CHAPTER 8. Aromatic and Phenolic Compounds. Jones & Bartlett Learning, LLC. Jones & Bartlett Learning, LL NOT FOR SALE OR DISTRIBUT

Transcription

1 APTE 8 Aromatic and Phenolic ompounds T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI APTE UTLIE T F SALE DISTIBUTI 8.1 verview Jones & 8. Bartlett Shikimic Learning, Acid Pathway LL T F SALE Phenylalanine DISTIBUTI and Tyrosine Synthesis Tryptophan Synthesis 8.3 Phenylpropanoid Pathway Synthesis of Trans-innamic Acid Lignin Synthesis Polymerization Jones of Monolignols & Bartlett Learning, LL Genetic Engineering T F of Lignin SALE DISTIBUTI 8.4 atural Products Derived from the Phenylpropanoid Pathway atural Products from Monolignols T F SALE DISTIBUTI 8.5 Synthesis and Properties of Polyketides Synthesis of halcones Jones & Bartlett Synthesis Learning, of Flavanones LLand Derivatives T F SALE Synthesis DISTIBUTI and Properties of Flavones Synthesis and Properties of Anthocyanidins Synthesis and Properties of Isofl avonoids Examples of ther Plant Polyketide Synthases Synthesis and Activity of oumarins T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI 119. T F SALE DISTIBUTI.

2 of lignin precursors and is considered a primary Jones & Bartlett Learning, 8.1 verview LL Jones & metabolic Bartlett Learning, pathway terrestrial LL plants. oumaroyl-oa DISTIBUTI is also the initial compound for a T F SALE DISTIBUTI Plants are autotrophic organisms and T must F SALE synthesize all their metabolites from the intermediates of the -3 cycle and other pathways of primary carbon and nitrogen metabolism. variety of natural products, including flavonoid synthesis. Flavonoids are found in most plants and This includes the 0 common amino acids used give rise to colored compounds such as anthocyanins. They function Jones to protect & Bartlett plants from Learning, LL in protein Jones synthesis. & Bartlett Many of these Learning, amino acids LL are synthesized T F directly SALE from precursors DISTIBUTI derived intense light and ultraviolet T F (UV) SALE radiation and DISTIBUT from primary metabolism. For example, the amino acid glutamic acid is produced in a single reaction: transfer of an amino group to -oxoglutarate can act as antioxidants. A vast number of additional natural products are synthesized from the aromatic amino acids and their precursors. ( -ketoglutaric acid), an intermediate nly a small number of these are presented in Jones in & the Bartlett citric acid Learning, cycle. Synthesis LLof the aromatic this chapter. T F amino SALE acids, phenylalanine, DISTIBUTI tyrosine, and tryptophan, T F SALE DISTIBUTI is more complex. In addition to their function in protein synthesis, most amino acids serve as precursors for the synthesis of other 8. Shikimic Acid Pathway All three aromatic amino acids are derived from metabolites. This is especially true for phenylalanine, which LL is the precursor for the phen- Jones & shikimic Bartlett acid Learning, pathway. Synthesis LL starts with ery- intermediates of the same series of reactions, the Jones & Bartlett Learning, T F SALE DISTIBUTI ylpropanoid pathway. emoval of the T amino F SALE throse-4-phosphate DISTIBUTI and phosphoenolpyruvic group from phenylalanine yields trans-cinnamic acid followed by addition of an and coenzyme A, which produces p-coumaroyl-oa. acid (PEP). The pathway is named after the first intermediate that is unique to aromatic amino acid synthesis, shikimic acid. The reactions in This key intermediate is essential in synthesis this pathway are similar in plants, fungi, and bacteria but are absent in Jones animals. & The Bartlett component Learning, LL T F SALE DISTIBUTI enzymes are homologous T F in all these SALE kingdoms. DISTIBUT Phosphoenol pyruvate Erythrose-4-phosphate In plants, the enzymes are found in plastids and are presumably all soluble in the stroma. The genes for these enzymes are encoded in the nucleus. An overview of the pathway is shown Jones & Bartlett Learning, LL in FIGUE Jones 8.1. In & addition Bartlett to Learning, the three aromatic LL T F SALE DISTIBUTI amino T acids, F the SALE intermediates DISTIBUTI of the shikimic Phenolics acid pathway are also used in the synthesis of a variety of other metabolites, including the plant hormone salicylic acid, vitamins such as folic acid, and a number of species-specific natural 3 Shikimic acid T F SALE DISTIBUTI 10 APTE 8 Aromatic and Phenolic ompounds Jones & products. Bartlett Learning, LL T F SALE The erythrose-4-phosphate DISTIBUTIprecursor is an intermediate in the -3 cycle and the pentose phosphate pathway in the chloroplast stroma Salicylic acid (see hapters and 3). PEP is an intermediate in glycolysis and may be imported into Benzoic acid Jones & Bartlett Learning, Volatiles LL chloroplasts. Alternatively, Jones it can & Bartlett be a product Learning, LL of chloroplast pyruvate metabolism via pyruvate kinase and pyruvate, Pi dikinase. The first T F SALE Quinones (rings) DISTIBUTI T F SALE DISTIBUT Para aminobenzoic acid reaction in the pathway is an aldol condensation that results in the synthesis of a 7-carbon (folic acid precursor) ketose, 3-deoxy-arabino-heptulosonic acid- horismic acid Jones & Bartlett Learning, LL T F SALE DISTIBUTI 3 3 T F SALE DISTIBUTI Tryptophan Phenylalanine Tyrosine 7-phosphate Jones &(DAP), Bartlett shown Learning, FIGUE LL8.. T F SALE DISTIBUTI FIGUE 8.1 Simplified overview of the shikimic acid pathway. This diagram highlights the main intermediates and products of the shikimic acid pathway. The precursors are phosphoenolpyruvate (PEP) and erythrose-4-phosphate. Shikimic Jones acid and & chorismic Bartlett acid Learning, are key intermediates. LL These intermediates can be removed from the pathway and used to synthesize a variety of additional phenolic T F compounds. SALE DISTIBUTI. T F SALE DISTIBUTI.

3 T F SALE DISTIBUTI P Erythrose-4-P PEP P Jones & Bartlett Learning, LL T F SALE DISTIBUTI FIGUE 8. Synthesis of 3-deoxy-arabinoheptulosonic acid-7-phosphate (DAP). The DAP synthase catalyzes an aldol-type condensation. The phosphate is removed from PEP, and its methylene carbon then forms a bond with the carbonyl carbon of erythrose-4-phosphate. T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE 4 DISTIBUTI P Deoxy-arabino-heptulosonic acid-7-p T F SALE DISTIBUTI DAP synthases in plants and bacteria show only about 0% amino acid identity. The plant enzyme, however, complements bacterial mutants lacking this synthase. The bacterial enzyme exhibits feedback inhibition by the aromatic T F SALE DISTIBUTI Dehydroquinic acid is the substrate for a dehydratase that catalyzes removal of water and introduces a double bond into the ring. This is followed by reduction of the ketone to an alco- Jones & Bartlett Learning, hol forming LL shikimic acid, the first unique Jones intermediate (FIGUE & Bartlett Learning, LL amino acid end products, phenylalanine and T F SALE DISTIBUTI 8.4). In some plants, T shikimic F SALE DISTIBUT tyrosine. This does not appear to be true of the acid is the starting material for the synthesis of plant synthases. Expression of different isozymes of DAP synthase in plants, however, common are the water-soluble gallotannins, a variety of phenolic natural products. The most is influenced by environmental factors such as which are complexes of phenolics with sugars, high light Jones intensity & Bartlett or wounding Learning, and the pres- LL usually glucose. Plants Jones make & these Bartlett compounds Learning, LL ence T of hormones, F SALE gibberellic acid DISTIBUTI and jasmonic acid. The intermediates and end products of the shikimic acid pathway are often intermediates in the production of phytoalexins and other components of plant defense reactions. The second step in the pathway is the forma- tion of a cyclic intermediate, 3-dehydroquinic T F SALE DISTIBUTI acid, from DAP (FIGUE 8.3). The enzyme, dehydroquinate synthase, is an oxidoreductase that requires AD as a cofactor. The enzyme in this case catalyzes both an oxidation followed by a reduction, thereby regenerating the oxidized cofactor. P i T F SALE DISTIBUTI to protect their tissues T from F UV SALE damage and DISTIBUTI to deter herbivores. Tannins can bind irreversibly to proteins and inhibit enzyme activity. umans have traditionally used tannic acid rich plant extracts to tan and preserve animal hides. Some humans also like the bitter flavor of gallotan- nins in beverages such as tea. T F SALE DISTIBUTI Shikimic acid is further metabolized to chorismic acid, another key intermediate in this pathway. A kinase adds a phosphate group to one of the meta hydroxyls to produce shikimic acid-3-phosphate. This intermedi- ate is then condensed with another molecule T F SALE DISTIBUTI T F SALE DISTIBUT 1 reduced Jones & Bartlett Learning, LL 3 AD 1 T F SALE DISTIBUTI P Jones & Bartlett oxidizedlearning, LL T F SALE DISTIBUTI DAP 3-Dehydroquinic acid P i T F SALE DISTIBUTI FIGUE 8.3 eactions catalyzed by dehydroquinate synthase. The phosphate is removed from -7 of DAP and the resulting carbocation forms a bond with -. The enzyme then catalyzes the oxidation of the hydroxyl () group on -6 to a ketone and a reduction of the ketone on - to an to yield 3-dehydroquinic acid. The redox cofactor for this enzyme is AD, which is regenerated during the reaction. T F SALE DISTIBUTI 8. Shikimic Acid Pathway 11. T F SALE DISTIBUTI.

4 T F SALE DISTIBUTI 3-Dehydroquinic acid dehydratase 3-Dehydroshikimic acid reductase ADP FIGUE 8.4 Synthesis of shikimic acid. A Jones & Bartlett dehydratase-reductase Learning, LL binds dehydroquinic acid. It catalyzes removal of the hydroxyl next to the T F SALE DISTIBUTI carboxyl group on -1 and a proton from the adjacent -6 carbon, generating a double bond in the ring. The ADP-dependent reductase activity then reduces the keto group to a hydroxyl group to generate shikimic acid. ADP T F SALE DISTIBUTI Shikimic acid T F SALE DISTIBUT of PEP to produce 5-enolpyruvyl-shikimate- 3-phosphate (EPSP) (FIGUE 8.5). The enzyme, Jones EPSP & Bartlett synthase, Learning, is inhibited LL by an amino acid analog, -phosphonomethyl glycine, also T F known SALE as glyphosate. DISTIBUTI Although the structures do not appear to be similar, glyphosate competes with PEP for the same binding site on the synthase. Jones & As Bartlett shown in Learning, FIGUE 8.6, the LL synthase consists of two globular domains. Binding of shikimate-3-phosphate triggers a T F SALE DISTIBUTI global T F SALE DISTIBUTI 1 P PEP 4 P Shikimate-3-phosphate T F SALE DISTIBUTI Glyphosate P FIGUE 8.5 Synthesis of 5-enol-pyruvylshikimate-3-phosphate (EPSP). The EPSP synthase catalyzes removal of the phosphate from PEP. The resulting three-carbon fragment is added to the hydroxyl group on -3 of shikimate-3-phosphate. The amino acid analog, glyphosate (inset), is a competitive inhibitor of PEP. T F SALE DISTIBUTI T F SALE DISTIBUT P Enolpyruvylshikimate-3-phosphate 1 APTE 8 Aromatic and Phenolic ompounds T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI P i T F SALE DISTIBUTI T F SALE DISTIBUTI FIGUE 8.6 Structure of EPSP synthase. The enzyme consists of two domains shown in blue-green and red-yellow. In the absence of substrate, a wide gap separates the two domains. After binding shikimate- 3-phosphate (shown as purple sticks), the enzyme undergoes a large conformational shift to narrow the gap between the domains as shown. The constricted active site can now bind pyruvate or glyphosate, shown as red sticks. The EPSP synthase is Jones & a good Bartlett example of Learning, induced fit where LL binding of one substrate induces a T F conformational SALE change DISTIBUTI in the protein to accommodate the second substrate and subsequent catalysis. (Structure from Protein Data Bank 1G6S. E. Schonbrunn, S. Eschenburg, W. Shuttleworth, J. V. Schloss,. Amrhein, J.. S. Evans, and W. Kabsch, Proc. atl. Acad. Sci. 98 [001]: ) T F SALE DISTIBUTI T F SALE DISTIBUT. T F SALE DISTIBUTI.

