8.2 Shikimic Acid Pathway

CHAPTER 8 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FORAromatic SALE OR DISTRIBUTION and NOT FOR SALE OR DISTRIBUTION Phenolic Compounds © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

8.1 Overview 8.5 Synthesis and Properties of Polyketides Synthesis of Chalcones © Jones &8.2 Bartlett Shikimic Learning, Acid Pathway LLC © Jones & BartlettSynthesis Learning, of Flavanones LLC and Derivatives NOT FOR SALE ORPhenylalanine DISTRIBUTION and Tyrosine Synthesis NOT FOR SALESynthesis OR DISTRIBUTION and Properties of Flavones Tryptophan Synthesis Synthesis and Properties of Anthocyanidins Synthesis and Properties of Isoﬂ avonoids 8.3 Phenylpropanoid Pathway Examples of Other Plant Polyketide Synthases Synthesis of Trans-Cinnamic Acid Synthesis and Activity of Coumarins Lignin Synthesis Polymerization© Jonesof Monolignols & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC Genetic EngineeringNOT FOR of Lignin SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 8.4 Natural Products Derived from the Phenylpropanoid Pathway Natural Products from Monolignols © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

119

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. 8.1 Overview of lignin precursors and is considered a primary © Jones & Bartlett Learning, LLC © Jones & metabolicBartlett Learning,pathway in terrestrialLLC plants. Cou- NOT FOR SALE OR DISTRIBUTIONPlants are autotrophic organisms andNOT must FOR SALEmaroyl-CoA OR DISTRIBUTIONis also the initial compound for a synthesize all their metabolites from the inter- variety of natural products, including flavonoid mediates of the C-3 cycle and other pathways synthesis. of primary carbon and nitrogen metabolism. Flavonoids are found in most plants and This includes the 20 common amino acids used give rise to colored compounds such as antho- in protein© Jones synthesis. & BartlettMany of these Learning, amino acids LLC cyanins. They function© Jones to protect & Bartlett plants from Learning, LLC are synthesizedNOT FOR directly SALE from OR precursors DISTRIBUTION derived intense light and ultravioletNOT FOR (UV) SALE radiation OR and DISTRIBUTION from primary metabolism. For example, the can act as antioxidants. A vast number of addi- amino acid glutamic acid is produced in a single tional natural products are synthesized from reaction: transfer of an amino group to 2-oxo- the aromatic amino acids and their precursors. glutarate (␣-ketoglutaric acid), an intermediate Only a small number of these are presented in © Jonesin & the Bartlett citric acid Learning, cycle. Synthesis LLC of the aromatic this© chapter. Jones & Bartlett Learning, LLC NOT FORamino SALE acids, OR phenylalanine, DISTRIBUTION tyrosine, and tryp- NOT FOR SALE OR DISTRIBUTION tophan, is more complex. In addition to their 8.2 Shikimic Acid Pathway function in protein synthesis, most amino acids serve as precursors for the synthesis of other All three aromatic amino acids are derived from metabolites. This is especially true for phen- intermediates of the same series of reactions, the © Jones & Bartlett Learning,ylalanine, whichLLC is the precursor for the© phen-Jones & shikimicBartlett acid Learning, pathway. Synthesis LLC starts with ery- NOT FOR SALE OR DISTRIBUTIONylpropanoid pathway. Removal of theNOT amino FOR SALEthrose-4-phosphate OR DISTRIBUTION and phosphoenolpyruvic group from phenylalanine yields trans-cinnamic acid (PEP). The pathway is named after the first acid followed by addition of an OH and coen- intermediate that is unique to aromatic amino zyme A, which produces p-coumaroyl-CoA. acid synthesis, shikimic acid. The reactions in This key intermediate is essential in synthesis this pathway are similar in plants, fungi, and bac- © Jones & Bartlett Learning, LLC teria but are absent© in Jones animals. & The Bartlett component Learning, LLC NOT FOR SALE OR DISTRIBUTION enzymes are homologousNOT FORin all these SALE kingdoms. OR DISTRIBUTION 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 O O– nucleus. An overview of the pathway is shown © Jones &C Bartlett Learning, LLC in ©FIGURE Jones 8.1. In & addition Bartlett to Learning, the three aromatic LLC NOT FOR SALE OR DISTRIBUTION aminoNOT acids, FOR the SALE intermediates OR DISTRIBUTION of the shikimic Phenolics acid pathway are also used in the synthesis of a variety of other metabolites, including the plant HO OH hormone salicylic acid, vitamins such as folic OH acid, and a number of species-specific natural Shikimic acid © Jones & Bartlett Learning, LLC © Jones & products.Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALEThe ORerythrose-4-phosphate DISTRIBUTION precursor is an intermediate in the C-3 cycle and the pentose O O– phosphate pathway in the chloroplast stroma Salicylic acid C (see Chapters 2 and 3). PEP is an intermedi- Benzoic acid ate in glycolysis and may be imported into H C ©2 JonesO & Bartlett Learning,Volatiles LLC chloroplasts. Alternatively,© Jones it can& Bartlett be a product Learning, LLC Quinones (rings) of chloroplast pyruvate metabolism via pyru- ONOT FOR− SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION OH O vate kinase and pyruvate, Pi dikinase. The first Para aminobenzoic acid reaction in the pathway is an aldol condensa- Chorismic acid (folic acid precursor) tion that results in the synthesis of a 7-carbon O ketose, 3-deoxy-arabino-heptulosonic acid- O O © Jones & Bartlett Learning, LLC 7-phosphate© Jones &(DAHP), Bartlett shown Learning, in FIGURE LLC 8.2. + CH C O− + CH C O− H N NOT FOR SALE+ OR CDISTRIBUTIONO− 3 NOT FOR SALE OR DISTRIBUTION H3N CH H3N CH2 CH2 H C 2 FIGURE 8.1 Simpliﬁ ed 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. © JonesHN & Bartlett Learning, LLC Shikimic© Jones acid and & chorismicBartlett acid Learning, are key intermediates. LLC These intermediates OH can be removed from the pathway and used to synthesize a variety of additional NOT FORTryptophan SALE OR DISTRIBUTIONPhenylalanine Tyrosine phenolicNOT FORcompounds. SALE OR DISTRIBUTION

120 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. H+ FIGURE 8.2 Synthesis of 3-deoxy-arabino- © Jones & Bartlett Learning, LLC O– © Jones &heptulosonic Bartlett acid-7-phosphate Learning, (DAHP).LLC O NOT FORThe SALE DAHP synthaseOR DISTRIBUTION catalyzes an aldol-type NOT FOR SALEO OR DISTRIBUTIONH C condensation. The phosphate is removed H2C O C CH from PEP, and its methylene carbon then –O H C O P C C forms a bond with the carbonyl carbon of – H2 OH O PEP erythrose-4-phosphate. –O OH O O P Erythrose-4-P © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE– OOR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

