl-CoA reductase, an enzyme in

the acetate/mevalonate pathway, is highly regulated

n The resulting product, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA), is

subsequently reduced by HMG-CoA reductase in two coupled

reactions that form mevalonic acid.

n Two sequential ATP-dependent phosphorylations of mevalonic acid

and a subsequent phosphorylation/elimination-assisted

decarboxylation yield IPP.

HMG-CoA reductase

n HMG-CoA reductase is one of the most highly regulated enzymes in animals, being largely responsible for the control of cholesterol biosynthesis.

n Accumulated evidence indicates that the plant enzyme, which is located in the ER membrane, is also highly regulated.

n In many cases, small gene families, each containing multiple members, encode this reductase. These gene families are expressed in complex patterns, with individual genes exhibiting constitutive, tissue- or development-specific, or hormone-inducible expression.

HMG-CoA reductase

n Specific HMG-CoA reductase genes can be induced by wounding or pathogen infection.

n The activity of HMG-CoA reductase may be subject to post translational regulation, for example, by a protein kinase cascade that phosphorylates and thereby inactivates the enzyme.

n Allosteric modulation probably also plays a regulatory role. Proteolytic degradation of HMG-CoA reductase protein and the rate of turnover of the corresponding mRNA transcripts may also influence enzyme activity.

n Researchers have not arrived at a unified scheme that explains how the various mechanisms that regulate HMG-CoA reductase facilitate the production of different terpenoids families.

n The precise biochemical controls that influence activity have been difficult to assess in vitro because the enzyme is associated with the ER membrane.

In plastids, IPP is synthesized from pyruvate and glyceraldehyde 3-phosphate

Prenyltransferase and terpenesynthase reactions

n Prenyltransferase enzymes generate the allylic diphosphate esters geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP).

n Reactions that these compounds undergo (often cyclizations), which are catalyzed by terpene synthases, yield a wide variety of terpenoid compounds.

n Both prenyltransferases and terpene synthases utilize electrophilic reaction mechanisms involving carbocationic intermediates, a feature of terpenoid biochemistry.

n Enzymes in both groups share similar properties and contain conserved sequence elements, such as an aspartate - rich DDxxD motif involved in substrate binding, which may participate in initiating divalent metal ion–dependent ionizations.

異戊烯基轉移酶

Representative terpenoids biosynthesized by plants. Monoterpenes, sesquiterpenes, and diterpenes are derived from the prenyl diphosphate substrates geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP), respectively, and are produced in both angiosperms and gymnosperms. (-)-Copalyl diphosphate and ent-kaurene are sequential intermediates in the biosynthesis of gibberellin plant growth hormones. Taxa-4(5), 11(12)-diene is the first dedicated intermediate in the biosynthesis of Taxol.

Enzyme Limonene Synthase

n The families of enzymes responsible for the formation of terpenoids from GPP, FPP, and GGPP are known as monoterpene, sesquiterpene, and diterpene synthases, respectively.

n These synthases use the corresponding prenyl diphosphates as substrates to form the enormous diversity of carbon skeletons characteristic of terpenoids.

n Most terpenoids are cyclic, and many contain multiple ring systems, the basic structures of which are determined by the highly specific synthases.

n Terpenoid synthases that produce cyclic products are also referred to as “cyclases,” although examples of synthases producing acyclic products are also known.

Enzyme for Limonene Synthase• A diverse array of monoterpene

synthases has been isolated from essential oil producing angiosperm species and resin producing gymnosperms.

• These enzymes use a common mechanism in which ionization of GPP leads initially to the tertiary allylic isomer linalyl diphosphate (LPP).

Enzyme for Limonene Synthase

n This isomerization step is required because GPP cannot cyclize directly, given the presence of the trans-double bond.

n Ionization of the enzyme-bound LPP intermediate promotes cyclization to a six-membered ring carbocation (the α-terpinyl cation), which may undergo additional electrophilic cyclizations, hydride shifts, or other rearrangements before the reaction is terminated by deprotonation of the carbocation or capture by a nucleophile (e.g., water).

Enzyme for Limonene Synthase

n Variations on this simple mechanistic scheme, involving subsequent reactions of the α-terpinyl carbocation, are responsible for the enzymatic formation of most monoterpene skeletons.

n The simplest monoterpene synthase reaction is catalyzed by limonene synthase, a useful model for all terpenoid cyclizations. The electrophilic mechanism of action used by limonene synthase can be viewed as an intramolecular equivalent of the prenyltransferase reaction.

Enzyme for Limonene Synthase

n Synthases that produce acyclic olefin products (e.g., myrcene) and bicyclic products (α- and β-pinene) from GPP are also known, as are enzymes that transform GPP to oxygenated derivatives such as 1,8-cineole and bornyl diphosphate, the precursor of camphor.

n An interesting feature of the monoterpene synthases is the ability of these enzymes to produce more than one product; for example, pinene synthase from several plant sources produces both α- and β-pinene. The pinenes are among the most common monoterpenes produced by plants and are principal components of turpentine of thepines, spruces, and firs.

Cloning and characterization of

Pinene synthase from Chamaecyparis

formosensis Matsum

氨基酸序列之相似度比較圖

628Car, (+)-3-carene synthase Lim, (-)-limonene synthase Lin, (-)-linalool synthase Myr, myrcene synthase Phe, β-phellandrene synthase Pin, pinene synthase

Pa, Picea abies Ag , Abies grandis

不同針葉樹mono-TPS基因的親緣關係

Pa, Picea abies Ag , Abies grandis

Car, (+)-3-carene synthase Lim, (-)-limonene synthase Lin, (-)-linalool synthase Myr, myrcene synthase Phe, β-phellandrene synthase Pin, pinene synthase

52%

52.2%

51.7%53.3%

52.9%

54.3%

52.4%

Phylogenetic Tree Showing Cf-Pinene and Other

Reported Coniferous Monoterpene Synthase.

於 E. coli pQE31表現之蛋白質

63 KDa

GC-MS Analysis of Monoterpene Products of Recombinant

Protein Product of Cf-Pin with Geranyl Diphosphate as the

Substrate