5 conformational change to the more closed structure & Bartlett seen in Figure Learning, 8.6. PEP LL then binds to the tives Jones and also & Bartlett salicylic acid Learning, some LL plants. precursor for benzoic acid and its deriva- Jones T F active SALE site formed DISTIBUTI at the interface of the two Methylated T F benzoic SALE and salicylic DISTIBUTI acids are volatile and are responsible for many floral scents domains. Both kinetic and crystal structure analyses show that glyphosate competes with that attract pollinators. Salicylic acid is also a PEP for this same site. The unique nature of this signaling molecule that initiates responses to structural change is further illustrated by the abiotic stress and pathogen invasion, a process fact that glyphosate does Jones not compete & Bartlett with PEP Learning, referred LLto as systemic acquired Jones resistance & Bartlett Learning, LL in any of the many other T reactions F SALE that use PEP DISTIBUTI (SA). Additionally, chorismic acid T in F some SALE DISTIBUT as a substrate. Glyphosate is a highly effective, plants provides the ring portion of quinones broad-spectrum herbicide and is the active and para-amino benzoic acid, the precursor component in Monsanto s oundup. for folic acid synthesis. In many plants some EPSP is the substrate for a synthase that of these compounds can also be derived from removes Jones the phosphate & Bartlett from Learning, -3 and introduces T a second F double SALE bond DISTIBUTI into the ring. T F SALE DISTIBUTI LL phenylalanine. This results in the production of chorismic Phenylalanine and Tyrosine Synthesis acid (FIGUE 8.7). horismic acid can be a horismic acid is the branch point between synthesis of phenylalanine and tyrosine or tryptophan (Figure 8.1). horismate mutase Jones & Bartlett Learning, LL catalyzes Jones the & transfer Bartlett of the Learning, pyruvyl side LL chain T F SALE DISTIBUTI to T -1 to F produce SALE prephenic DISTIBUTI acid, the precursor of phenylalanine and tyrosine (Figure 8.7). This mutase is allosterically regulated by the EPSP P three aromatic amino acids. Allosteric regulators are generally small molecules that can either Jones & Bartlett Learning, activate LL or inhibit enzyme activity, Jones but these & Bartlett Learning, LL synthase T F SALE DISTIBUTI regulators do not bind to the enzyme s T active F site. SALE DISTIBUT Most allosterically regulated enzymes are composed of two or more subunits, and the regu- lators generally act by changing the subunit association. In the case of chorismate mutase, the enzyme s activity Jones is inhibited & Bartlett by phenylalanine and tyrosine T and activated F SALE by tryptophan, DISTIBUTI Learning, LL T F SALE DISTIBUTI Pi thereby ensuring balanced pools of all three horismic acid amino acids. In the next step, an amino group is added mutase to prephenic acid to produce arogenic acid (Figure Jones 8.7). & The Bartlett transaminase Learning, that catalyzes LL T F SALE DISTIBUTI T F SALE DISTIBUTI Prephenic acid FIGUE 8.7 Synthesis of tyrosine and phenylalanine from chorismic acid. horismate synthase catalyzes the removal of the phosphate Jones & Bartlett Learning, LL group from EPSP and introduces a second T F SALE 3 DISTIBUTI double bond into the ring to generate chorismic Glu acid. The prephenate mutase reaction results in α-ketoglutarate transaminase the relocation of the pyruvyl moiety to -1 of the ring producing prephenic acid. A transaminase ADP then catalyzes transfer of an amino group from Tyrosine glutamic acid to - of the side chain resulting Jones & Bartlett ADP Learning, in the amino acid, arogenic acid. In most plants, LL T F arogenic acid is the precursor for both tyrosine SALE DISTIBUTI T F SALE DISTIBUTI and phenylalanine. To produce tyrosine, an 3 ADP-dependent dehydrogenase oxidatively decarboxylates -1 of the ring and catalyzes 3 formation of a third double bond to generate an dehydratase aromatic ring. In phenylalanine synthesis, a Phenylalanine dehydratase removes both the -1 carboxyl group and a water molecule from the ring, Arogenic acid T F SALE thereby DISTIBUTI generating the aromatic amino acid. T F SALE DISTIBUTI dehydrogenase T F SALE DISTIBUT 8. Shikimic Acid Pathway 13. T F SALE DISTIBUTI.

6 this reaction uses glutamic acid as the amino the ketose derivative. Decarboxylation of the Jones & Bartlett Learning, donor. xidative-decarboxylation LL of arogenic Jones & aromatic Bartlett ring Learning, and dehydration LL and cyclization T F SALE DISTIBUTI acid by an ADP-dependent dehydrogenase T F SALE produce the DISTIBUTI five-membered ring of indole yields tyrosine. To synthesize phenylalanine, glycerol-phosphate. decarboxylation of arogenic acid is followed The final step in this branch of the pathway is catalyzed by tryptophan synthase. by a dehydration to produce the aromatic ring. This enzyme cleaves the glycerol-p moiety Tryptophan Jones Synthesis & Bartlett Learning, LL from the indole ring Jones and replaces & Bartlett it with Learning, LL horismic T F acid SALE is also the DISTIBUTI precursor for serine (Figure 8.8). T Tryptophan F SALE synthase DISTIBUT is the synthesis of tryptophan (Figure 8.1). A an heterotetramer in plants. It is an synthase catalyzes removal of the pyruvyl unusual example of a bifunctional enzyme. side chain and a transamination to produce Indole glycerol-phosphate binds to an active anthranilic acid (FIGUE 8.8). The amino donor site located on the subunit. Binding causes Jones in & this Bartlett reaction Learning, is glutamine. LLThe next step a conformational Jones & Bartlett change Learning, that closes LLthe T F involves SALE a glycosylation, DISTIBUTI that is, addition active T site. F After SALE removal of DISTIBUTI glyceraldehydeof 5-phospho-ribosyl-1-diphosphate to the 3-phosphate, the indole ring diffuses through amino group of anthranilic acid. The sugardiphosphate is a good leaving group, and units to the active site in the subunit. a long protein tunnel (~5Å) between sub- the sugar is bonded to anthranilate by an Serine then binds and closes the active site Jones & Bartlett Learning, -glycosidic LL bond as seen in nucleosides. Jones An & in Bartlett the Learning, subunit. The LL hydroxyl group is T F SALE DISTIBUTI isomerase then catalyzes the formation T of F SALE removed from DISTIBUTI serine, and the beta-carbon is bonded to the indole ring. Formation of tryptophan opens the active sites on both subunits, and the products are released. horismic acid Anthranilic acid Pyruvic acid Although crystal structures of several tryptophan synthases with Jones bound & intermediates Bartlett Learning, LL Jones & Bartlett Learning, LL Glu T F SALE Gln DISTIBUTI are available, it is T still not F known SALE how the DISTIBUT two active sites communicate to coordinate synthase their activity, but this enzyme does not produce free indole. Some plants, such as maize, produce indole-based phytoalexins. In this Jones case an & enzyme Bartlett resembling Learning, only LLthe T F SALE P DISTIBUTI subunit T F of tryptophan SALE synthase DISTIBUTI is found in PP the cytoplasm. A similar enzyme is the most 5-P-ribosyl-PP likely source of the volatile indole released P from many plants. Jones & 5-P-ibosyl Bartlett anthranilic Learning, acid LL T F SALE DISTIBUTI T F SALE DISTIBUTI isomerase synthase P transferase Trp synthase α Trp synthase Indole glycerol phosphate Jones & Bartlett β Learning, LL T F SALE DISTIBUTI Serine T F SALE DISTIBUTI T F SALE DISTIBUTI Tryptophan 14 APTE 8 Aromatic and Phenolic ompounds P Glyceraldehyde-3-P FIGUE 8.8 Synthesis of tryptophan. Anthranilate synthase binds chorismic acid and catalyzes removal of the pyruvyl moiety followed by transfer of an amino group from glutamine to the ring to form anthranilic acid. The sugar, 5-phosphate-ribosyldiphosphate, is coupled to the amino group in anthranilate by an -glycosidic bond and pyrophosphate is released. The 5-phospho-ribosyl moiety then undergoes isomerization to a ketose. The ring of the intermediate is decarboxylated, and a third enzyme catalyzes a ring closure of the ketose to generate the fi ve-membered ring in indole glycerol-3-phosphate. This intermediate is the substrate for the subunit of tryptophan synthase. Binding of indole glycerol-phosphate to the subunit closes this Jones subunit active & site, Bartlett and the enzyme Learning, cleaves the LL glyceryl-phosphate T F moiety SALE from the indole DISTIBUTI ring. The products are not released from the enzyme. The indole intermediate is transported through a channel in the enzyme to the subunit. Serine also binds to the subunit, and binding closes this second active site. The hydroxyl group is removed from serine, and the remaining three-carbon fragment is coupled to the indole ring to generate tryptophan. pening of the active sites on both subunits releases tryptophan from the subunit and glyceraldehyde-3-phosphate from the -subunit site. T F SALE DISTIBUTI T F SALE DISTIBUT. T F SALE DISTIBUTI.

7 The enzyme that catalyzes this initial step is Jones 8.3 & Bartlett Phenylpropanoid Learning, LL Pathway phenylalanine Jones & Bartlett ammonia Learning, lyase (PAL). LL PAL is T F Many SALE complex DISTIBUTI plant products are synthesized found T in F angiosperms SALE and DISTIBUTI gymnosperms and from the aromatic amino acids, including both also in some mosses, algae, fungi, and bacteria. primary and secondary metabolites (natural Several PAL isozymes occur in angiosperms. products). Phenylalanine occupies a particularly prominent position in plant metabolism most likely associated with the outer face of The plant enzymes are found in the cytoplasm, as it is the starting material Jones for & the Bartlett synthesis Learning, the endoplasmic LL reticulum. PAL from Jones various & Bartlett Learning, LL of a large number T of aromatic F SALE compounds. DISTIBUTI plant sources is a tetramer with a T mass F of 5 SALE DISTIBUT These metabolites are referred to as phenylpropanoids and include primary products such specific for phenylalanine. Some dicots also to 330 kda. In dicotyledonous plants, PAL is as the monolignol precursors of lignin and a have another enzyme that deaminates tyrosine large number of species-specific natural products. Lignin Jones is a & major Bartlett component Learning, of second- LL accepts both phenylalanine Jones & and Bartlett tyrosine Learning, as LL (tyrosine ammonia lyase). In monocots, PAL ary cell T walls F and SALE the second DISTIBUTI most abundant substrates, although T the F catalytic SALE efficiency DISTIBUTI is polymer in the biosphere after cellulose. Its lower for tyrosine. synthesis is vital to the survival of all terrestrial Plant PAL belongs to the amino acid plants. Lignin is a polymer of phenylpropanoid ammonia lyase family of enzymes. The plant precursors that are exported to the cell wall enzyme is homologous to the bacterial histidine Jones ammonia & Bartlett lyase (AL) Learning, and has the LL same Jones & matrix Bartlett of mature Learning, plant tissues. LL In the wall the T F precursors SALE are DISTIBUTI polymerized and form crosslinks to components of the primary cell wall. of PAL from parsley (Petroselinum crispum) mechanism T F of SALE action. The DISTIBUTI crystal structure Phenylpropanoids are also the precursors for has been determined. The protein is mainly phytoalexins in some plants and volatiles such composed of -helices, and its overall tertiary structure is very similar to the PAL as eugenol found in cloves. Another major role of phenylpropanoids Jones is in the & Bartlett production Learning, from LL hodosporidium toruloides and Jones histidine & Bartlett Learning, LL of polyketides, including T F flavonoids, SALE which DISTIBUTI ammonia lyase from the bacterium, T Pseudomonas syringae. All these enzymes are unique F SALE DISTIBUT are found in most plants and enable them to interact and cope with environmental factors. in that they generate their own cofactor at All this complex variety of end products starts the active site by cyclization and dehydration with a phenylalanine precursor. of an alanine-serine-glycine sequence to generate 4-methylidene-imidazole-5-one Jones & Bartlett (MI) Learning, LL Synthesis T F of Trans-innamic SALE DISTIBUTI Acid (FIGUE 8.9). The T methylidene F SALE group of DISTIBUTI the The synthesis of phenylpropanoids starts MI cofactor binds the amino group of with the removal of the amino group of the substrate, phenylalanine, and weakens the phenylalanine to produce trans-cinnamic acid. bond on the -carbon of the substrate T F SALE DISTIBUTI (a) (b) Ala 04 3 T F SALE DISTIBUTI 3 T F SALE DISTIBUTI Gly 0 Ala 04 Ala 04 Gly 0 Gly 0 T F SALE DISTIBUTI FIGUE 8.9 Activity of phenylalanine ammonia lyase (PAL). (a) PAL catalyzes the removal of the amino group from the -carbon and a hydrogen from the -carbon of phenylalanine to produce trans-cinnamic acid and ammonia. (b) The enzyme generates a unique cofactor at its active site by cyclization of three residues, alanine-04, serine-03, and glycine-0 (residue numbering is from the bacterial enzyme), followed by a dehydration. The amino group in the peptide bond between ala-04 and ser-03 attacks the carbonyl carbon in the peptide bond between ser-03 and gly-0. Subsequent removal of two molecules generates the 4-methylidene-imidazole-5-one (MI) cofactor that binds the substrate, phenylalanine, and facilitates removal of the amino group to generate 3 (shown in Figure 8.10). T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI 8.3 Phenylpropanoid Pathway 15. T F SALE DISTIBUTI.