OH O O© Jones & HBartlett Learning, LLC © Jones & Bartlett Learning, LLC O C6 C – – 7 H CH 3 O O NOT FOR SALE OR4 DISTRIBUTION2 + Pi NOT FOR SALE OR DISTRIBUTION P C C5 C C 1 H2 OH H2 –O OH O 3-Deoxy-arabino-heptulosonic acid-7-P © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION DAHP synthases in plants and bacteria show Dehydroquinic acid is the substrate for a only about 20% amino acid identity. The plant dehydratase that catalyzes removal of water and enzyme, however, complements bacterial mu- introduces a double bond into the ring. This is tants lacking this synthase. The bacterial enzyme followed by reduction of the ketone to an alco- exhibits feedback inhibition© Jones by & the Bartlett aromatic Learning, hol forming LLC shikimic acid, the first ©unique Jones inter- & Bartlett Learning, LLC amino acid end products,NOT phenylalanineFOR SALE OR and DISTRIBUTIONmediate (FIGURE 8.4). In some plants,NOT shikimic FOR SALE OR DISTRIBUTION tyrosine. This does not appear to be true of the acid is the starting material for the synthesis of plant synthases. Expression of different iso- a variety of phenolic natural products. The most zymes of DAHP synthase in plants, however, common are the water-soluble gallotannins, 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-LLC usually glucose. Plants© Jones make &these Bartlett compounds Learning, LLC ence NOTof hormones, FOR SALE gibberellic OR acid DISTRIBUTION and jasmonic to protect their tissuesNOT from FOR UV SALE damage OR and DISTRIBUTION to acid. The intermediates and end products of the deter herbivores. Tannins can bind irreversibly shikimic acid pathway are often intermediates to proteins and inhibit enzyme activity. Humans in the production of phytoalexins and other have traditionally used tannic acid–rich plant components of plant defense reactions. extracts to tan and preserve animal hides. Some © Jones & BartlettThe second Learning, step in the pathwayLLC is the forma- humans© Jones also & like Bartlett the bitter Learning, flavor of gallotan-LLC tion of a cyclic intermediate, 3-dehydroquinic nins in beverages such as tea. NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION acid, from DAHP (FIGURE 8.3). The enzyme, dehy- Shikimic acid is further metabolized to droquinate synthase, is an oxidoreductase that chorismic acid, another key intermediate in requires NAD as a cofactor. The enzyme in this this pathway. A kinase adds a phosphate group case catalyzes both an oxidation followed by a to one of the meta hydroxyls to produce reduction, thereby regenerating© Jones & the Bartlett oxidized Learning, shikimic LLC acid-3-phosphate. This© intermedi- Jones & Bartlett Learning, LLC cofactor. NOT FOR SALE OR DISTRIBUTIONate is then condensed with anotherNOT molecule FOR SALE OR DISTRIBUTION

–O O C O 1 –O reduced C © Jones & Bartlett Learning, LLCHO FIGURE 8.3© JonesReactions & catalyzed Bartlett by de- Learning, LLC 2 CH C 3 2 1 O P hydroquinate synthase. The phosphate is NOT– FOR SALE OR+ NAD DISTRIBUTION2 + i NOT FOR SALE OR DISTRIBUTION O 4 O H2 5 CH 7 3 removed from C-7 of DAHP and the resulting C P H HC 64 carbocation forms a bond with C-2. The O 7 OH 5 enzyme then catalyzes the oxidation of the –O C6 O OH OH hydroxyl (OH) group on C-6 to a ketone and OH OH a reduction of the ketone on C-2 to an OH to yield 3-dehydroquinic acid. The redox cofactor © Jones & Bartlettoxidized Learning, LLC © Jones & Bartlett Learning, LLC for this enzyme is NAD, which is regenerated NOT FOR SALEDAHP OR DISTRIBUTION 3-Dehydroquinic acidNOT FORduring SALE the reaction. OR DISTRIBUTION

8.2 Shikimic Acid Pathway 121

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. O– O– O– FIGURE 8.4 Synthesis of shikimic acid. A © JonesHO & Bartlett Learning, LLC © Jones & Bartlettdehydratase-reductase Learning, LLC binds dehydroquinic acid. H C C C It catalyzes removal of the hydroxyl next to the NOTH FOR SALEO OR DISTRIBUTION O NOT FOR SALEO ORcarboxyl DISTRIBUTION group on C-1 and a proton from the C HC HC dehydratase reductase adjacent C-6 carbon, generating a double bond –H O NADPH in the ring. The NADPH-dependent reductase O 2 OH O OH HO OH activity then reduces the keto group to a hydroxyl NADP OH OH OH group to generate shikimic acid. 3-Dehydroquinic acid ©3-Dehydroshikimic Jones & Bartlett acid Learning, LLCShikimic acid © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

of PEP to produce 5-enolpyruvyl-shikimate- do not appear to be similar, glyphosate com- 3-phosphate (EPSP) (FIGURE 8.5). The enzyme, petes with PEP for the same binding site on © JonesEPSP & Bartlett synthase, Learning, is inhibited LLC by an amino acid the© synthase. Jones &As Bartlett shown in Learning, FIGURE 8.6, the LLC syn- analog, N-phosphonomethyl glycine, also NOT FOR SALE OR DISTRIBUTION thaseNOT consists FOR of SALE two globular OR DISTRIBUTION domains. Bind- known as glyphosate. Although the structures ing of shikimate-3-phosphate triggers a global

–O O H C FIGURE 8.5 Synthesis of 5-enol-pyruvyl- 2 O– C O shikimate-3-phosphate (EPSP). The EPSP © Jones & Bartlett Learning, LLC O © Jones & Bartlett Learning,synthase catalyzes LLC removal of the phos- C P NOT FOR SALE OR DISTRIBUTION1 NOT FOR SALE OR DISTRIBUTIONphate from PEP. The resulting three-carbon 62 –O C O O– –O fragment is added to the hydroxyl group on 3 5 –O O 4 PEP O C-3 of shikimate-3-phosphate. The amino O P OH O acid analog, glyphosate (inset), is a P – O OH H – competitive inhibitor of PEP. N O O Shikimate-3-phosphate C © Jones & Bartlett Learning,–O LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONGlyphosate NOT FOR SALE OR DISTRIBUTION

–O O C © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONCH2 NOT FOR SALE OR DISTRIBUTION –O C + Pi O O O P O C – OH O O– © Jones & Bartlett Learning,Enolpyruvylshikimate-3-phosphate LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

FIGURE 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 © Jones & Bartlett Learning, LLC the two© domains. Jones After & binding Bartlett shikimate- Learning, LLC NOT FOR SALE OR DISTRIBUTION 3-phosphateNOT (shown FOR as SALEpurple sticks), OR the DISTRIBUTION 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 & Bartlett Learning, LLC © Jones &a good Bartlett example ofLearning, “induced ﬁ t” where LLC binding of one substrate induces a NOT FOR SALE OR DISTRIBUTION NOT FORconformational SALE OR change DISTRIBUTION 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, N. Amrhein, J. N. S. Evans, © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,and W. Kabsch, LLC Proc. Natl. Acad. Sci. 98 NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION[2001]: 1376–1380.)

122 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. conformational change to the more closed struc- precursor for benzoic acid and its deriva- © Jones ture& Bartlett seen in FigureLearning, 8.6. PEP LLC then binds to the tives© Jones and also & Bartlettsalicylic acidLearning, in some LLC plants. NOT FORactive SALE site OR formed DISTRIBUTION at the interface of the two MethylatedNOT FOR benzoic SALE and OR salicylic DISTRIBUTION acids are vol- domains. Both kinetic and crystal structure atile and are responsible for many floral scents 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 LLC to as systemic acquired© Jonesresistance & Bartlett Learning, LLC in any of the many otherNOT reactions FOR SALEthat use OR PEP DISTRIBUTION(SAR). Additionally, chorismic acidNOT in FOR some SALE OR DISTRIBUTION 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 Roundup®. 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, C-3 and intro- LLC phenylalanine. © Jones & Bartlett Learning, LLC ducesNOT a second FOR doubleSALE bondOR DISTRIBUTION into the ring. NOT FOR SALE OR DISTRIBUTION This results in the production of chorismic Phenylalanine and Tyrosine Synthesis acid (FIGURE 8.7). Chorismic acid can be a Chorismic acid is the branch point between synthesis of phenylalanine and tyrosine or tryptophan (Figure 8.1). Chorismate mutase – © Jones & Bartlett Learning,O O LLC catalyzes© Jones the & transfer Bartlett of the Learning, pyruvyl side LLC chain C NOT FOR SALE OR DISTRIBUTION toNOT C-1 toFOR produce SALE prephenic OR DISTRIBUTION acid, the precur-