8 T F SALE DISTIBUTI Ala 04 B: Jones & Bartlett Learning, LL T F SALE DISTIBUTI Ala 04 T F SALE Gly0 DISTIBUTI T F SALE DISTIBUTI Gly 0 Step 1 Step Step 3 Step 4 FIGUE 8.10 Mechanism of action of phenylalanine ammonia lyase (PAL). This enzyme is the gateway to the phenylpropanoid pathway, and its mechanism has been thoroughly investigated. Step 1: The methylidene carbon of the MI cofactor (Figure 8.9B) in PAL binds to the amino group of phenylalanine. Step : A basic group in the enzyme, B, stereospecifi cally removes one of the protons from the -carbon of phenylalanine. Step 3: Jones earrangement & Bartlett of the resulting Learning, anion generates LLa double bond in the substrate and facilitates cleavage of the bond between the -carbon of phenylalanine and its amino group. Step 4: Trans-cinnamic acid is released from the enzyme. The protonated B T residue F in the SALE active site donates DISTIBUTI its to the aminated cofactor and facilitates removal of the 3 and regeneration of MI. (Mechanism adapted from: alabrese, J. et al., rystal Structure of Phenylalanine Ammonia Lyase: Multiple elix Dipoles Implicated in atalysis, Biochemistry 43: [004] ) B T F SALE DISTIBUTI B Jones & Bartlett Learning, LL T F SALE DISTIBUT Ala 04 Ala Jones & Bartlett Learning, 04 LL T F Gly SALE 0 DISTIBUTI Gly 0 (FIGUE 8.10). Jones A proton & Bartlett is removed Learning, from this LL The monooxygenases Jones in this & Bartlett pathway Learning, are LL -carbon T with F the SALE help of a basic DISTIBUTI group in often referred to as T hydroxylases. F SALE The hydroxyl DISTIBUT the active site, most likely a histidine residue. () group is added at the para-position with Breaking of the very stable bond in this respect to the side chain to produce 4-coumaric reaction is facilitated by the electrophilic environment around the active site created by the be produced in some plants by direct deami- acid (p-coumaric acid). oumaric acid can also Jones aligned & Bartlett helix dipoles Learning, of the enzyme. LL emoval of nation Jones of tyrosine & Bartlett in a TAL-catalyzed Learning, reaction LL T F the SALE proton generates DISTIBUTI an anion that rearranges (FIGUE T 8.11). F SALE DISTIBUTI and results in the cleavage of the bond oumaric acid can be ligated to coenzyme between phenylalanine and the MI cofactor. A by a ligase; this reaction requires two equivalents of ATP to activate coumaric acid and The product, trans-cinnamic acid, is released from the active site. The cofactor is regenerated by LL donation from an acidic residue Jones in & oumaroyl-oa Bartlett Learning, is a major LLbranch point in the catalyze the formation of a thioester bond. Jones & Bartlett Learning, T F SALE DISTIBUTI the enzyme and ammonia is produced. T Transcinnamic acid is the precursor for most of the panoids. There is experimental evidence that all F SALE synthesis of DISTIBUTI monolignols and other phenylpro- phenylpropanoids, including the monolignols, the monolignols are present as oa esters until used to synthesize lignin. a final reduction to the alcohols, but some of the modifying enzymes can use the free acids Lignin Jones Synthesis & Bartlett Learning, LL or other derivatives as Jones substrates. & Bartlett In addition, Learning, LL Lignin T is a F highly SALE irregular polymer DISTIBUTI formed recent analyses have T also F shown SALE that monolignols are present as complexes with shikimic or DISTIBUT from aromatic alcohols referred to as monolignols. The major monolignols are coumaroyl, quinic acids, which are intermediates in the shikimic acid pathway (FIGUE 8.1). Because these coniferyl, and sinapyl alcohols. These precursors are synthesized from trans-cinnamic acid acids are synthesized in plastids, it is assumed by enzymes associated with the endoplasmic that they Jones must & be Bartlett specifically Learning, transported LL to the T F reticulum. SALE The DISTIBUTI monolignols are exported to cytoplasm T F to serve SALE as monolignol DISTIBUTI carriers. the cell wall, and polymerization of the monolignols then occurs outside the cell membrane. or the free acid, undergoes reduction to cou- Para-coumaroyl-oA, or other conjugate maroyl alcohol by two reductases that use Synthesis of p-oumaroyl Alcohol The aromatic ADP as a cofactor. The mechanism of action Jones & Bartlett Learning, ring of trans-cinnamic LL acid can be hydroxylated Jones & of Bartlett these two Learning, enzymes is LL slightly different. The T F SALE DISTIBUTI by a specific cytochrome P450 monooxygenase. T F SALE first enzyme, DISTIBUTI coumaroyl-oa reductase (), 16 APTE 8 Aromatic and Phenolic ompounds. T F SALE DISTIBUTI.

9 T F SALE DISTIBUTI ADP ADP trans-innamic acid para-oumaric acid TAL 3 T F SALE DISTIBUTI FIGUE 8.11 Synthesis of p-coumaric acid. A hydroxyl () group is added to trans-cinnamic acid by a cytochrome P450 monooxygenase (hydroxylase). The enzyme adds the -group at the para position with respect to the side chain. oumaric acid can also be synthesized by direct deamination of tyrosine catalyzed by tyrosine ammonia lyase (TAL) in some plants. T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI Tyrosine 3 T F SALE DISTIBUTI T F SALE DISTIBUTI ADP P i Jones & Bartlett Learning, LL T F SALE DISTIBUTI transferase p-oumaric acid transferase T F SALE DISTIBUTI ATP ligase FIGUE 8.1 Para-coumaric acid derivatives active in monolignol synthesis. oumaric acid generally occurs in vivo as a oa-thioester. In some plants, conjugates with shikimic acid and quinic acid have also been found, and these may function as alternate precursors in monolignol synthesis. T F SALE DISTIBUTI S-oA Jones & Bartlett Learning, LL T F SALE DISTIBUTI p-oumaroyl-oa p-oumaroyl-shikimic acid T F SALE DISTIBUT T F SALE DISTIBUTI p-oumaroyl-quinic acid T F SALE DISTIBUTI reduces the acid to the aldehyde. It is referred component found in complexes with other to as a type B reductase because it transfers the types of lignin. B hydride from ADP Jones (FIGUE& 8.13). Bartlett The second reductase, coumaroyl T aldehyde F SALE dehydro- DISTIBUTI Synthesis of oniferyl and Sinapyl Alcohols T F ou-sale DISTIBUT Learning, LL genase (AD), is a type A reductase. It transfers maric acid, either as the free acid or a derivative, the A hydride from the cofactor and reduces is hydroxylated by a second monooxygenase the aldehyde to an alcohol. These reductases are to produce the dihydroxy derivative, caffeic highly specific and cannot substitute for each acid. This second group in the aromatic other. This Jones has been & Bartlett demonstrated Learning, genetically LL ring is then methylated Jones by & Bartlett an -methyl Learning, LL altered T plants F designed SALE to generate DISTIBUTI different pools of monolignols. oumaroyl alcohol is one of the monolignol precursors. It can be polymerized into a complex referred to as p-hydroxyphenyl lignin, or Jones & -lignin. Bartlett -lignin Learning, is common LLin some monocots, SALE but in most DISTIBUTI plants it is often a T F minor transferase that requires T F S-adenosyl SALE methionine (SAM) as the methyl donor. Monohy- DISTIBUTI droxy substituted rings are usually not good substrates for the methyl transferases, but they can bind a number of different hydroxylated Jones substrates. & Bartlett The methylated Learning, caffeic LLacid is T ferulic F acid SALE (FIGUE 8.14). DISTIBUTI Ferulic acid is then 8.3 Phenylpropanoid Pathway 17. T F SALE DISTIBUTI.

10 T F SALE DISTIBUTI ADP A B ADP A ADP A B B T F SALE DISTIBUTI T F SALE DISTIBUT S-oA A B B AD FIGUE 8.13 eductases that reduce p-coumaroyl-oa and other monolignol Jones precursors. & Bartlett The fi rst enzyme, Learning, p-coumaroyl-oa LL reductase (, also called cinnamate-oa reductase), transfers the B hydride from ADP and reduces the thioester to the aldehyde. It is referred to as a type B reductase. The second enzyme, coumaroyl T F aldehyde SALE dehydrogenase DISTIBUTI (AD), transfers the A hydride from the cofactor to produce coumaroyl alcohol; it is a type A reductase. Both and AD can also catalyze reduction of other monolignol precursors; ADP is the reducing agent in both reactions. T F SALE DISTIBUTI ADP T F SALE DISTIBUTI T F SALE DISTIBUTI S-oA ADP p-oumaroyl-oa ADP affeoyl-oa 3 T A S F SALE DISTIBUTI T F SALE S-oA DISTIBUTI T F SALE DISTIBUT S-Adenosyl methionine (SAM) T F SALE DISTIBUTI -Methyl transferase T F SALE DISTIBUTI S A S-oA T F SALE DISTIBUTI S-Adenosyl homocysteine (SA) T F SALE DISTIBUTI 3 Guaiacyl lignin (G-lignin) 18 APTE 8 Aromatic and Phenolic ompounds ADP ADP Feruloyl-oA oas T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI ADP oniferyl aldehyde ADP AD 3 oniferyl alcohol FIGUE 8.14 Synthesis of coniferyl alcohol. oumaroyl-oa is hydroxylated by a specifi c cytochrome P450 monooxygenase at the meta position (with respect to the side chain) to generate caffeic acid or a caffeoyl conjugate. A caffeoyl -methyl transferase then adds a methyl group to this meta-hydroxyl group to produce ferulic acid. The methyl donor is S-adenosyl methionine (SAM). emoval of the methyl group from SAM yields S-adenosyl homocysteine (SA), which can be remethylated. Ferulic acid and its conjugates are reduced by the p-coumaroyl-oa reductase () and coumaroyl aldehyde dehydrogenase (AD) reductases described in Figure 8.13 to yield coniferyl alcohol. This product is a lignin precursor that can be polymerized to guaiacyl or G-lignin, which is the major lignin type found in gymnosperms, especially conifers. T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI. T F SALE DISTIBUTI.

11 reduced to the alcohol by the same redox Polymerization of Monolignols Jones enzymes, & Bartlett Learning, and AD, LL described in Figure The monolignol precursors, coumaroyl, T F 8.13 SALE to yield DISTIBUTI coniferyl alcohol. This alcohol coniferyl, T F and SALE sinapyl alcohols, DISTIBUTI are exported to is the major component of guaiacyl lignin the exterior of the cell. They may be packaged (G-lignin). into Golgi vesicles or transported directly. In Another cytochrome P450 monooxygenase some mature plant tissues monolignols are introduces a third hydroxyl group into the aromatic ring to produce 5-hydroxy Jones & ferulic Bartlett acid. Learning, LL found as glycosylated derivatives, presumably for storage until secondary wall synthesis is This third hydroxyl T group F is methylated SALE by DISTIBUTI induced. Polymerization of the T monolignols F SALE DISTIBUT a SAM-dependent transferase, as shown in occurs outside the cell membrane, between it FIGUE 8.15, to produce sinapic acid. Sinapic acid is and the primary cell wall. The polymerization then reduced to sinapyl alcohol, a major monolignol made in dicotyledonous angiosperms and enzymatic free radical formation. Two types of process is nonenzymatic but is initiated by an the precursor Jones of & syringyl Bartlett lignin Learning, (S-lignin). LL oxidases, peroxidases and laccases, are found T F SALE DISTIBUTI T F SALE DISTIBUTI S-oA T F SALE DISTIBUTI 3 Feruloyl-oA ADP ADP SAM T F SALE DISTIBUTI SA 3 5-ydroxyl feruloyl-oa -methyl transferase S-oA T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI 3 S-oA T F SALE DISTIBUTI 3 Sinapoyl-oA T F SALE DISTIBUTI ADP Jones & oas Bartlett Learning, LL T ADPF SALE DISTIBUTI Sinapyl aldehyde ADP ADP T F SALE DISTIBUTI AD T F SALE DISTIBUT Syringyl lignin Jones & Bartlett Learning, (S-lignin) LL T F SALE DISTIBUTI 3 Sinapyl alcohol FIGUE 8.15 Synthesis of sinapyl alcohol. Ferulic acid, or a derivative, is hydroxylated by a cytochrome P450 monooxygenase to 5-hydroxy ferulic acid. An -methyl transferase adds a methyl group to this Jones third hydroxyl & group Bartlett to produce Learning, sinapic acid, which LLis reduced to the alcohol by p-coumaroyl-oa reductase () and coumaroyl aldehyde dehydrogenase (AD). Sinapyl alcohol is a primary monolignol that is polymerized into syringyl or S-lignin, a major component of secondary cell walls in dicotyledonous plants. T F SALE DISTIBUTI 3 T F SALE DISTIBUTI T F SALE DISTIBUTI 8.3 Phenylpropanoid Pathway 19. T F SALE DISTIBUTI.