CH2 sor of phenylalanine and tyrosine (Figure 8.7). –O This mutase is allosterically regulated by the C O O EPSP O P O C three aromatic amino acids. Allosteric regulators – are generally small molecules that can either O OH – © JonesO & Bartlett Learning, activate LLC or inhibit enzyme activity,© Jonesbut these & Bartlett Learning, LLC synthaseNOT FOR SALE OR DISTRIBUTION regulators do not bind to the enzyme’sNOT active FOR site. SALE OR DISTRIBUTION Most allosterically regulated enzymes are com- –O O C posed of two or more subunits, and the regulators generally act by changing the subunit CH2 association. In the case of chorismate mutase, © Jones & Bartlett Learning, LLC the enzyme’s activity© Jones is inhibited & Bartlett by phenylala- Learning, LLC C O Pi NOT FOR SALEO COR DISTRIBUTION+ nine and tyrosineNOT and activated FOR SALE by tryptophan, OR DISTRIBUTION OH thereby ensuring balanced pools of all three O– Chorismic acid amino acids. In the next step, an amino group is added mutase to prephenic acid to produce arogenic acid © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O (Figure 8.7). The transaminase that catalyzes –O O NOT FOR SALE OR DISTRIBUTIONH2 NOT FOR SALE OR DISTRIBUTION C C C C O– Prephenic acid O FIGURE 8.7 Synthesis of tyrosine and phenylalanine from chorismic acid. Chorismate syn- O thase catalyzes the removal of the phosphate OH © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC + group from EPSP and introduces a second NOT FOR SALEH3 NORCH DISTRIBUTIONC double bond intoNOT the ringFOR to generate SALE chorismic OR DISTRIBUTION Glu O– acid. The prephenate mutase reaction results in α-Ketoglutarate transaminase CH2 the relocation of the pyruvyl moiety to C-1 of the ring producing prephenic acid. A transaminase NADPH then catalyzes transfer of an amino group from Tyrosine glutamic acid to C-2 of the side chain resulting © Jones & Bartlett Learning,NADP LLC © Jones & Bartlett Learning, LLC – O CO2 in the amino acid, arogenic acid. In most plants, O O O arogenic acid is the precursor for both tyrosine NOT FORH2 SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION C C H OH + C H N and phenylalanine. To produce tyrosine, an C 3 CH C NADP-dependent dehydrogenase oxidatively O– dehydrogenase O– + H O decarboxylates C-1 of the ring and catalyzes CO2 2 CH NH3 2 formation of a third double bond to generate an dehydratase aromatic ring. In phenylalanine synthesis, a dehydratase removes both the C-1 carboxyl © Jones & BartlettOH Learning, LLC © JonesPhenylalanine & Bartlett Learning, LLC group and a water molecule from the ring, NOT FOR SALEArogenic OR acidDISTRIBUTION NOT FOR SALE ORthereby DISTRIBUTION generating the aromatic amino acid.

8.2 Shikimic Acid Pathway 123

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. this reaction uses glutamic acid as the amino the ketose derivative. Decarboxylation of the © Jones & Bartlett Learning,donor. Oxidative-decarboxylation LLC of arogenic© Jones & aromaticBartlett ring Learning, and dehydration LLC and cyclization NOT FOR SALE OR DISTRIBUTIONacid by an NADP-dependent dehydrogenaseNOT FOR SALEproduce OR the DISTRIBUTION 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 path- by a dehydration to produce the aromatic ring. way is catalyzed by tryptophan synthase. This enzyme cleaves the glycerol-P moiety Tryptophan© Jones Synthesis & Bartlett Learning, LLC from the indole ©ring Jones and replaces& Bartlett it with Learning, LLC ChorismicNOT FOR acid SALEis also OR the DISTRIBUTIONprecursor for serine (Figure 8.8).NOT Tryptophan FOR SALE synthase OR DISTRIBUTIONis the synthesis of tryptophan (Figure 8.1). A an ␣2␤2 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 (FIGURE 8.8). The amino donor site located on the ␣ subunit. Binding causes © Jonesin & this Bartlett reaction Learning, is glutamine. LLC The next step a ©conformational Jones & Bartlett change Learning, that closes LLC the NOT FORinvolves SALE aOR glycosylation, DISTRIBUTION that is, addition activeNOT site. FOR After SALE removal OR of DISTRIBUTION glyceraldehyde- of 5-phospho-ribosyl-1-diphosphate to the 3-phosphate, the indole ring diffuses through amino group of anthranilic acid. The sugar- a long protein tunnel (~25Å) between sub- diphosphate is a good leaving group, and units to the active site in the ␤ subunit. the sugar is bonded to anthranilate by an Serine then binds and closes the active site © Jones & Bartlett Learning,N-glycosidic LLC bond as seen in nucleosides.© Jones An & inBartlett the ␤ Learning,subunit. The LLC hydroxyl group is NOT FOR SALE OR DISTRIBUTIONisomerase then catalyzes the formationNOT ofFOR SALEremoved OR from DISTRIBUTION 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. Chorismic acid Anthranilic acid Pyruvic acid Although crystal structures of several tryp- O O– O O– © Jones & Bartlett Learning, LLC tophan synthases ©with Jones bound & intermediatesBartlett Learning, LLC Glu NOT FOR SALE OR DISTRIBUTIONCH2 NOT FOR SALE OR DISTRIBUTION NH2 are available, it is still not known how the Gln O CH two active sites communicate to coordinate 2 synthase HO O O– their activity, but this enzyme does not O produce free indole. Some plants, such as OH O– maize, produce indole-based phytoalexins. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O In this case an enzyme resembling only the HO O – NOT FOROH SALE OR PDISTRIBUTION– O ␣ subunitNOT FOR of tryptophan SALE OR synthase DISTRIBUTION is found in O O−PP H transferase the cytoplasm. A similar enzyme is the most N HO 5-P-ribosyl-PP likely source of the volatile indole released O OH O−P from many plants.

−CO2 FIGURE 8.8 Synthesis of tryptophan. Anthranilate synthase binds chorismic acid and catalyzes removal of the pyruvyl synthase −H2O moiety followed by transfer of an amino group from glutamine to the ring to form anthranilic acid. The sugar, 5-phosphate-ribosyl- HO © Jones & Bartlett Learning, LLCdiphosphate, is coupled to the© amino Jones group &in anthranilate Bartlett Learning, LLC HO by an N-glycosidic bond and pyrophosphate is released. O−P Trp synthase NOTα FOR SALE OR DISTRIBUTIONThe 5-phospho-ribosyl moietyNOT then undergoes FOR SALEisomerization OR DISTRIBUTION to a ketose. The ring of the intermediate is decarboxylated, and a third enzyme catalyzes a ring closure of the ketose to generate N the ﬁ ve-membered ring in indole glycerol-3-phosphate. This N O intermediate is the substrate for the ␣ subunit of ␣2␤2 tryptophan Trp synthase synthase. Binding of indole glycerol-phosphate to the ␣ subunit Indole glycerol phosphate β © Jones & BartlettHO Learning, LLC © Jones & Bartlett Learning, LLC OH closes this subunit active site, and the enzyme cleaves the NOT FOR SALE NHOR DISTRIBUTIONO glyceryl-phosphateNOT FOR moiety SALE from the OR indole DISTRIBUTION ring. The products 2 OH are not released from the enzyme. The indole intermediate is Serine transported through a channel in the enzyme to the ␤ subunit. Serine also binds to the ␤ subunit, and binding closes this NH 2 O O−P second active site. The hydroxyl group is removed from serine, + OH and the remaining three-carbon fragment is coupled to the © Jones & Bartlett Learning, LLC N © Jonesindole & Bartlett ring to generate Learning, tryptophan. OpeningLLC of the active sites Glyceraldehyde-3-P on both subunits releases tryptophan from the ␤ subunit and NOT FOR SALE OR DISTRIBUTIONTryptophan NOT FORglyceraldehyde-3-phosphate SALE OR DISTRIBUTION from the ␣-subunit site.

124 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. 8.3 Phenylpropanoid Pathway The enzyme that catalyzes this initial step is © Jones & Bartlett Learning, LLC phenylalanine© Jones & Bartlettammonia Learning,lyase (PAL). LLC PAL is NOT FORMany SALE complex OR DISTRIBUTION plant products are synthesized foundNOT in FOR angiosperms SALE OR and DISTRIBUTION 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 particu- The plant enzymes are found in the cytoplasm, larly prominent position in plant metabolism most likely associated with the outer face of as it is the starting material© Jones for & the Bartlett synthesis Learning, the endoplasmic LLC reticulum. PAL from© Jones various & Bartlett Learning, LLC of a large number NOTof aromatic FOR SALEcompounds. OR DISTRIBUTIONplant sources is a tetramer with aNOT mass FORof 225 SALE OR DISTRIBUTION These metabolites are referred to as phenyl- to 330 kDa. In dicotyledonous plants, PAL is propanoids and include primary products such specific for phenylalanine. Some dicots also as the monolignol precursors of lignin and a have another enzyme that deaminates tyrosine large number of species-specific natural prod- (tyrosine ammonia lyase). In monocots, PAL ucts. ©Lignin Jones is a & major Bartlett component Learning, of second- LLC accepts both phenylalanine© Jones & and Bartlett tyrosine Learning, as LLC ary cellNOT walls FOR and SALE the second OR DISTRIBUTIONmost abundant substrates, althoughNOT the FOR catalytic SALE efficiency OR DISTRIBUTION 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 histi- © Jones &matrix Bartlett of mature Learning, plant tissues. LLC In the wall the dine© Jones ammonia & Bartlettlyase (HAL) Learning, and has the LLC same NOT FORprecursors SALE OR are DISTRIBUTION polymerized and form cross- mechanismNOT FOR of SALE action. OR The DISTRIBUTION crystal structure links to components of the primary cell wall. of PAL from parsley (Petroselinum crispum) 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 ter- as eugenol found in cloves. Another major tiary structure is very similar to the PAL role of phenylpropanoids© Jones is in the & Bartlettproduction Learning, from LLCRhodosporidium toruloides and© Jones histidine & Bartlett Learning, LLC of polyketides, includingNOT FORflavonoids, SALE which OR DISTRIBUTIONammonia lyase from the bacterium,NOT Pseudo-FOR SALE OR DISTRIBUTION are found in most plants and enable them to monas syringae. All these enzymes are unique 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 gen- © Jones & Bartlett Learning, LLC erate 4-methylidene-imidazole-5-one© Jones & Bartlett (MIO) Learning, LLC SynthesisNOT FOR of Trans SALE-Cinnamic OR DISTRIBUTIONAcid (FIGURE 8.9). The NOTmethylidene FOR SALE group OR of DISTRIBUTIONthe The synthesis of phenylpropanoids starts MIO 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. C–H bond on the ␤-carbon of the substrate © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONO NOTH FORO SALE OR DISTRIBUTION – O O– (a) H + + NH3 NH3 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC CH NOT FOR SALE OR DISTRIBUTIONHO 2NOT FOR SALE OR DISTRIBUTION O HO O N H O H N (b) –H2O Ala204 N N –H2O N N Gly N H H H 202 H O Ala HN © Jones & Bartlett Learning, LLC HN © Jones204 & Bartlett Learning, LLC Ala204 Gly202 NOT FOR SALE OR DISTRIBUTION Gly202 NOT FOR SALE OR DISTRIBUTION FIGURE 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-204, serine-203, and glycine-202 (residue numbering is from the bacterial enzyme), followed by a dehydration. The amino group in the peptide bond between ala-204 and ser-203 attacks the carbonyl carbon in the peptide bond between ser-203 and gly-202. Subsequent removal of two H2O © Jones &molecules Bartlett generates Learning, the 4-methylidene-imidazole-5-one LLC (MIO) cofactor© that Jones binds the & substrate, Bartlett phenylalanine, Learning, and facilitates LLC NOT FORremoval SALE of the OR amino DISTRIBUTION group to generate NH3 (shown in Figure 8.10). NOT FOR SALE OR DISTRIBUTION

8.3 Phenylpropanoid Pathway 125

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. B: H O © Jones & Bartlett Learning, LLC © Jones & BartlettBH Learning, LLC NOT FOR SALE OR DISTRIBUTIONH H O H H NOTO FOR SALE ORH DISTRIBUTIONO H OH OH OH + + H N H H BH 2 H2N H H N 2 NH2

CH2 H2C © Jones & Bartlett Learning, LLC H2C © Jones & BartlettH2C Learning, LLC − − − O O O O NOTN FOR SALE OR DISTRIBUTIONN N NOT FOR SALEN OR DISTRIBUTION N N N N H H H H HN Ala204 HN Ala204 HN Ala HN © Jones & Bartlett Learning, LLC Ala© 204Jones & Bartlett Learning,204 LLC Gly Gly202 Gly NOT FOR SALE OR 202DISTRIBUTION NOT FORGly SALE202 OR DISTRIBUTION202 Step 1 Step 2 Step 3 Step 4 FIGURE 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 MIO cofactor (Figure 8.9B) in PAL binds to the amino group of phenylalanine. Step 2: A basic group in the enzyme, B, stereospeciﬁ cally removes one of © Jones & Bartlett Learning,the protons from LLC the ␤-carbon of phenylalanine. Step© 3:Jones Rearrangement & Bartlett of the resulting Learning, anion generates LLC a double bond in the substrate and facilitates cleavage of the bond between the ␣-carbon of phenylalanine and its amino group. Step 4: Trans-cinnamic NOT FOR SALE OR DISTRIBUTIONacid is released from the enzyme. The protonated BHNOT residue FOR in the SALE active site OR donates DISTRIBUTION its H to the aminated cofactor and facilitates removal of the NH3 and regeneration of MIO. (Mechanism adapted from: Calabrese, J. et al., “Crystal Structure of Phenylalanine Ammonia Lyase: Multiple Helix Dipoles Implicated in Catalysis,” Biochemistry 43: [2004] 11403–416.)

(FIGURE© 8.10 Jones). A proton & Bartlett is removed Learning, from thisLLC The monooxygenases© Jones in this & Bartlettpathway Learning,are LLC ␤-carbonNOT with FOR the SALE help of OR a basicDISTRIBUTION group in often referred to as NOThydroxylases. FOR SALE The hydroxyl OR DISTRIBUTION the active site, most likely a histidine residue. (OH) group is added at the para-position with Breaking of the very stable C–H bond in this respect to the side chain to produce 4-coumaric reaction is facilitated by the electrophilic envi- acid (p-coumaric acid). Coumaric acid can also ronment around the active site created by the be produced in some plants by direct deami- © Jonesaligned & Bartlett helix dipoles Learning, of the enzyme.LLC Removal of nation© Jones of tyrosine & Bartlett in a TAL-catalyzed Learning, reaction LLC NOT FORthe SALE proton ORgenerates DISTRIBUTION an anion that rearranges (FIGURENOT 8.11 FOR). SALE OR DISTRIBUTION and results in the cleavage of the C–N bond Coumaric acid can be ligated to coenzyme between phenylalanine and the MIO cofactor. A by a ligase; this reaction requires two equiva- The product, trans-cinnamic acid, is released lents of ATP to activate coumaric acid and from the active site. The cofactor is regener- catalyze the formation of a thioester bond. © Jones & Bartlett Learning,ated by H LLC donation from an acidic residue© Jones in & Coumaroyl-CoABartlett Learning, is a major LLC branch point in the NOT FOR SALE OR DISTRIBUTIONthe enzyme and ammonia is produced.NOT Trans FOR- SALEsynthesis OR of DISTRIBUTIONmonolignols and other phenylpro- cinnamic acid is the precursor for most of the panoids. There is experimental evidence that all phenylpropanoids, including the monolignols, the monolignols are present as CoA 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, LLC or other derivatives© as Jones substrates. & Bartlett In addition, Learning, LLC LigninNOT is aFOR highly SALE irregular OR polymer DISTRIBUTION formed recent analyses haveNOT also FOR shown SALE that monoli- OR DISTRIBUTION from aromatic alcohols referred to as monolignols are present as complexes with shikimic or gnols. The major monolignols are coumaroyl, quinic acids, which are intermediates in the shi- coniferyl, and sinapyl alcohols. These precur- kimic acid pathway (FIGURE 8.12). Because these acids are synthesized in plastids, it is assumed © Jonessors & Bartlett are synthesized Learning, from LLCtrans-cinnamic acid © Jones & Bartlett Learning, LLC by enzymes associated with the endoplasmic that they must be specifically transported to the NOT FORreticulum. SALE ORThe DISTRIBUTION monolignols are exported to cytoplasmNOT FOR to serve SALE as monolignol OR DISTRIBUTION carriers. the cell wall, and polymerization of the mono- Para-coumaroyl-CoA, or other conjugate lignols then occurs outside the cell membrane. or the free acid, undergoes reduction to coumaroyl alcohol by two reductases that use Synthesis of p-Coumaroyl Alcohol The aromatic NADPH as a cofactor. The mechanism of action © Jones & Bartlett Learning,ring of trans LLC-cinnamic acid can be hydroxylated© Jones & ofBartlett these two Learning, enzymes is LLC slightly different. The NOT FOR SALE OR DISTRIBUTIONby a specific cytochrome P450 monooxygenase.NOT FOR SALEfirst enzyme, OR DISTRIBUTION coumaroyl-CoA reductase (CCR),