12 in the cell wall that can act as free radical Unlike the carbohydrates of the primary cell Jones & Bartlett Learning, initiators. Peroxidases LL are heme-fe containing Jones & wall, Bartlett lignin Learning, is a relatively LL inflexible polymer. It T F SALE DISTIBUTI enzymes that use a variety of substrates T and F SALE makes cell walls DISTIBUTI rigid and provides support for hydrogen peroxide to generate a radical: plant stems and vascular tissues. It is especially important in xylem vessels where it maintains A A the vessel structure under negative water pressure. Lignin also provides a barrier to pathogen Plants contain a number of isoforms of these enzymes; Jones the peroxidases & Bartlett in the Learning, cell wall are designated T class F III peroxidases. SALE They DISTIBUTI can generate chemical bonds is highly T resistant F SALE to both enzy- DISTIBUT LL invasion. The lignin structure Jones & with Bartlett its random Learning, LL reactive oxygen species (S, see Appendix 5) matic and chemical degradation. nly a few associated with the wound response. Peroxidases are also capable of generating monolignol use a free radical mechanism to generate random organisms, mostly fungi, can degrade lignin; they radicals. The source of the hydrogen peroxide breaks in the polymer, essentially reversing the Jones in & the Bartlett cell wall Learning, is unknown LL but could presumably SALE be generated DISTIBUTI by the peroxidase itself from T F SALE DISTIBUTI polymerization Jones & process. Bartlett Learning, LL T F molecular oxygen. Genetic Engineering of Lignin The second type of radical initiator, the laccases, are u -containing proteins belonging 130 APTE 8 Aromatic and Phenolic ompounds The properties of lignin in plants depend on the concentration of monolignols exported to the wall, which determines the different types,, G, or S. Gymnosperm wood is mainly to the phenol oxidase enzyme family. They Jones & Bartlett Learning, generate radicals LL from molecular oxygen: T F SALE DISTIBUTI T F SALE G-lignin; the DISTIBUTI monolignol pool contains mostly 4A 4A coniferyl alcohol with smaller amounts of An unpaired electron that is introduced into sinapyl alcohol. This pool gives rise to a type a monolignol can be delocalized over the aromatic ring and side chain and subsequently more stable than the ether linkages that of lignin with mainly linkages that are result couple with Jones another & Bartlett radical to Learning, form a bond LL in from a higher concentration Jones of & sinapyl Bartlett alcohol Learning, LL a number T of F different SALE linkages. DISTIBUTI Some of the precursors (FIGUE T 8.17). F Plants SALE containing DISTIBUT a possible linkages between monolignols are high content of G-lignin are more difficult for shown in FIGUE A similar random radical herbivores, such as domestic cattle and sheep, mechanism links lignin to carbohydrates in the to digest. G-lignin also requires harsher chemical treatment in the pulping process to matrix of the primary cell wall (see hapter 4). make T F SALE DISTIBUTI T F SALE DISTIBUTI 5-5 Linkage paper Jones and presents & Bartlett a barrier Learning, to the production LL of T ethanol-based F SALE biofuels. DISTIBUTI Many research groups have attempted to genetically alter the composition of lignin with the goal of producing a product that is easier to degrade, either enzymatically or chemically. Jones & Examples Bartlett of Learning, these manipulations LL include a gene T F SALE knock-out or DISTIBUTI down-regulation of the enzyme that methylates caffeoyl-oa (oamt). This should result in a pool of monolignols with a higher concentration of coumaroyl alcohol (use FIGUE 8.18 as a guide). The engineered lignin Jones & Bartlett 4--5 Learning, Linkage LL that results from this manipulation Jones & Bartlett should have Learning, LL T F SALE DISTIBUTI less of both the G- T and F S-lignin SALE components DISTIBUT Jones & Bartlett Learning, LL T F SALE DISTIBUTI Jones & Bartlett Learning, LL T F SALE β--4 DISTIBUTI Linkage β-5 Linkage FIGUE 8.16 Some common linkages found in lignin. The types of linkages found Jones in lignin & Bartlett depend on the Learning, pool of monolignols LLthat are synthesized T and F exported SALE from the cell. DISTIBUTI In conifers, coniferyl alcohol is the most abundant precursor, and the resulting G-lignin contains mainly bonds such as the 5-5 linkage shown. Dicotyledonous angiosperms produce mainly sinapyl alcohol precursors and the resulting S-lignin contains more ether linkages such as the 4--5 and --4 linkages. Because most plants produce some of each type of monolignol, all the above linkages and many mixed linkages such as the -5 confi guration with both and bonds are possible. T F SALE DISTIBUTI. T F SALE DISTIBUTI.

13 Jones & Bartlett Learning, LL T F SALE DISTIBUTI T F SALE DISTIBUTI Me 3 ( δ Me Me Jones & Bartlett MeLearning, LL (ABYDATE) T F SALE DISTIBUTI T F SALE DISTIBUT Me Me Me Me Me Me T F SALE DISTIBUTI T F SALE DISTIBUTI Me Me Me Me Me T F SALE DISTIBUTI T F SALE DISTIBUTI Me Me Me Me Me Me T F SALE DISTIBUTI T F SALE DISTIBUT Me Me Me Jones & Bartlett Learning, LL T F SALE DISTIBUTI T F SALE DISTIBUTI ( 4 Me Me FIGUE 8.17 Model of linkages found in conifer lignin. These are some of the major linkages that can be found in G-lignin formed predominantly from coniferyl alcohol. Both and bonds are Jones formed, but & the Bartlett linkages Learning, are more common LLin this type of secondary cell wall polymer. (eproduced from: M. Baucher, B. Monties, M. Van Montagu, et al. rit. ev. in Plant Sci. 17 [1998]: , Figure 7. ourtesy of Marc Van Montagu, Ghent University.) T F SALE DISTIBUTI T F SALE DISTIBUTI and therefore fewer recalcitrant bonds. The (see Figure 8.18). The mutant plants contain less lignin in transgenic tobacco and poplar plants, S-lignin in their walls as expected but again however, actually had Jones a higher & Bartlett content of Learning, compensated LL with a higher content of Jones hydroxyferulic alcohol. The mutant maize T leaves F and SALE DISTIBUT & Bartlett Learning, LL 5-hydroxyguaiacyl units T (i.e., F more SALE coumaroyl DISTIBUTI alcohol was incorporated into the polymer), but stalks are more digestible by ruminants. Grain additional 5-hydroxyferulic alcohol was also yield is lower in mutant plants, however, and found. Apparently, the other -methyl transferases can adequately substitute for oamt break easily in the wind). they are more susceptible to lodging (i.e., stems in these Jones plants, and & Bartlett the resulting Learning, modified lignin LL In the model plant, Jones Arabidopsis, & Bartlett mutants Learning, LL still contains T F a high SALE proportion DISTIBUTI of linkages that with knock-out T mutations F in SALE the gene DISTIBUTI for are resistant to degradation. ferulic acid monooxygenase (F5) produce aturally occurring mutations in the monolignol enzyme genes were found in maize (Zea gene for this monooxygenase leads to pro- no S-lignin. onversely, overexpression of the mays) and were designated bm1,,3,4 (brown duction of cell walls that are predominantly Jones & mid-rib). Bartlett The Learning, bm3 mutant LL was found to have S-lignin, Jones suggesting & Bartlett that Learning, manipulation LL of the T F reduced SALE -methyl DISTIBUTI transferase (MT) activity expression T F of the SALE ferulate-5-hydroxylase DISTIBUTI gene 8.3 Phenylpropanoid Pathway 131. T F SALE DISTIBUTI.

14 T F SALE DISTIBUTI T F SALE DISTIBUTI 3 Phenylalanine PAL T F SALE DISTIBUTI trans-innamic acid T F SALE DISTIBUT 4 T F SALE DISTIBUTI p-oumaric acid 4L T F SALE DISTIBUTI T F SALE DISTIBUTI S-oA Jones & p-oumaroyl-oa Bartlett Learning, LL T F SALE DISTIBUTI 3 affeoyl-oa T F SALE DISTIBUTI Feruloyl-oA oamt F5 T F SALE DISTIBUT 5-ydroxy feruloyl-oa p-oumaroyl aldehyde T F SALE DISTIBUTI AD oniferyl aldehyde AD MT Jones & Sinapoyl-oA Bartlett Learning, LL T F SALE DISTIBUTI Sinapyl aldehyde T F SALE DISTIBUTI oumaroyl alcohol 3 oniferyl alcohol AD T F SALE 3 DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT 3 Sinapyl alcohol T F SALE DISTIBUTI T F SALE DISTIBUTI 13 APTE 8 Aromatic and Phenolic ompounds Peroxidases, laccases ell membrane Jones & Bartlett Learning, ell wall LL Lignin T F SALE DISTIBUTI FIGUE 8.18 Simplified map of monolignol and lignin synthesis in vascular plants. The most direct route to the three major monolignols synthesized from p-coumaric acid and some of the enzymes required are shown. In most plants, alternate reactions/ enzymes are present, providing additional routes to the major monolignols and other compounds that may be found in lignin. The in vivo pathway for lignin synthesis is more grid-like than this simple linear diagram. The mechanism by which monolignols are transported across the cell membrane is unknown. AD, coumaroyl alcohol dehydrogenase; oamt, caffeoyl-oa--methyl transferase; 3, p-coumarate-3-hydroxylase (monooxygenase); Jones 4, & Bartlett coumaroyl-4-hydroxylase; Learning,, LL coumaroyl-oa reductase; 4L, 4-coumarate oa ligase; MT, caffeic acid/5-hydroxy ferulic acid -methyltransferase; F5, ferulate-5-hydroxylase; PAL, phenylalanine ammonia lyase. T F SALE DISTIBUTI. T F SALE DISTIBUTI.

15 may be a key step in increasing the digestibility 3 Jones of & forage Bartlett crops. Learning, LL Jones & Bartlett Learning, LL T F SALE The results DISTIBUTI in any one plant system, however, may not be applicable to other species. T F SALE DISTIBUTI Down-regulation of the gene for 4-coumarate oa ligase (4L) in tobacco produced plants 8 8 with less overall lignin in the cell walls and a polymer with a higher Jones content & of Bartlett coumaroyl Learning, LL alcohol. onversely, in T Arabidopsis F SALE the total lig-distibutinin content in 4-coumarate oa ligase mutant plants was reduced but the walls still contained Pinoresinol high levels of S-lignin. This suggests that linkage alternate pathways for monolignol synthesis are present Jones in some & Bartlett species and Learning, can compensate T for alterations F SALE in the main DISTIBUTI sequence of T F SALE DISTIBUTI LL 8 5 monolignol production, but these alternatives may be species specific. The striking results in poplar (Populus tremuloides) illustrate the 3 flexibility of plants in producing lignins. 3 Jones & Down-regulation Bartlett Learning, of the LL aldehyde reductase Jones & Bartlett Licarin Learning, A LL T F (AD) SALE synthesis DISTIBUTI results in plants with a higher T F SALE 8-5 linkage DISTIBUTI content of aldehydes in their cell walls. Aldehyde incorporation produces a pronounced 3 red coloration in the vascular tissues of the woody stems. Although genomics and genetic 8 engineering techniques Jones have greatly & Bartlett 4 increased Learning, 3 LL our knowledge of lignin T synthesis, F SALE application DISTIBUTI of these techniques to manipulate the quantity and properties of lignin is still problematic Virolin because of our overall lack of understanding of how cell walls are made. Methods for FIGUE 8.19 Structures of some common lignans. Lignans linkage analyzing Jones lignin & and Bartlett linkages, Learning, such as nuclear LL are dimers of monolignols with precise linkages formed magnetic T resonance F SALE (M) spectroscopy, DISTIBUTI are a between the precursors. T They are F synthesized SALE by free DISTIBUTI radical initiated bond formation guided by dirigent proteins. valuable asset to lignin engineers and are currently used to provide a more directed path to linkages found in lignin (see Figure 8.16). The dilignol bonds in these compounds correspond to lignin manipulation. T F SALE DISTIBUT T F SALE DISTIBUT Jones & 8.4 Bartlett atural Learning, Products LL Derived from T F SALE the Phenylpropanoid DISTIBUTIPathway (FIGUE T 8.0). F As SALE in the case DISTIBUTI of lignin synthesis, the free radical initiator is a peroxidase. atural Products from Monolignols Lignan dirigent proteins are found in the Lignans Lignans are dimeric phenylpropanoids cytoplasm, but similar dirigents have been that are synthesized from monolignols, mainly localized in the cell walls of some plants. It has coniferyl alcohol. They Jones are found & Bartlett in the seed Learning, been proposed LL that these extracellular Jones dirigent & Bartlett Learning, LL coats, flowers, and epidermal T F layers SALE of stems DISTIBUTI proteins may be active in lignin formation. T F The SALE DISTIBUT and leaves where they serve to defend plant binding of monolignols to dirigents with different sites could account for the regular bond- tissues against pathogens. They are also precursors for a variety of complex natural products. ing patterns found in some lignin polymers. Lignans are synthesized by a free radical Although a large number of different dirigent coupling Jones mechanism, & Bartlett but Learning, the linkages LL are proteins and their genes Jones have & Bartlett been predicted Learning, LL specific T rather F than SALE random as DISTIBUTI in lignin. The from genomic analyses, T F an even SALE larger number would presumably be required to guide DISTIBUTI structures of some common lignans are shown in FIGUE The monolignol substrates are total lignin synthesis. The dirigent proteins in held in position by proteins called dirigents cell walls may determine the bonding in the initial products of polymerization to which addi- (guide proteins). These proteins have no enzymatic Bartlett activity; Learning, they serve LL to bind the lignols tional Jones monolignols & Bartlett are then Learning, added a random, LL Jones & T F and SALE guide the radical-mediated DISTIBUTI bond formation radical-coupled T F SALE process. DISTIBUTI 8.4 atural Products Derived from the Phenylpropanoid Pathway 133. T F SALE DISTIBUTI.