126 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. H O H O FIGURE 8.11 Synthesis of p-coumaric © Jones & Bartlett Learning, LLC © Jones & Bartlettacid. A hydroxyl Learning, (OH) group is LLC added to – O – trans-cinnamic acid by a cytochrome NOT FOR SALE OR DISTRIBUTIONO 2 NOTO FOR SALE OR DISTRIBUTION H H P450 monooxygenase (hydroxylase). NADPH The enzyme adds the OH-group at the HO + H2O para position with respect to the side NADP trans para chain. Coumaric acid can also be -Cinnamic acid -Coumaric acid synthesized by direct deamination of tyrosine catalyzed by tyrosine ammonia © Jones & Bartlett Learning, LLC lyase (TAL) in some plants.© Jones & Bartlett Learning, LLC NOT FOR SALETAL OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION NH3 O

O– + © Jones & Bartlett Learning, LLCNH3 © Jones & Bartlett Learning, LLC NOT FOR SALE ORHO DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Tyrosine

H O

S-CoA © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning,H LLC 2 ADP + 2Pi NOT FOR SALE OR DISTRIBUTION NOT FORHO SALE OR DISTRIBUTION p-Coumaroyl-CoA 2 ATP H O ligase H O OH OH © Jones & Bartlett Learning,O– LLC O© Jones & Bartlett Learning, LLC H H NOT FOR SALE OR DISTRIBUTIONtransferase NOT FOR SALE OR DISTRIBUTION HO HO transferase –O O p-Coumaric acid p-Coumaroyl-shikimic acid © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC H O OH NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION OH O H FIGURE 8.12 Para-coumaric acid derivatives active in monolignol synthesis. HO OH Coumaric acid generally occurs in vivo as a CoA-thioester. In some plants, –O O © Jones &conjugates Bartlett with shikimicLearning, acid and LLCquinic acid have also been found, and© theseJones & Bartlett Learning, LLC NOT FORmay SALE function OR as alternate DISTRIBUTION precursors in monolignol synthesis. NOT FOR SALE ORp -Coumaroyl-quinicDISTRIBUTION acid

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. HB hydride from NADPH© Jones (FIGURE &8.13 Bartlett). The sec- Learning, LLC © Jones & Bartlett Learning, LLC ond reductase, coumaroylNOT aldehydeFOR SALE dehydro- OR DISTRIBUTIONSynthesis of Coniferyl and Sinapyl AlcoholsNOT FORCou- SALE OR DISTRIBUTION genase (CAD), is a type A reductase. It transfers maric acid, either as the free acid or a derivative, the HA 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 OH group in the aromatic other.© This Jones has been & Bartlett demonstrated Learning, in genetically LLC ring is then methylated© Jones by& Bartlettan O-methyl Learning, LLC alteredNOT plants FOR designed SALE toOR generate DISTRIBUTION different transferase that requiresNOT FOR S-adenosyl SALE ORmethio- DISTRIBUTION pools of monolignols. nine (SAM) as the methyl donor. Monohy- Coumaroyl alcohol is one of the monolignol droxy substituted rings are usually not good precursors. It can be polymerized into a com- substrates for the methyl transferases, but plex referred to as p-hydroxyphenyl lignin, or they can bind a number of different hydrox- © Jones &H-lignin. Bartlett H-lignin Learning, is common LLC in some mono- ylated© Jones substrates. & Bartlett The methylated Learning, caffeic LLC acid NOT FORcots, SALE but ORin most DISTRIBUTION plants it is often a minor isNOT ferulic FOR acid SALE(FIGURE 8.14 OR). DISTRIBUTIONFerulic acid is then

8.3 Phenylpropanoid Pathway 127

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. NADPH NADP NADPH NADP © Jones & Bartlett Learning, LLC HA © JonesHA & Bartlett Learning,HA LLC H H B NOT FOR SALE OR DISTRIBUTION B O NOT FORO SALE OR DISTRIBUTIONHB O O C NH C NH C C 2 2 NH2 NH2 + + N N N N R R R R © JonesH &O Bartlett Learning, LLC © Jones & Bartlett Learning, LLC H O H OH NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR HDISTRIBUTION S-CoA A HB H H H H B HO CCR CAD HO HO © JonesFIGURE & Bartlett 8.13 Reductases Learning, that reduce LLC p-coumaroyl-CoA and other monolignol© Jones precursors. & Bartlett The ﬁ rst enzyme, Learning, p-coumaroyl-CoA LLC reductase (CCR, also called cinnamate-CoA reductase), transfers the HB hydride from NADPH and reduces the thioester to the NOT FORaldehyde. SALE It is ORreferred DISTRIBUTION to as a type B reductase. The second enzyme, coumaroylNOT FOR aldehyde SALE dehydrogenase OR DISTRIBUTION (CAD), transfers the HA hydride from the cofactor to produce coumaroyl alcohol; it is a type A reductase. Both CCR and CAD can also catalyze reduction of other monolignol precursors; NADPH is the reducing agent in both reactions.

O O © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONS-CoA NADP NOT FOR SALES-CoA OR DISTRIBUTION NADPH HO O HO 2 + H2O p-Coumaroyl-CoA OH Caffeoyl-CoA H©3C Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC H2 A NOTS CFORO SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION H

S-Adenosyl methionine HO OH O-Methyl transferase (SAM) NH O 2 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O− NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION O

H2 S C O A S-CoA

HO © Jones & Bartlett Learning, LLCHO OH © Jones & Bartlett Learning, LLC NOT FORS-Adenosyl SALE homocysteine OR DISTRIBUTIONNH2 NOTO FOR SALE OR DISTRIBUTION (SAH) O CH3

O− Feruloyl-CoA FIGURE 8.14 Synthesis of coniferyl alcohol. NADPH CoASH © Jones & Bartlett Learning, LLC Coumaroyl-CoA© Jones is hydroxylated & Bartlett by a speciﬁ Learning, c LLC CCR cytochrome P450 monooxygenase at the meta NOT FOR SALE OR DISTRIBUTIONNADP position (withNOT respect FOR to the SALE side chain) OR to DISTRIBUTION Coniferyl aldehyde generate caffeic acid or a caffeoyl conjugate. A caffeoyl O-methyl transferase then adds a methyl group to this meta-hydroxyl group to NADPH CAD produce ferulic acid. The methyl donor is NADP S-adenosyl methionine (SAM). Removal of the © Jones & Bartlett Learning, LLC © Jonesmethyl & Bartlettgroup from SAM Learning, yields S-adenosyl LLC NOT FOR SALE OR DISTRIBUTION NOT FORhomo SALEcysteine (SAH), OR whichDISTRIBUTION can be remethyl- OH ated. Ferulic acid and its conjugates are reduced by the p-coumaroyl-CoA reductase (CCR) and coumaroyl aldehyde dehydroge- HO nase (CAD) reductases described in Figure 8.13 Guaiacyl lignin O to yield coniferyl alcohol. This product is a lignin © Jones & Bartlett Learning,(G-lignin) LLC © JonesCH & Bartlett Learning,precursor that can LLC be polymerized to guaiacyl 3 or G-lignin, which is the major lignin type NOT FOR SALE OR DISTRIBUTION ConiferylNOT FOR alcohol SALE ORfound DISTRIBUTION in gymnosperms, especially conifers.

128 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. reduced to the alcohol by the same redox Polymerization of Monolignols © Jones & Bartlett Learning, LLC © Jones enzymes,& Bartlett CCR Learning, and CAD, LLCdescribed in Figure The monolignol precursors, coumaroyl, NOT FOR8.13 SALE to yield OR DISTRIBUTIONconiferyl alcohol. This alcohol coniferyl,NOT FOR and SALEsinapyl alcohols,OR DISTRIBUTION 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 aro- © Jones & Bartlett Learning,found LLC as glycosylated derivatives,© presumably Jones & Bartlett Learning, LLC matic ring to produce 5-hydroxy ferulic acid. for storage until secondary wall synthesis is This third hydroxyl NOTgroup FOR is methylated SALE OR by DISTRIBUTIONinduced. Polymerization of the NOTmonolignols FOR SALE OR DISTRIBUTION a SAM-dependent transferase, as shown in occurs outside the cell membrane, between it FIGURE 8.15, to produce sinapic acid. Sinapic acid is and the primary cell wall. The polymerization then reduced to sinapyl alcohol, a major mono- process is nonenzymatic but is initiated by an lignol made in dicotyledonous angiosperms and © Jones & Bartlett Learning, LLC enzymatic free radical© Jones formation. & Bartlett Two types Learning, of LLC the precursor of syringyl lignin (S-lignin). oxidases, peroxidases and laccases, are found NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

O O HO S-CoA NADP S-CoA © Jones & Bartlett Learning, LLC NADPH © Jones & Bartlett Learning, LLC HO NOT FOR SALE OR DISTRIBUTION O2 NOTHO FOR SALE OR +DISTRIBUTIONH2O O O CH 3 CH3 Feruloyl-CoA 5-Hydroxyl feruloyl-CoA

O H3C O S-CoA © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION HO NOT FOR SALE OR DISTRIBUTION O CH3 Sinapoyl-CoA

Sinapyl aldehyde

NADPH © Jones & Bartlett Learning, LLC CAD © Jones & Bartlett Learning, LLC NADP NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

H3C

O OH © Jones & BartlettSyringyl Learning, lignin LLC © Jones & Bartlett Learning, LLC (S-lignin) HO NOT FOR SALE OR DISTRIBUTION O NOT FOR SALE OR DISTRIBUTION

CH3 Sinapyl alcohol FIGURE 8.15 Synthesis of sinapyl alcohol. Ferulic acid, or a derivative, is hydroxylated by a cytochrome P450 monooxygenase to © Jones &5-hydroxy Bartlett ferulic Learning,acid. An O-methyl LLC transferase adds a methyl group to this© Jonesthird hydroxyl & groupBartlett to produce Learning, sinapic acid, whichLLC is reduced to the alcohol by p-coumaroyl-CoA reductase (CCR) and coumaroyl aldehyde dehydrogenase (CAD). Sinapyl alcohol is a NOT FORprimary SALE monolignol OR DISTRIBUTIONthat is polymerized into syringyl or S-lignin, a major componentNOT FOR of secondary SALE cell OR walls DISTRIBUTIONin dicotyledonous plants.

8.3 Phenylpropanoid Pathway 129

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. in the cell wall that can act as free radical Unlike the carbohydrates of the primary cell © Jones & Bartlett Learning,initiators. PeroxidasesLLC are heme-Fe containing© Jones & wall,Bartlett lignin Learning, is a relatively LLC inflexible polymer. It NOT FOR SALE OR DISTRIBUTIONenzymes that use a variety of substratesNOT and FOR SALEmakes cellOR walls DISTRIBUTION 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 2AH H O → 2A• 2H O 2 2 2 the vessel structure under negative water pres- Plants contain a number of isoforms of these sure. Lignin also provides a barrier to pathogen enzymes;© Jones the peroxidases & Bartlett in the Learning, cell wall are des-LLC invasion. The lignin© structureJones &with Bartlett its random Learning, LLC ignatedNOT class FORIII peroxidases. SALE OR They DISTRIBUTION can generate chemical bonds is highlyNOT resistantFOR SALE to both OR enzy- DISTRIBUTION reactive oxygen species (ROS, see Appendix 5) matic and chemical degradation. Only a few associated with the wound response. Peroxi- organisms, mostly fungi, can degrade lignin; they dases are also capable of generating monolignol use a free radical mechanism to generate random radicals. The source of the hydrogen peroxide breaks in the polymer, essentially reversing the © Jonesin & the Bartlett cell wall Learning, is unknown LLC but could presum- polymerization© Jones & process. Bartlett Learning, LLC NOT FORably SALE be generated OR DISTRIBUTION by the peroxidase itself from NOT FOR SALE OR DISTRIBUTION molecular oxygen. Genetic Engineering of Lignin The second type of radical initiator, the lac- The properties of lignin in plants depend cases, are Cu-containing proteins belonging on the concentration of monolignols exported to the phenol oxidase enzyme family. They to the wall, which determines the different © Jones & Bartlett Learning,generate radicals LLC from molecular oxygen:© Jones & types,Bartlett H, G, Learning, or S. Gymnosperm LLC wood is mainly NOT FOR SALE OR DISTRIBUTION NOT FOR SALEG-lignin; OR the DISTRIBUTION monolignol pool contains mostly 4AH O → 4A• 2H O 2 2 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 aro- of lignin with mainly C–C linkages that are matic ring and side chain and subsequently more stable than the ether linkages that result couple© with Jones another & Bartlett radical to Learning, form a bond LLC in from a higher concentration© Jones of & sinapyl Bartlett alcohol Learning, LLC a numberNOT of FOR different SALE linkages. OR DISTRIBUTION Some of the precursors (FIGURE NOT8.17). FORPlants SALE containing OR DISTRIBUTIONa possible linkages between monolignols are high content of G-lignin are more difficult for shown in FIGURE 8.16. 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 chemi- matrix of the primary cell wall (see Chapter 4). cal treatment in the pulping process to make © Jones & Bartlett Learning, LLC paper© Jones and presents & Bartlett a barrier Learning, to the production LLC NOT FOR SALE OR DISTRIBUTION of NOTethanol-based FOR SALE biofuels. OR DISTRIBUTION Many research groups have attempted to R genetically alter the composition of lignin with R the goal of producing a product that is easier R to degrade, either enzymatically or chemically. R R © Jones & Bartlett Learning, LLC © Jones & ExamplesBartlett ofLearning, these manipulations LLC include a gene R NOT FOR SALER OR DISTRIBUTION NOT FOR SALEknock-out OR or DISTRIBUTION down-regulation of the enzyme R O R that methylates caffeoyl-CoA (CCoAOMT). This R should result in a pool of monolignols with a R higher concentration of coumaroyl alcohol (use R FIGURE 8.18 as a guide). The engineered lignin 5-5 Linkage© Jones & Bartlett4-O-5 Learning, Linkage LLC that results from this© manipulationJones & Bartlett should have Learning, LLC NOT FOR SALE OR DISTRIBUTION less of both the G-NOT and FORS-lignin SALE components OR DISTRIBUTION

R R R HO FIGURE 8.16 Some common linkages found in lignin. The types ©HO Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O HO of linkages found in lignin depend on the pool of monolignols that NOT FOR SALE OR DISTRIBUTION are synthesizedNOT andFOR exported SALE from ORthe cell. DISTRIBUTION In conifers, coniferyl alcohol is the most abundant precursor, and the resulting G-lignin R R R O contains mainly C–C bonds such as the 5-5 linkage shown. Dicotyledonous angiosperms produce mainly sinapyl alcohol precursors and the resulting S-lignin contains more ether linkages R R R such as the 4-O-5 and ␤-O-4 linkages. Because most plants © Jones & BartlettR Learning, LLC R © Jonesproduce & Bartlett some of each Learning, type of monolignol, LLC all the above linkages and many mixed linkages such as the ␤-5 conﬁ guration with both NOT FOR SALE ORβ-O-4 DISTRIBUTION Linkage β-5 Linkage NOT FORC–C SALE and C–O bondsOR DISTRIBUTIONare possible.