16 T F SALE DISTIBUTI 3 T F SALE DISTIBUTI oniferyl alcohol peroxidase T F SALE DISTIBUTI T F SALE DISTIBUT Dirigent protein T F SALE DISTIBUTI 3 3 T F SALE DISTIBUTI 8 8 radical coupling T F SALE DISTIBUTI 3 3 T F SALE DISTIBUTI T F SALE DISTIBUTI cyclization 3 Dirigent protein T F SALE DISTIBUT T F SALE DISTIBUTI 8 T F SALE DISTIBUTI 8 T F SALE DISTIBUTI In some T plants, F lignans SALE are modified DISTIBUTI to produce species-specific natural products. Some of potential anticancer agent but is too toxic to of the spindle apparatus. T F It is, SALE therefore, DISTIBUT a these modified lignans are useful to humans as be taken internally. It is used topically to treat anticancer agents and nutritional supplements. some skin cancers. A synthetic derivative, etoposide, is less toxic and can be taken internally The structures of three examples are shown in Jones FIGUE & Bartlett 8.1. Sesamin Learning, is an LL antioxidant found to treat Jones certain & Bartlett types of Learning, lung and testicular LL T F in SALE the seed oil DISTIBUTI of the sesame plant (Sesamum cancers. T Etoposide F SALE acts by inhibiting DISTIBUTI topoisomerase activity and DA synthesis rather than indicum). Gomisin A, from Schisandra chinensis, a tropical vine from Asia, is used in traditional binding to tubulin. herbal medicines as a treatment for liver disorders. Podophyllotoxin from May apple Volatiles Many plants produce volatile compounds Bartlett from Learning, monolignols. LLVolatiles in flowers Jones & Bartlett Learning, (Podophyllum LL peltatum) inhibits cell division Jones by & T F SALE DISTIBUTI binding to tubulin and preventing the formation T F SALE and fruits are DISTIBUTI attractive to pollinators and her- 134 APTE 8 Aromatic and Phenolic ompounds Jones & Bartlett () Pinoresinol Learning, LL 3 T F SALE DISTIBUTI FIGUE 8.0 Dirigent proteins guide the synthesis of lignans. The dirigent proteins are represented by brackets. The proteins bind two molecules of coniferyl alcohol in this example and hold them in a defi ned position. A radical is generated by a cytoplasmic peroxidase. Because of binding to the dirigents, radical coupling occurs only between the 8-8 -positions. The product is the lignan, pinoresinol, a compound that is common in the leaceae (olive family).. T F SALE DISTIBUTI.

17 T F SALE DISTIBUTI 8 Sesamin 8' 8 8' ' 8 8' Gomisin T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI FIGUE 8.1 Structures of modified lignans that are useful to humans. Sesamin is an antioxidant found in sesame seed oil that is marketed as a neutraceutical. Gomisin-A has an unusual eight-membered ring. It is the active ingredient in some traditional herbal medicines used to treat liver disorders. Podophyllotoxin inhibits tubulin polymerization and prevents cell division. Although podophyllotoxin is too toxic for therapeutic use, it is the natural model for etoposide, a synthetic anticancer drug. T F SALE DISTIBUTI Jones 8 & Bartlett Learning, LL T F SALE DISTIBUTI 8' T F SALE DISTIBUT T F SALE DISTIBUTI Jones 3 & Bartlett Learning, LL T F SALE DISTIBUTI 3 T F SALE DISTIBUT Podophyllotoxin Etoposide Anethole 3 bivores, while volatile compounds in leaves and stems T repel F insects SALE or attract insect DISTIBUTI predators. T F SALE DISTIBUTI A common pathway for the synthesis of some volatiles is shown in FIGUE 8.. The alcohol Propenyl cation group of monolignols such as coniferyl alcohol 3 can be removed by a reductase to generate a Jones & resonance-stabilized Bartlett Learning, carbocation LL intermediate. 3 T F eduction SALE of the DISTIBUTI allylic cation yields eugenol, T oniferyl F alcohol SALE DISTIBUTI the aromatic compound found in cloves (Syzgium aromaticum), which are used by humans Allyl cation as both a flavoring agent and an analgesic (pain reliever). Similarly, reduction of the corresponding propenyl cation and Jones dehydration & Bartlett produces Learning, LL 3 ADP ADP anethole, a volatile found T in F many SALE plants such DISTIBUTI T F SALE DISTIBUT FIGUE 8. Examples of volatile natural products produced from monolignols. emoval of the hydroxyl group from coniferyl alcohol generates Jones two & resonance- Bartlett stabilized Learning, carbocations. LL eduction T of the F allylic SALE cation generates DISTIBUTI the methylene group in eugenol, the volatile compound in cloves. eduction of the propenyl cation and subsequent dehydration generates anethole, the volatile found in many plants such as fennel. emoval of the hydroxyl group of sinapyl alcohol and reduction of the resulting allylic cation to a methylene group initiates the formation of myristicin, which also requires ring closure to form the methylene dioxy ring. Myristicin is the volatile that adds to the fragrance of nutmeg seeds. T F SALE DISTIBUTI Sinapyl alcohol 3 Jones & Bartlett Learning, Eugenol LL T F SALE DISTIBUTI T F SALE DISTIBUTI Myristicin 8.4 atural Products Derived from the Phenylpropanoid Pathway 135. T F SALE DISTIBUTI.

18 Jones & Bartlett Learning, 8.5 Synthesis and Properties of LL Polyketides T F SALE DISTIBUTI T F SALE DISTIBUTI As described in the previous section, the phenylpropanoid pathway furnishes monolignols 3 3 that are essential for lignin synthesis and, ydroxyferulic acid therefore, can be considered part of the primary metabolism of plants. Jones This & same Bartlett pathway Learning, LL reverse aldol T F SALE DISTIBUTI also provides precursors T F for many SALE different DISTIBUT aromatic compounds that are unique to a particular plant, such as eugenol and myristicin (Figure 8.), and are generally regarded as secondary metabolites or natural products. In Jones & Bartlett Learning, LL addition, Jones coumaroyl-oa & Bartlett from Learning, the phenylpropanoid T pathway F SALE is the starting DISTIBUTI material for the LL T F SALE DISTIBUTI 3 synthesis of polyketides such as the flavonoids, Vanillin which have traditionally been considered natural products. FIGUE 8.3 Synthesis of vanillin. Vanillin is produced by a fermentation process that alters ferulic acid derivatives found in secondary cell walls. ydration of the side chain of Many flavonoids are unique to a particular Bartlett family Learning, of plants. Both LLtheir synthesis in a Jones & Bartlett Learning, hydroxyferulic LL acid is followed by a reverse aldol reaction Jones to & T F SALE DISTIBUTI generate the aldehyde, vanillin. T F SALE specific plant DISTIBUTI and molecular details have been used to construct biochemically based phylogenies. Most land plants and many aquatic species, however, contain some class of flavonoids as fennel (Foeniculum vulgare). If the starting material is sinapyl alcohol, a similar reduction that function to protect the organisms against and subsequent formation of a methylene dioxy UV radiation, free-radical Jones damage, & Bartlett pathogens, Learning, LL ring yields T myristicin F SALE (Figure 8.), DISTIBUTI the volatile and herbivores or to T attract F pollinators, SALE mycorrhizal fungi, and symbiotic microorganisms. DISTIBUT compound found in nutmeg (Myristica fragrans). Vanillin is a natural product derived from Given their essential roles in enabling plants hydroxyferulic acid. In the pathway illustrated to interact with both biotic and abiotic factors in FIGUE 8.3, the double bond in hydroxyferulic in their environment, flavonoids and other acid is hydrated followed by a reverse aldol reaction SALE that cleaves DISTIBUTI the side chain to yield vanillin. tance T in plant F metabolism. SALE DISTIBUTI polyketides Jones are & Bartlett definitely Learning, of primary LL impor- T F In some plants an alternate route uses the oa Synthesis of halcones ester of ferulic acid as a precursor. ommercial vanilla extract from the pods of the vanilla oumaroyl-oa is a key intermediate in the orchid (Vanilla planiflora) is produced after a long phenylpropanoid pathway, and its synthesis Jones & Bartlett Learning, fermentation LL period. The pods are then Jones dried & from Bartlett phenylalanine Learning, is described LL in Figures 8.11 T F SALE DISTIBUTI and extracted with ethanol. eal vanilla extract contains vanillin and a number of additional compounds. Imitation vanilla extracts can be produced by fermentation of the ferulic acid 136 APTE 8 Aromatic and Phenolic ompounds T F SALE and 8.1. It is DISTIBUTI a substrate for the enzyme, chalcone synthase (S). S is found in most plants and provides the precursor for a large number of flavonoids. A cysteine thiolate anion in the enzyme s active site attacks the carbonyl found in some woody stems. carbon of coumaroyl-oa, Jones displacing & Bartlett the oa Learning, LL Salicylic T Acid F The plant SALE hormone, DISTIBUTI salicylic acid, and binding the coumaroyl T F moiety SALE (FIGUES 8.4 DISTIBUT can be synthesized by a variety of pathways. It is and 8.6). The second substrate is malonylderived in some plants from coumaroyl-oa. oa, which is produced by an acyl-oa carboxylase (A) complex found in the cytoplasm. In bacteria and other plants, such as Arabidopsis, salicylic acid can be produced from chorismic Unlike the plastid enzyme complex, the A Jones acid, & Bartlett an intermediate Learning, the LL shikimic acid pathway SALE (Figures 8.1 DISTIBUTI and 8.7). Salicylic acid is a protein. T Its F mechanism SALE of action DISTIBUTI is the same as complex Jones in the & Bartlett cytoplasm Learning, is one large LL fusion T F hormone produced after pathogen invasion. It the multisubunit A found in chloroplasts; it is transported to undamaged plant tissues where uses bicarbonate, ATP, and acetyl-oa to produce the three-carbon acid, malonyl-oa (see it mediates systemic acquired resistance (SA). It is also the active ingredient in aspirin (acetyl hapter 6). Jones & Bartlett Learning, salicylic acid), LL although the modern commercial Jones & Bartlett Malonyl-oA Learning, coordinates LL with an asparagine (Asn) DISTIBUTI residue in the S active T F SALE DISTIBUTI product is made by chemical synthesis. T F SALE site,. T F SALE DISTIBUTI.

19 is T F SALE DISTIBUTI which facilitates its decarboxylation. The remaining two-carbon fragment, an acetyl- oa cation, attacks the carbonyl carbon of the coumaroyl moiety and displaces it from the cysteine active site. T The F product SALE is cou-distibutimaroyl diketide (Figure 8.4). This diketide intermediate is reattached to the cysteine thiolate in the active site of S and a second equivalent of malonyl-oa is coordinated to the Jones asparagine & Bartlett residue, Learning, decarboxylated, LL and T added F to the SALE diketide. The DISTIBUTI resulting triketide intermediate undergoes another round of binding to the active site cysteine and acetate addition to yield a tetraketide. This final ketide intermediate undergoes cycliza- Jones & tion Bartlett and aromatization Learning, while LL still bound to T F S SALE to produce DISTIBUTI the product called a chalcone ys S Jones & Bartlett Learning, LL T F SALE DISTIBUTI S-oA p-oumaroyl-oa T F Asn SALE DISTIBUTI S-oA Malonyl-oA T F SALE DISTIBUTI Jones & Bartlett Learning, LL T F SALE DISTIBUTI p-oumaroyl diketide T F SALE DISTIBUTI FIGUE 8.4 Mechanism of action of chalcone synthase (S). All polyketide synthases have a conserved catalytic triad consisting of histidine (is), cysteine (ys), and asparagine (Asn) residues in their active sites. The histidine imidazole- acts as a base and removes a proton from the cysteine sulfhydryl group generating a reactive thiolate anion. This anion attacks the carbonyl carbon of coumaroyl-oa and displaces the oa. The second substrate, malonyl-oa, binds near the asparagine residue. The Asn amide- forms an bond with the carbonyl oxygen of malonyl-oa and facilitates its decarboxylation. The resulting acetyl cation attacks the carbonyl carbon of the coumaroyl cysteine complex and produces a diketide product. T F SALE DISTIBUTI T F SALE DISTIBUTI oas S S-oA S-oA T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI (FIGUE 8.5). The overall reaction catalyzed by S is as follows: oumaroyl-oa 3 Malonyl-oA Jones & Bartlett Learning, LL halcone 4 oa-s 3 T F SALE DISTIBUT halcone synthases s are found in all vascular plants and some bryophytes. They also occur in fungi and certain classes of algae. Most Ss have a monomer molecular weight of approximately 40 kda and Jones function & Bartlett as dimers. Learning, The LL Ss found in plants T are F part SALE of a large family DISTIBUTI of enzymes that can catalyze the synthesis of an aromatic ring from fatty acid precursors, usually malonyl-oa. The plant synthases are part of a larger superfamily, the polyketide synthases. Polyketides Jones are & Bartlett produced Learning, by many organisms, LL including T F bacteria SALE such as DISTIBUTI Streptomyces, and 8.5 Synthesis and Properties of Polyketides 137. T F SALE DISTIBUTI.