130 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. H COH H COH 2 © Jones & Bartlett Learning, LLC 2 © Jones & Bartlett Learning,CH LLC CH 2 CH NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION2 OH CH H COH OMe 2 H COH HO CH CH 2 3 CH HC (O δ CH MeO MeO O CH © Jones & BartlettMeO Learning,HC LLC (CARBOHYDRATE) © Jones & Bartlett Learning, LLC HCOH OH OHC CH CH OH NOT HCFOR SALEO OR DISTRIBUTION NOT FOR SALEH COH OR DISTRIBUTION 2 OMe 2 OMe H COH H COH O 2 2 HC O OH MeO MeO OMe MeO HC O CH O CH © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O HC CH H COH NOTHC FORO SALE OR DISTRIBUTION 2 NOT FOR SALE OR2 DISTRIBUTION OMe HC CH H COH HC O HC MeO 2 H C CH O CH HCOH HOCH OH 2 H COH 2 O 2 OMe HOCH HC O CHO © Jones & Bartlett Learning,HOCH LLC © Jones & Bartlett Learning, LLC HC H COH CHO MeO 2 OMe NOT FOR SALE OR DISTRIBUTION O NOT FORCH SALE OR DISTRIBUTIONO H C CH MeO CH 2 O O CH HC O CH OHC CH CH OH MeO HOCH 2 2 HOCH O H COH MeO O CH 2 HC © Jones & Bartlett Learning, LLC OM MeO HOCH© Jones & Bartlett Learning,OMe LLC e 2 CO NOT FOR SALE OR DISTRIBUTIONOH NOT FOR SALEHC ORO DISTRIBUTION O CH H COH CO MeO MeO HOCH 2 2 HC O CH HC CH 2 OMe HO CH HC HC CH O O 2 H COH © Jones & Bartlett Learning, LLC © Jones2 & BartlettHCOH Learning, LLC HCOH NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION HCOH H C (O 4 MeO 2 OH OMe OH FIGURE 8.17 Model of linkages found in conifer lignin. These are some of the major linkages that can be found in G-lignin © Jones &formed Bartlett predominantly Learning, from coniferyl LLC alcohol. Both C–C and C–O bonds© are Jones formed, but& theBartlett C–C linkages Learning, are more common LLC in NOT FORthis SALE type of secondaryOR DISTRIBUTION cell wall polymer. (Reproduced from: M. Baucher,NOT B. Monties, FOR M. SALE Van Montagu, OR etDISTRIBUTION al. Crit. Rev. in Plant Sci. 17 [1998]: 125–197, Figure 7. Courtesy of Marc Van Montagu, Ghent University.)

and therefore fewer recalcitrant C–C 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© Jonesa higher & Bartlettcontent ofLearning, compensated LLC with a higher content© of Jones hydroxy- & Bartlett Learning, LLC 5-hydroxyguaiacyl unitsNOT (i.e., FOR more SALE coumaroyl OR DISTRIBUTIONferulic alcohol. The mutant maizeNOT leaves FOR and SALE OR DISTRIBUTION 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 O-methyl trans- they are more susceptible to lodging (i.e., stems ferases can adequately substitute for CCoAOMT break easily in the wind). in these© Jones plants, and & Bartlett the resulting Learning, modified ligninLLC In the model© plant, Jones Arabidopsis & Bartlett, mutants Learning, LLC still containsNOT FOR a high SALE proportion OR DISTRIBUTION of linkages that with knock-out NOTmutations FOR inSALE the geneOR DISTRIBUTIONfor are resistant to degradation. ferulic acid monooxygenase (F5H) produce Naturally occurring mutations in the mono- no S-lignin. Conversely, overexpression of the lignol enzyme genes were found in maize (Zea gene for this monooxygenase leads to pro- mays) and were designated bm1,2,3,4 (brown duction of cell walls that are predominantly © Jones &mid-rib Bartlett). The Learning, bm3 mutant LLC was found to have S-lignin,© Jones suggesting & Bartlett that Learning,manipulation LLC of the NOT FORreduced SALE O-methyl OR DISTRIBUTION transferase (COMT) activity expressionNOT FOR of theSALE ferulate-5-hydroxylase OR DISTRIBUTION gene

8.3 Phenylpropanoid Pathway 131

Phenylalanine PAL © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONtrans-Cinnamic acid NOT FOR SALE OR DISTRIBUTION

C4H

p-Coumaric acid © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 4CL NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION H O

S-CoA H p © Jones & Bartlett Learning, LLC HO © Jones & Bartlett-Coumaroyl-CoA Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION C3H

Caffeoyl-CoA © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC CCoAOMT NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION CCR F5H Feruloyl-CoA 5-Hydroxy feruloyl-CoA

COMT CCR © Jones & Bartlettp-Coumaroyl Learning, aldehyde LLC © Jones & Bartlett Learning, LLC Coniferyl aldehyde Sinapoyl-CoA NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION CAD CCR CAD Sinapyl aldehyde

CAD © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC H NOT FOR SALE OR DISTRIBUTION NOTH FOR SALEH OR3C DISTRIBUTION H O OH OH OH H H H HO HO HO O O © Jones & Bartlett Learning, LLC CH© Jones & Bartlett Learning, LLC CH3 3 CoumaroylNOT FOR alcohol SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Coniferyl alcohol Sinapyl alcohol

Cell membrane

Cell wall © Jones & Bartlett Learning, LLCPeroxidases, laccases © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOTLignin FOR SALE OR DISTRIBUTION FIGURE 8.18 Simpliﬁ ed 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. CAD, coumaroyl alcohol dehydrogenase; CCoAOMT, caffeoyl-CoA-O-methyl © Jones & Bartlett Learning,transferase; C3H, LLC p-coumarate-3-hydroxylase (monooxygenase);© Jones C4H,& Bartlett coumaroyl-4-hydroxylase; Learning, CCR, LLC coumaroyl-CoA reductase; 4CL, 4-coumarate CoA ligase; COMT, caffeic acid/5-hydroxy ferulic acid O-methyltransferase; F5H, ferulate-5-hydroxylase; PAL, NOT FOR SALE OR DISTRIBUTIONphenylalanine ammonia lyase. NOT FOR SALE OR DISTRIBUTION