20 oa-s form the core structures of certain antibiotics, Jones & Bartlett Learning, LL Jones & such Bartlett as erythromycin. Learning, Although LL they catalyze T F SALE DISTIBUTI T F SALE similar reactions, DISTIBUTI the polyketide synthases of S-oA S-oA plants have very little amino acid sequence similarity to their bacterial counterparts. The plant enzymes probably evolved from fatty p-oumaroyl-oa Malonyl-oA acid synthases (see hapter 6). The x-ray crystal structure Jones & of Bartlett the S from Learning, LL T F SALE DISTIBUTI alfalfa (Medicago sativa) T has F been SALE solved to 1.5-Å DISTIBUT resolution (FIGUE 8.6A). The homodimer has two S-oA structurally separate active sites. oumaroyl- S-oA oa binds first in a cleft that excludes other phenolics due to its size and the charged residues Jones & Bartlett Learning, oa-s LL in the Jones vicinity & of Bartlett the active Learning, site. As shown LL in T F SALE DISTIBUTI FIGUE T 8.6B, F the SALE active site consists DISTIBUTI of a catalytic triad of histidine, cysteine, and asparagine residues. The histidine acts as a general base and S-oA S-oA removes a proton from the cysteine sulfhydryl oa-s group to produce an active thiolate anion that Jones & Bartlett Learning, LL Jones & binds Bartlett coumarate Learning, by displacing LL the oa. Malonyl-oA enters DISTIBUTI the active site near the aspara- T F SALE DISTIBUTI T F SALE gine residue, which provides hydrogen bonds to this substrate by way of its amide- ( -amino S-oA group). Decarboxylation of malonyl-oa produces the cation that attacks the coumaroyl moi- oa-s ety, producing a oa-bound Jones diketide. & Bartlett This cycle Learning, LL T F SALE DISTIBUTI is repeated two more T times F to yield SALE a tetraketide DISTIBUT intermediate that then folds and is desaturated to produce an aromatic ring. aringenin chalcone diffuses out of the active site, which is not large enough to allow further condensation to longer Jones & Bartlett halcone Learning, LL polyketides. Jones The & Bartlett S from Learning, alfalfa has LL a high FIGUE 8.5 T halcone F synthesis SALE catalyzed DISTIBUTI by chalcone synthase (S). S degree T of F sequence SALE similarity DISTIBUTI to other polyketide binds p-coumaroyl-oa fi rst and then adds a two-carbon fragment resulting from the decarboxy lation of malonyl-oa (see Figure 8.4). Loss of drives the reaction synthases found in plants, including those that toward synthesis. The cycle repeats two more times to generate a tetraketide produce different products, such as stilbenes and intermediate. The tetraketide undergoes cyclization and generation of double bonds pyrones. Most of the plant synthases seem to to produce a second aromatic ring in the fi nal product, a tetrahydroxy-chalcone. have evolved from a common ancestor. T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI (a) (b) FIGUE 8.6 X-ray crystal structure of S from alfalfa. (a) S occurs as a homodimer with two independent active sites. The tunnel into the active site is occupied by a coenzyme A analog shown as spheres. (b) The active site of S is shown with a bound narigenin chalcone in purple sticks. The active site histidine-303 (blue) is placed at the top of the binding cavity. It acts as a base to remove a proton from the cysteine-164 shown in yellow. The ketide intermediates are bound to this cysteine residue. Jones Asparagine-336 & Bartlett in red sticks Learning, is the binding site LL for malonyl-oa. (Structure a from Protein Data Bank 1W. J.-L. Ferrer, J. Jez, M. E. Bowman,. Dixon, and J. P. oel, at. Struct. Biol. 6 [1999]: Structure b from Protein Data Bank 1GK. J.-L. Ferrer, J. Jez, M. E. Bowman,. Dixon, and J. P. oel, at. Struct. Biol. 6 [1999]: ) T F SALE DISTIBUTI 138 APTE 8 Aromatic and Phenolic ompounds T F SALE DISTIBUTI T F SALE DISTIBUTI. T F SALE DISTIBUTI.

21 (a) T F SALE DISTIBUTI T F SALE DISTIBUTI chalcone isomerase Tetrahydroxychalcone T F SALE DISTIBUTI 6 (b) Jones & Bartlett Learning, A LL T F SALE DISTIBUTI 5 B 4 3 aringenin T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI aringenin flavanone T F SALE DISTIBUTI FIGUE 8.7 Synthesis of naringenin flavanone. (a) A specific chalcone isomerase (I) catalyzes formation of the pyran ring in the S-confi guration. (b) The letter designations and numbering system for the general fl avonoid molecule are illustrated. Synthesis of Flavanones and Derivatives Some flavonoids are acylated, usually with The chalcones are the precursors for most methyl or acetyl groups, but other additions are flavonoid compounds. Further Jones ring & Bartlett closure to Learning, possible. LL The acyl groups are added to Jones an in & Bartlett Learning, LL produce a pyran ring in T naringenin F SALE occurs spontaneously. Plants, however, have an isomercosylated flavonoids. Acylation can change the DISTIBUTI one of the rings or to the sugar moiety T in F gly-sale DISTIBUT ase that catalyzes this reaction. The enzymatic solubility of the flavonoids and cause a shift in ring closure is at least 100 times faster than their absorption spectra. the uncatalyzed reaction and also produces A sulfotransferase can catalyze the sulfation the biologically Jones active & Bartlett S-isomer Learning, (FIGUE 8.7). LL The of the B ring of some Jones flavonoids. & Bartlett The sulfur Learning, LL uncatalyzed T F reaction SALE results in DISTIBUTI a racemic mixture. The product of isomerization is classified T F SALE DISTIBUTI 5 6 as a flavanone and is the key intermediate in B the synthesis of other classes of flavonoids The major differences in these compounds are A 3 Jones & changes Bartlett in the Learning, oxidation state LLof the pyran () Jones & Bartlett 6 5 Learning, 4 LL T F ring SALE (FIGUE 8.8). DISTIBUTI T F SALE DISTIBUTI Many of the modifications shown in Figure 8.8 are catalyzed by oxygenases that add hydroxyl groups or a double bond to the ring of naringenin flavanone. These enzymes are associated with the outer Jones face & of Bartlett the endoplasmic reticulum. Additional T F enzymatic SALE reac- DISTIBUTI T F SALE DISTIBUT Learning, LL Jones & Bartlett Learning, LL tions can also modify the A and B rings to produce a large array of derivatives that are B collectively referred to as flavonoids. Most flavonoids are glycosylated for storage or transport. Jones The & sugar Bartlett attached Learning, to the flavonoid T is usually F glucose SALE but can also DISTIBUTI be galactose, T F SALE DISTIBUTI LL Flavanol Isoflavone Jones & Bartlett Flavone Learning, Dihydroflavonol LL arabinose, xylose, or rhamnose. These reactions FIGUE 8.8 Structures of the various are catalyzed by glycosyl transferases that use a flavonoid classes. Flavonoids are placed UDP-sugar substrate and make an ether bond in different classes, fl avonols, isofl avones, fl avones, and dihydrofl avonols, based on between the sugar and an group in the the modifi cations shown in the ring. Jones & flavonoid. Bartlett Additional Learning, sugars LL are often added to Jones & Bartlett Learning, Within each class, further additions are LL T F the SALE first one. DISTIBUTI often T made F to the A SALE and B rings. DISTIBUTI Flavonol Leucoanthocyanidin 8.5 Synthesis and Properties of Polyketides 139. T F SALE DISTIBUTI.

22 donor is phosphoadenosine phosphosulfate formation of a double bond as in the fatty acid Jones & Bartlett Learning, (PAPS). Sulfated LL flavonoids are often signaling molecules used by plants such as legumes T F SALE As illustrated DISTIBUTI in FIGUE 8.9, the iron in Jones & desaturases. Bartlett Learning, LL T F SALE DISTIBUTI to attract the appropriate species of symbiotic flavone synthase I is ligated to histidine and bacteria (see hapter 5). aspartic acid residues in the active site. This Flavonoids can be prenylated, usually with Fe 3 is reduced by ascorbic acid. The ferrous dimethylallyldiphosphate but occasionally form of the enzyme then binds oxoglutarate and molecular oxygen. Jones The & Bartlett oxoglutarate Learning, LL with longer Jones terpene & Bartlett precursors. Learning, This increases LL the hydrophobicity T F SALE of the products. DISTIBUTI Prenylated undergoes oxidative T decarboxylation F SALE to succinate, which is released from the enzyme. This DISTIBUT flavonoids generally function as phytoalexins. reaction generates an active ferryl intermediate, Synthesis and Properties of Flavones and the enzyme can now bind and catalyze the Flavones are made from naringenin flavanone & Bartlett by addition Learning, of a double LL bond to the -ring naringenin Jones flavanone. & Bartlett Additional Learning, reactions LLin T F between SALE - and DISTIBUTI -3 (Figure 8.8). Two dif- flavonoid T F modification SALE are catalyzed DISTIBUTI by DDs, formation of a double bond in the substrate, Jones ferent flavone synthases (FS) can catalyze and genomic analyses suggest that this type of this desaturation. In many angiosperms, flavone synthase II (FS II) is a cytochrome thought. The Arabidopsis genome has approxi- dioxygenase is more common than previously P450 monooxygenase. In others, such as some mately 64 genes for putative DDs, and some Jones & Bartlett Learning, species LL the Apiaceae (parsley, carrot), Jones the & oxygenase-catalyzed Bartlett Learning, reactions LL may use this T F SALE DISTIBUTI synthase (flavone synthase I [FS I]) is T an oxoglutarate-dependent dioxygenase (DD). The monooxygenases. F SALE type of enzyme DISTIBUTI rather than cytochrome P450 DDs are nonheme iron containing enzymes Flavones are colorless compounds with that can bind and transfer one oxygen atom an absorption maximum in the UV region to oxoglutarate ( -ketoglutarate) and the other of the spectrum. They are generally stored oxygen atom Jones to & a Bartlett substrate Learning, or catalyze LL the in cells as 7--glycosides Jones and & can Bartlett be found Learning, LL T F SALE DISTIBUTI in all tissues of most T plants, F except SALE for the DISTIBUT Brassicaceae, which do not produce flavones. EZYME Flavones function to protect plant cells from Asp UV radiation and may also act as allelochemicals (i.e., compounds that inhibit growth of is is Jones & Fe Bartlett 3 Learning, LLAscorbate competing Jones photosynthetic & Bartlett Learning, organisms). LL They T F SALE DISTIBUTI may T also F serve SALE to attract and DISTIBUTI guide pollinators. As seen in FIGUE 8.30, colorless flavones fluoresce under UV light, and the patterns can EZYME Asp be detected by bees and some other potential Dehydroascorbate is is pollinators. The flavones serve as a nectar guide Jones & Bartlett Learning, Fe LL Jones & but Bartlett also ensure Learning, that pollen LLis transferred onto T F SALE DISTIBUTI T F SALE the insect. DISTIBUTI Synthesis and Properties of Anthocyanidins Anthocyanidins are the precursors of plant Succinate -xoglutarate pigments collectively referred to as anthocyanins. Their colorless precursors, Jones leuco-anthocyanidins, & Bartlett Learning, LL EZYMET F SALE DISTIBUTI are synthesized from T dihydroflavonols, F SALE which DISTIBUT Asp are the products of another DD that introduces an group at -3 in the ring of is is 3 Fe IV = naringenin flavanone. The keto group in the Jones & Bartlett Learning, FlavanoneLL T F SALE DISTIBUTI T F SALE DISTIBUTI FIGUE 8.9 Mechanism of action of flavone synthase I (FS I). FS I belongs to the -oxoglutarate dependent nonheme iron family of dioxygenases (DD). Iron is liganded to histidine and aspartic acid residues in the protein. The ferric iron is EZYME reduced by ascorbate. The Fe form of the enzyme binds oxoglutarate and catalyzes Asp 3 is is an oxidative-decarboxylation to succinate. The resulting Fe IV form of the enzyme Fe 3 then catalyzes formation of a double bond the fl avanone substrate between - and -3 in the ring. In a more generalized reaction, an DD transfers the Fe-bound T F SALE DISTIBUTI Flavone oxygen T to a second F substrate SALE to generate DISTIBUTI a hydroxylated product. 140 APTE 8 Aromatic and Phenolic ompounds. T F SALE DISTIBUTI.

23 T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI (a) (b) FIGUE 8.30 olorless flavones are visible under UV light. Flower (oreopsis leavenworthii, top; Bidens mitis, bottom) images T F SALE DISTIBUTI are shown photographed under visible (a) and UV (b) light. The UV images are transduced into blue. UV-absorbing fl avones are concentrated in the center of the flowers. The patterns are visible to some insects, such as honeybees, and may increase attraction to the fl owers and help guide the pollinator after it lands. Jones & Bartlett ring is then Learning, reduced, LL and a second DD, T F anthocyanidin SALE DISTIBUTI synthase, introduces two double bonds into the ring to produce the anthocyanidin flavylium cation (FIGUE 8.31). The final steps in anthocyanin synthesis include glycosylation hanges Jones in cell & Bartlett shape influence Learning, stacking LL of the aromatic T F rings SALE of the anthocyanins DISTIBUTI and lead to variations in the perceived colors. The presence of cations, mainly Mg and Al 3, can also influence the final color of flower pigments. A of the group in the ring and further modifications of the A and B Jones rings. & Bartlett Learning, LL particularly dramatic example are the colors of The unmodified T anthocyanidin F SALE flavylium DISTIBUTI T F SALE DISTIBUT cation can be generated in vitro and exhibits a FIGUE 8.31 Examples of anthocyanidins. reddish hue at neutral p. The color is influenced both by the type of solvent and p. In further modifi ed to produce different colored The product of anthocyanidin synthase is the fl avylium cation shown at the top that is flower petals the color of the anthocyanins can products. The hydroxyl () group at 1 vary depending Jones & on Bartlett the chemical Learning, modifications, LL in most anthocyanidins is glycosylated Jones & to Bartlett Learning, LL 3 stabilize the ring structure. ydroxyl groups including T further F SALE glycosylation, DISTIBUTI acylation, and T F SALE DISTIBUTI in the A and B rings are also often glycosylated. The colors shown above can be 1 prenylation. Anthocyanins are often stored in special vacuoles modified for pigment storage. generated in vitro from purifi ed compounds. These vacuoles are acidic and generally give The actual colors of anthocyanidins in vivo Pelargonidin: 1 =, =, 3 = depend on a number of physiological rise to a blue color. hanges in the vacuolar p, Luteolinidin: parameters such as p of the compartment 1 =, =, 3 = Jones & such Bartlett as raising Learning, the p to 7 LL or higher, can shift Jones & Bartlett Learning, in which the pigments are stored and Delphinidin: LL 1 =, =, 3 = T F the SALE color toward DISTIBUTI the red end of the spectrum. presence T of F metal ions SALE and copigments. DISTIBUTI yanidin: 1 =, =, 3 = 8.5 Synthesis and Properties of Polyketides 141. T F SALE DISTIBUTI.

24 hydrangea flowers, which are generally pink The attenuation of oxygen evolution in photosystem Bartlett II Learning, by anthocyanins LLhelps prevent the Jones & Bartlett Learning, when grown LL in alkaline soils but can change Jones & T F SALE DISTIBUTI to blue after application of an acidic mixture T of F SALE formation of DISTIBUTI oxygen free radicals. The anthocyanins themselves are also very effective soluble aluminum salts to the soil (see photo on page 119). The presence of other flavonoids, antioxidants. They can readily dissipate reactive oxygen species such as the hydroperoxy such as the colorless flavones, act as copigments and can also modulate color. Anthocyanins in flowers Jones can & obviously Bartlett have Learning, a variety LL of peroxisomes (see Appendix Jones 5). & Bartlett Learning, LL radical and other radical species produced in colors T and are F a major SALE factor in DISTIBUTI the attraction Anthocyanidins T can F be polymerized SALE into DISTIBUT of pollinators. Genetic engineering techniques oligomers called proanthocyanidins or condensed tannins. The precursors accumulate have been used to try to alter flower colors for commercial purposes. Given the number of in vacuoles and are exported to the cell wall. factors that can influence the final petal colors, Subsequent oxidation by laccases produces Jones this & Bartlett is not readily Learning, accomplished LL by modifications SALE of a few genes. DISTIBUTI color T characteristic F SALE of some DISTIBUTI seeds, fruits, and linear Jones polymers & Bartlett with a deep Learning, purple to LL brown T F In contrast to flower petals, anthocyanins in tree bark. These compounds inhibit proteolytic enzymes in herbivores and also slow the leaves are usually a red color and are responsible for the bright red of autumn leaves, particularly in some maple species. Anthocyanins in growth of bacterial and fungal pathogens. Jones & Bartlett Learning, photosynthetic LLtissues are stored in vacuoles Jones & Bartlett Synthesis Learning, and Properties LLof Isofl avonoids T F SALE DISTIBUTI and protect cells against the damaging T effects F SALE Isoflavonoids DISTIBUTI are synthesized from naringenin flavanone by a cytochrome P450 mono- of intense sunlight. Their synthesis can be increased during periods of high light intensity and low temperatures, such as early spring the cleavage of the bond at -3 in the ring oxygenase. Isoflavone synthase (IFS) catalyzes in the northern hemisphere. Under these conditions, the Jones light & absorption Bartlett by Learning, anthocyanins LL then migrates to -3 Jones and the & enzyme Bartlett adds Learning, an LL and generates a radical intermediate. The B ring reduces T the F rate of SALE the light absorption DISTIBUTI and group to - (FIGUE T 8.3). F A dehydratase SALE can DISTIBUT Jones & Bartlett Learning, ADP LL ADP T F SALE DISTIBUTI transfer in photosystem II. In cool conditions, fixation is limiting and cannot recycle the cofactors required in the light reaction. T F SALE DISTIBUTI 14 APTE 8 Aromatic and Phenolic ompounds remove from this initial product to yield a specific isoflavone such as genistein. Isoflavone synthase can also act on deoxyflavonones that lack the hydroxyl group on -5 of ring A to produce Jones an additional & Bartlett class Learning, of isoflavonoids. LL As T in the F other SALE flavonoids, DISTIBUTI modifying groups such as sugars can then be added to the basic isoflavonoid ring system. Isoflavonoids are not widespread among plants but are commonly found in legumes. Jones & Isoflavonoid Bartlett Learning, synthesis LL is inducible, and the T F SALE products act DISTIBUTI as phytoalexins in leaf tissues to protect against pathogens. Isoflavonoids are excreted from legume roots and serve as attractants for nitrogen-fixing symbiotic bacteria (see hapter 5). Jones & Bartlett Learning, LL The isoflavone ring Jones system & bears Bartlett a resemblance to the mammalian T F sex SALE hormones, DISTIBUT Learning, LL T F SALE DISTIBUTI the estradiols (FIGUE 8.33). Isoflavonoids are able to bind weakly to mammalian estrogen Jones & Bartlett Learning, LL dehydratase T F SALE DISTIBUTI T F SALE DISTIBUTI Genistein T F SALE DISTIBUTI FIGUE 8.3 Synthesis of genistein isoflavone. The enzyme, isofl avone synthase, is a specifi c cytochrome P450 monooxygenase. It binds the S-isomer of naringenin fl avanone and catalyzes removal of an atom at -3 in the ring, producing a radical intermediate. Migration of the aromatic B ring to -3 and hydroxylation of - yield an isofl avone intermediate. In soybean (Glycine max), a dehydratase removes from the ring, producing the isofl avone, genistein. Jones & Bartlett Learning, LL T F SALE DISTIBUTI. T F SALE DISTIBUTI.

25 T F SALE DISTIBUTI T F SALE DISTIBUTI Daidzein: = Genistein: = Isoflavones in soy T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI Jones & Bartlett Learning, LL Estradiol T F SALE DISTIBUTI oumestrol Isoflavone in clover, alfalfa sprouts FIGUE 8.33 Some isoflavonoids resemble estradiol. The ring structure of isofl avones in many legumes resembles the mammalian sex hormone, estradiol, and can bind to estrogen receptors. The critical property for receptor binding is the distance between the hydroxyl groups. T F SALE DISTIBUTI T F SALE DISTIBUTI receptors and can function as phytoestrogens. urcumin is produced mainly in the roots of The phytoestrogen in clover, coumestrol, is the plant as a phytoalexin. umans use the known to have negative Jones effects & on Bartlett the reproductive cycles of ruminants T F such as SALE sheep. The DISTIBUTI color to foods. urcumin also inhibits T the F secre-sale DISTIBUT Learning, roots to LL provide a pungent taste and Jones yellow & Bartlett Learning, LL phytoestrogens in soy-rich foods, such as tofu, tion of mucus in mammalian lungs and may be may have beneficial effects in humans. linical trials have shown that a soy-rich diet can and cystic fibrosis. useful in treatment of the symptoms of asthma reduce blood cholesterol levels and the risk of heart Jones attacks & in Bartlett postmenopausal Learning, women. LL Pyrone Synthases The Jones pyrone & Bartlett synthases Learning, are LL The T U.S. Food F and SALE Drug Administration DISTIBUTI recommends an intake of 5 g per day of soy restricted distribution among plants. The another type of T polyketide F SALE synthase with DISTIBUTI protein to diminish the effects of menopause basic pyrone synthase uses acetyl-oa as its and reduce cholesterol levels. The effects are initial substrate and adds malonyl-oa in a variable and may depend on the activity of gut condensation reminiscent of fatty acid synthesis Jones (FIGUE& 8.35). Bartlett Pyrone Learning, synthases LL catalyze Jones & flora Bartlett that can Learning, modify the LL phytoestrogens and T F promote SALE uptake DISTIBUTI from the intestines. the T production F SALE of a triketide DISTIBUTI intermediate and its subsequent cyclization to 6-methyl- Examples of ther Plant Polyketide Synthases 4-hydroxy-pyrone. S is ubiquitous in plants and provides Like the Ss, the pyrone synthases are the naringenin precursor for a wide variety dimers with two active sites. The chalcone and of flavonoids. ther types Jones of polyketide & Bartlett synthases have been described T F that SALE are often DISTIBUTI amino acid similarity and the same catalytic T F triad SALE DISTIBUT Learning, basic pyrone LL synthases exhibit 30% Jones to 50% & Bartlett Learning, LL species specific, leading to the production of of histidine, cysteine, and asparagine. A comparison of the x-ray crystal structures of S unique natural products. nly a few of these are described here to illustrate how small from Medicago sativa and the pyrone synthase changes in enzyme structure can lead to a from Gerbera hybrida is shown in FIGUE The variety Jones of different & Bartlett products. Learning, ne example LLis structural data clearly Jones illustrate & Bartlett that production Learning, LL the synthesis T F of curcumin SALE in urcuma DISTIBUTI longa, a of different products T is a F result SALE of minor residue DISTIBUTI plant in the Zingiberaceae (ginger) family. In. longa, a unique diketide synthase adds malonyl-oa to the oa ester of ferulic acid (FIGUE 8.34). A second feruloyl-oa is added Jones & to Bartlett the diketide Learning, product LL to yield curcumin, T F the SALE main ingredient DISTIBUTI in the spice, turmeric. mutations that affect the size and hydrophobicity of the ketide binding cavity. Stilbene Synthase An especially perplexing example Jones of polyketide & Bartlett synthesis Learning, is the LL production T of stilbenes. F SALE DISTIBUTI 8.5 Synthesis and Properties of Polyketides 143. T F SALE DISTIBUTI.

26 T F SALE DISTIBUTI p-oumaroyl-oa T F SALE DISTIBUTI T F SALE DISTIBUTI S-oA S-oA ADP SAM 3 Feruloyl-oA ADP SA Jones & Bartlett Learning, LL diketide synthase T F SALE DISTIBUT S-oA Malonyl-oA T F SALE DISTIBUTI 3 S-oA T F SALE DISTIBUTI T F SALE DISTIBUTI curcumin synthase T F SALE Feruloyl-oA DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI 3 3 urcumin FIGUE 8.34 Synthesis of curcumin. A unique diketide synthase binds the oa ester of ferulic acid (see Figure 8.14) and catalyzes the addition of malonyl-oa. The feruloyl-oa diketide intermediate is further modifi ed by a specifi c polyketide synthase that adds a second feruloyl moiety to the side chain to yield curcumin. This Jones particular polyketide & Bartlett synthase Learning, is unique to some LL plants of the Zingiberaceae (ginger) family. T F SALE DISTIBUTI T F SALE DISTIBUTI Stilbene synthases utilize coumaroyl-oa Jones & as Bartlett their initial Learning, substrate LL and add three units of T oa-s F SALE 3 oa-s DISTIBUTI 3 oa-s T F 3 SALE malonyl-oa DISTIBUTI to produce the same tetraketide Acetyl-oA intermediate that is found in chalcone synthesis. The stilbene synthases, however, catalyze a oa-s Malonyl-oA oa-s subsequent decarboxylation of the intermediate and a - to -7 cyclization instead of the oa-s -1 to -6 cyclization Jones in chalcone & Bartlett synthases Learning, LL Malonyl-oA T F SALE DISTIBUTI (FIGUE 8.37). Both T types F of synthases SALE exhibit DISTIBUT over 50% amino acid similarity and have identical 3 residues in the active site and surrounding substrate entrance cavity. Yet they consistently catalyze the formation of different products from Jones the same & tetraketide Bartlett intermediate. Learning, LL Subtle T F SALE DISTIBUTI differences T F in the SALE active site DISTIBUTI region apparently 6-Methyl-4-hydroxy--pyrone FIGUE 8.35 Activity of pyrone synthase. The enzyme favor the formation of a larger molecule by Ss and decarboxylation of the tetraketide catalyzes the sequential condensation of two molecules of and synthesis of a smaller product by stilbene malonyl-oa with an acetyl-oa starter unit instead of the synthases. p-coumaroyl-oa starter in S. A triketide intermediate is Jones & Bartlett produced in this reaction and then cyclized into an unsaturated -lactone called a pyrone. T F SALE can be further DISTIBUTI modified, mainly by As in the Learning, case of the flavonoids, LL the stilbenes T F SALE DISTIBUTI glycosylation. 144 APTE 8 Aromatic and Phenolic ompounds. T F SALE DISTIBUTI.

27 T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI FIGUE 8.36 omparison of the active site structures of -pyrone synthase (-PS) (left) and chalcone synthase (S) (right). The major difference between these homologous, dimeric enzymes is the size of the binding cavity, which is considerably smaller in the case of -PS. In addition, -PS has an active site entrance that is constricted compared with S and lined with several hydrophobic residues. elatively small molecules have access to the active site cavity and result in the condensation and exit of relatively hydrophobic products. This is in contrast to the more open and hydrophilic Jones cavity & Bartlett of S (see Learning, also Figure 8.6). LL (eproduced from: J. M. Jez, M. B. Austin, J. Ferrer, M. E. Bowman, J. T Schröder, F and J. SALE P. oel, hemistry DISTIBUTI & Biology 7 [000]: , Figure 6. ourtesy of Joseph P. oel, oward oward ughes Medical Institute.) T F SALE DISTIBUTI They are widespread but not universally distributed in flowering plants. They have anti- Jones & Bartlett Learning, LL T F SALE DISTIBUTI S-oA bacterial and antifungal properties and act as T F SALE DISTIBUT 3 S-oA phytoalexins. esveratrol, the stilbene shown in Figure 8.37, is abundant in grapes. Based on 3 experiments in vitro and experiments in animals, it Jones acts as an & Bartlett antioxidant Learning, and can inhibit LL 3 oa-s blood platelet aggregation (clotting) in mammals. esveratrol is the compound in red wine T F SALE DISTIBUTI T F SALE DISTIBUTI that is presumably responsible for the French S-oA paradox, that is, how to consume a diet rich in saturated fats and cholesterol and still maintain Jones & a low Bartlett risk for Learning, atherosclerosis LL and heart attack. Jones & oa-s Bartlett Learning, LL, oa-s Experiments in mice have recently shown that T F 1 6 condensation 7 condensation consumption SALE of DISTIBUTI resveratrol protects mice from T F SALE DISTIBUTI chalcone synthase stilbene synthase the adverse effects of a high-calorie diet even though the animals were relatively obese compared with controls. Larger quantities of resvera- trol than are present in a normal diet, however, 4 9 Jones & Bartlett 9 Learning, LL must be consumed to obtain these health benefits. esveratrol is available in pure form and T F SALE DISTIBUTI T F SALE DISTIBUT clinical trials are currently being done in humans 7 3 suffering from type diabetes. Synthesis Jones and & Activity Bartlett of oumarins Learning, LL The T coumarins, F SALE like other DISTIBUTI products of the phenylpropanoid pathway, start with the synthesis of the key intermediate, coumaroyl-oa. oumarin synthesis requires a specific hydroxylase (cytochrome P450 monooxygenase) that adds Jones & an Bartlett to the Learning, aromatic ring LL at the ortho position. T F The SALE next step is DISTIBUTI a nonenzymatic isomerization of halcone (aringenin) Stilbene (esveratrol) FIGUE 8.37 omparison of the activity of chalcone and stilbene synthases. Both enzymes catalyze the condensation of three molecules of malonyl-oa with a p-coumaroyl-oa staring unit to generate the same tetraketide intermediate. In chalcone synthase S, the intermediate undergoes a -1 to -6 cyclization and aromatization to produce naringenin chalcone. In stilbene synthase, the tetraketide intermediate undergoes a decarboxylation before cyclization of - to -7 to produce the second aromatic ring to yield resveratrol. T F SALE DISTIBUTI T F SALE DISTIBUTI 8.5 Synthesis and Properties of Polyketides T F SALE DISTIBUTI.

28 S-oA T F SALE DISTIBUTI ADP ADP visible light Umbelliferone T F SALE 3 DISTIBUTI oumarin 7 usually substituted with 6 and 8,, 3 FIGUE 8.38 Synthesis of umbelliferone. oumarins are synthesized from p-coumaroyl-oa, the key phenylpropanoid pathway intermediate. A specifi c cytochrome P450 monooxygenase hydroxylates the ring at the ortho position with respect to the side chain and removes the oa. A light-catalyzed isomerization of the double bond in the side chain is followed by spontaneous formation of the -lactone. oumarins can be further modifi ed by hydroxylation and methylation. The inset shows the numbering system and substitution patterns of coumarins. the double bond in the side chain catalyzed by PP visible light, followed by spontaneous formation Jones of & a Bartlett -lactone. FIGUE Learning, 8.38 illustrates LLthe synthesis of T F umbelliferone SALE and DISTIBUTI the general structural designations T F SALE 6 DISTIBUTI for coumarins. oumarins have a restricted distribution among angiosperms and are found in some ADP members of the Fabaceae, Asteraceae, and Jones & Bartlett Learning, Apiaceae. The LLsweet smell of new-mown hay Jones is & Bartlett Learning, LL ADP T F SALE DISTIBUTI due to the release of coumarins from damaged T F SALE DISTIBUTI clover (Melilotus officinalis). oumarins can be dimerized to dicoumarol, which is a highly effective anticoagulant (FIGUE 8.39). onsumption of large amounts of sweet clover can cause ADP internal bleeding Jones in & ruminants. Bartlett Learning, Synthetic anticoagulants, LL Jones & ADP Bartlett Learning, LL T such F as SALE warfarin, are DISTIBUTI based on the T F SALE DISTIBUT dicoumarol structure. nce widely used as a rat poison, warfarin is now prescribed as an anticlotting agent for the treatment of humans with atherosclerosis. Psoralen Jones & oumarins Bartlett Learning, are often modified LL by the addition SALE of other chemical DISTIBUTI groups. The -7 posi- Umbelliferone FIGUE Jones 8.40 Synthesis & Bartlett of the furanocoumarin, Learning, psoralen. LL T F T F (7-hydroxycoumarin) SALE is DISTIBUTI prenylated at -6 with dimethylallyldiphosphate, which can then lead to formation tion is usually hydroxylated and glycosylated. of a fi ve-membered furan ring. The prenyl side chain is A specific prenyl transferase can use dimethyl hydroxylated by a cytochrome P450 monooxygenase. A allyl diphosphate to prenylate -6. The prenylated second P450 monooxygenase catalyzes removal of the side coumarin is then further modified chain and formation of a double bond in the furan ring to Jones & Bartlett Learning, form psoralen. The ring may be modifi ed by glycosylation. by two monooxygenases LL to yield a linear The linear furanocoumarins are phytophotosensitizers that T F SALE DISTIBUTI furano derivative called a psoralen (FIGUE T 8.40). F SALE cause severe dermatitis DISTIBUTI in humans. 146 APTE 8 Aromatic and Phenolic ompounds T F SALE DISTIBUTI oa-s T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI Dicoumarol Warfarin FIGUE 8.39 Structures of two coumarin derivatives. Dicoumarol is produced in damaged clover plants by dimerization of coumarol. It is a powerful anticoagulant in mammals. Warfarin is a synthetic coumarin with structural similarity to dicoumarol and is used therapeutically to prevent internal clot formation. 6 T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT PP. T F SALE DISTIBUTI.

29 The furanocoumarins are produced in some ing of the skin. In the United States, cow Jones crop & Bartlett plants such Learning, as celery LL in response to fungal SALE pathogens. DISTIBUTI These coumarin derivatives is T an invasive F SALE plant that produces DISTIBUTI furanocou- parsnip Jones or hogweed & Bartlett (eracleum Learning, sphondylium) LL T F act as phytophotosensitizers in humans. marins. The plant is often found growing in Workers who harvest the plants by hand can open fields and along highways. Its dermatitis-causing properties present a major barrier come in contact with the furanocoumarin; subsequent exposure of the skin to sunlight to removal and eradication of this invasive causes dermatitis followed Jones by severe & Bartlett blister- Learning, species. LL T F SALE DISTIBUTI T F SALE DISTIBUT A P T E S U M M A Y A general outline describing the synthesis of aromatic the central product of the shikimic acid pathway; compounds T F in plants SALE is illustrated DISTIBUTI in FIGUE Synthesis begins in plastids with two simple intermediates (PAL), a cytoplasmic enzyme and the gateway to it is the substrate T F for phenylalanine SALE DISTIBUTI ammonia lyase of primary carbon metabolism, erythrose-4-phosphate phenylpropanoid synthesis. The precursor for most and phosphoenolpyruvate (PEP). These feed into the phenylpropanoids is coumarate or an esterified derivative, & most Bartlett often Learning, coumaroyl-oa. LLThe synthesis of Jones & shikimic Bartlett acid Learning, pathway to LL produce chorismic acid and Jones the essential aromatic amino acids, tyrosine, phenylalanine, and tryptophan. Intermediates in the shikimic of coumaroyl-oa. atural products such as flavo- primary products such as lignin draws from the pool T F SALE DISTIBUTI T F SALE DISTIBUTI acid pathway can provide the building blocks for additional aromatics such as the gallotannins and the plant be considered primary metabolites because of their noids also draw from the same precursor pool and can hormone, salicylic acid (Figure 8.1). Phenylalanine is essential functions in plant environmental interactions. Many polyketide synthases Jones use & coumaroyl-oa Bartlett Learning, LL as a starting unit and produce unique natural products T F SALE DISTIBUTI T F SALE DISTIBUT Phosphoenol pyruvate such as curcumin (Figure 8.34). Erythrose-4-phosphate Additional aromatic compounds are synthesized by branches from the phenylpropanoid pathway, and only Phenolics a few of these have been covered in this chapter such as 3 Jones & Bartlett Learning, LL the coumarins. Aromatic metabolism plants comprises 3 3 a rich assortment of chemical reactions and products. T F SALE DISTIBUTI T F SALE DISTIBUTI Polyketides, flavonoids, stilbenes, etc. trans-innamic Acid coumarate synthase p-oumaric Acid Monolignols T F SALE DISTIBUTI Lignin Volatiles oumarins The structures and syntheses of many new compounds are still not fully resolved and others are waiting to be discovered. The aromatic amino acids are also precursors in other unique synthetic pathways, such as the glucosinolates covered in hapter 5. The alkaloids are a rich Tryptophan Jones & Bartlett Learning, Tyrosine Phenylalanine LL assortment of additional natural products that are also T F SALE DISTIBUTI T derived F SALE from amino DISTIBUTI acids, particularly the aromatic phenylalanine ammonia ayase (PAL) amino acids. A small number of these are described in hapter 9. T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT FIGUE 8.41 Summary of general aromatic metabolism in plants. The synthesis of aromatic compounds starts in plastids with the shikimic acid pathway and results in the production of the three aromatic amino acids, tyrosine, phenylalanine, and tryptophan. Phenylalanine occupies a key position as the precursor of the phenylpropanoid pathway. The cytoplasmic enzyme, PAL, removes the amino group from phenylalanine and yields trans-cinnamic acid, which is a substrate for a cytochrome P450 monooxygenase. The product is p-coumaric acid, which can be further modifi ed, usually by esterifi cation to coenzyme A. The resulting pool of coumaroyl-oa in the cytoplasm is a source for the synthesis of monolignols and lignin, which are primary products, and a large number of natural products, including the vital fl avonoids and their derivatives and defensive compounds such as coumarins. T F SALE DISTIBUTI T F SALE DISTIBUTI 8.5 Synthesis and Properties of Polyketides 147. T F SALE DISTIBUTI.

30 E F E E E S F F U T E I F M A T I Jones & Bartlett Boerjan, Learning, W., alph, J., LL & Baucher, M. (003). Lignin Jones Koes, & Bartlett., Verweij, Learning, W., & Quattrocchio, LL F. (005). biosynthesis. Annu. ev. Plant Biol. 54: Flavonoids: a colorful model for the regulation of T F SALE DISTIBUTI T F SALE DISTIBUTI Grotewold, E. (006). Genetics and biochemistry of evolution of biochemical pathways. Trends Plant Sci. floral pigments. Annu. ev. Plant Biol. 57: :36 4. errmann, K.M. (1995). The shikimate pathway: Tanaka, Y., Sasaki,., & hmiya, A. (008). early steps in the biosynthesis of aromatic compounds. Biosynthesis of plant pigments: anthocyanins, betalains Plant ell 7: and carotenoids. Plant J. 54: umphreys, J.M. T & happle, F SALE. (00). ewriting DISTIBUTI T F SALE DISTIBUT the lignin road map. urr. pin. Plant Biol. 5: T F SALE DISTIBUTI PLAT BIEMISTY on the web Visit the Plant Biochemistry student companion Web site at for additional online study resources on these topics. T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUT T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI T F SALE DISTIBUTI 148 APTE 8 Aromatic and Phenolic ompounds. T F SALE DISTIBUTI.