132 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. may be a key step in increasing the digestibility CH3 © Jones of& forageBartlett crops. Learning, LLC © Jones & Bartlett Learning,O LLC NOT FOR SALEThe results OR DISTRIBUTIONin any one plant system, how- NOT FOR SALE OR DISTRIBUTIONOH ever, may not be applicable to other species. O Down-regulation of the gene for 4-coumarate CoA ligase (4CL) in tobacco produced plants 88 with less overall lignin in the cell walls and a ’ polymer with a higher© Jonescontent &of Bartlettcoumaroyl Learning, LLC © Jones & Bartlett Learning, LLC alcohol. Conversely, inNOT Arabidopsis FOR SALE the total OR lig- DISTRIBUTION O NOT FOR SALE OR DISTRIBUTION nin content in 4-coumarate CoA ligase mutant HO O plants was reduced but the walls still contained Pinoresinol H3C high levels of S-lignin. This suggests that 8-8’ linkage alternate pathways for monolignol synthesis are present© Jones in some & Bartlett species andLearning, can compen- LLC © Jones & Bartlett Learning, LLC sate NOTfor alterations FOR SALE in the OR main DISTRIBUTION sequence of NOT FOR SALE OR DISTRIBUTION 85’ monolignol production, but these alternatives may be species specific. The striking results O in poplar (Populus tremuloides) illustrate the H3C O O CH3 flexibility of plants in producing lignins. HO © Jones &Down-regulation Bartlett Learning, of the LLCaldehyde reductase © Jones & BartlettLicarin Learning,A LLC NOT FOR(CAD) SALE synthesis OR DISTRIBUTION results in plants with a higher NOT FOR SALE8-5’ linkageOR DISTRIBUTION content of aldehydes in their cell walls. Alde- hyde incorporation produces a pronounced CH3 O red coloration in the vascular tissues of the O woody stems. Although genomics and genetic 8 © Jones & Bartlett Learning, LLC 4’ © Jones & Bartlett Learning, LLC engineering techniques have greatly increased H3C our knowledge of ligninNOT synthesis, FOR SALE application OR DISTRIBUTIONO NOT FOR SALE OR DISTRIBUTION OH of these techniques to manipulate the quan- tity and properties of lignin is still problematic O Virolin because of our overall lack of understand- H3C 8-O-4’ linkage ing of how cell walls are made. Methods for FIGURE 8.19 Structures of some common lignans. Lignans analyzing© Jones lignin & and Bartlett linkages, Learning, such as nuclear LLC are dimers of monolignols© withJones precise & linkages Bartlett formed Learning, LLC magneticNOT resonance FOR SALE (NMR) OR spectroscopy, DISTRIBUTION are a between the precursors.NOT They areFOR synthesized SALE by freeOR DISTRIBUTION radical–initiated bond formation guided by dirigent proteins. valuable asset to lignin engineers and are cur- The dilignol bonds in these compounds correspond to rently used to provide a more directed path to linkages found in lignin (see Figure 8.16). lignin manipulation. © Jones &8.4 Bartlett Natural Learning, Products LLC Derived from © Jones & Bartlett Learning, LLC NOT FOR SALEthe OR Phenylpropanoid DISTRIBUTION Pathway (FIGURENOT 8.20 FOR). As SALE in the ORcase DISTRIBUTIONof lignin synthesis, the free radical initiator is a peroxidase. Natural 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© Jonesare found & Bartlettin the seed Learning, been proposed LLC that these extracellular© Jones dirigent & Bartlett Learning, LLC coats, flowers, and epidermalNOT FOR layers SALE of stems OR DISTRIBUTIONproteins may be active in lignin formation.NOT FOR The SALE OR DISTRIBUTION and leaves where they serve to defend plant binding of monolignols to dirigents with differ- tissues against pathogens. They are also precur- ent sites could account for the regular bond- sors 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 LLC are proteins and their© genesJones have & Bartlettbeen predicted Learning, LLC specificNOT rather FOR than SALE random OR asDISTRIBUTION in lignin. The from genomic analyses,NOT FOR an even SALE larger OR num- DISTRIBUTION structures of some common lignans are shown ber would presumably be required to guide in FIGURE 8.19. 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 ini- (guide proteins). These proteins have no enzy- tial products of polymerization to which addi- © Jones &matic Bartlett activity; Learning, they serve LLC to bind the lignols tional© Jones monolignols & Bartlett are then Learning, added in a random,LLC NOT FORand SALE guide OR the radical-mediatedDISTRIBUTION bond formation radical-coupledNOT FOR SALE process. OR DISTRIBUTION

8.4 Natural Products Derived from the Phenylpropanoid Pathway 133

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. H © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC OH NOT FOR SALE OR DISTRIBUTION 2 NOT FOR SALE OR DISTRIBUTION H HO O Coniferyl alcohol CH3 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC peroxidase NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION Dirigent protein

H H 8 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O O NOT FOR SALE OR DISTRIBUTION OH NOT FOR SALE OR DISTRIBUTIONOH H H O O H H3C 3C radical coupling H3C H3C O H O H © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC

NOT FOR SALE OR DISTRIBUTIONO 8 NOT FOR SALE ORO DISTRIBUTION OH OH H H

Dirigent protein © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION cyclization CH3 O OH

O © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC

NOT FOR SALE OR DISTRIBUTION NOT8’8 FOR SALE OR DISTRIBUTION

HO © Jones & Bartlett Learning, LLC © JonesO & Bartlett(+) Pinoresinol Learning, LLC NOT FOR SALE OR DISTRIBUTION NOTH3 CFOR SALE OR DISTRIBUTION FIGURE 8.20 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 Oleaceae (olive family). © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC In someNOT plants, FOR lignansSALE areOR modified DISTRIBUTION to pro- of the spindle apparatus.NOT FOR It is, SALE therefore, OR DISTRIBUTIONa duce species-specific natural products. Some of potential anticancer agent but is too toxic to 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, eto- The structures of three examples are shown in poside, is less toxic and can be taken internally © JonesFIGURE & Bartlett 8.21. Sesamin Learning, is an LLCantioxidant found to ©treat Jones certain & Bartletttypes of Learning,lung and testicular LLC NOT FORin SALEthe seed OR oil DISTRIBUTIONof the sesame plant (Sesamum cancers.NOT EtoposideFOR SALE acts ORby inhibiting DISTRIBUTION topoisom- indicum). Gomisin A, from Schisandra chinensis, erase activity and DNA synthesis rather than 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 com- © Jones & Bartlett Learning,(Podophyllum LLC peltatum) inhibits cell division© Jones by & poundsBartlett from Learning, monolignols. LLC Volatiles in flowers NOT FOR SALE OR DISTRIBUTIONbinding to tubulin and preventing the formaNOTtion FOR SALEand fruits OR are DISTRIBUTION attractive to pollinators and her-

134 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. O FIGURE 8.21 Structures of modiﬁ ed lignans O © Jones & Bartlett Learning, LLCO © Jonesthat & Bartlettare useful to Learning,humans. Sesamin LLC is an antioxidant found in sesame seed oil that is O NOT FOR SALE OR DISTRIBUTION NOT FORmarketed SALE as aOR neutraceutical. DISTRIBUTION Gomisin-A has O an unusual eight-membered ring. It is the H C 3 8 active ingredient in some traditional herbal O 8' H C 2 medicines used to treat liver disorders. 3 2' 8' 8 O Podophyllotoxin inhibits tubulin polymeriza- OH H C tion and prevents cell division. Although O © Jones & 3Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O podophyllotoxin is too toxic for therapeutic NOT FOR SALE OR DISTRIBUTIONuse, it is the natural model forNOT etoposide, FOR a SALE OR DISTRIBUTION synthetic anticancer drug. O H3C O O

Sesamin Gomisin © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION O H3C O O OH O O 8 O 8 © Jones & Bartlett Learning,O LLC © Jones & Bartlett Learning, LLC O NOT FOR SALEO OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION 8' O O 8' HO

H3C O O H3C O © JonesCH3 & Bartlett Learning, LLCO © Jones & Bartlett Learning, LLC O CH3 NOT FOR SALE OR DISTRIBUTIONHO NOT FOR SALE OR DISTRIBUTION H3C

Anethole Podophyllotoxin Etoposide O H3C © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC+ bivores, while volatile compounds in leaves and CH2 stemsNOT repel FOR insects SALE or attract OR insectDISTRIBUTION predators. NOT FOR SALE OR DISTRIBUTION A common pathway for the synthesis of some OH HO Propenyl cation volatiles is shown in FIGURE 8.22. The alcohol O group of monolignols such as coniferyl alcohol HO H3C can be removed by a reductase to generate a © Jones & Bartlett Learning, LLC © JonesO & Bartlett Learning, LLC + resonance-stabilized carbocation intermediate. H3C CH NOT FORReduction SALE OR of theDISTRIBUTION allylic cation yields eugenol, NOTConiferyl FOR alcoholSALE OR DISTRIBUTION the aromatic compound found in cloves (Syz- Allyl cation gium aromaticum), which are used by humans HO as both a flavoring agent and an analgesic (pain O H C reliever). Similarly, reduction of the correspond- 3 NADPH © Jones & Bartlett Learning, LLC © Jones & BartlettNADP Learning, LLC ing propenyl cation and dehydration produces CH anethole, a volatile foundNOT in FOR many SALE plants ORsuch DISTRIBUTION NOT FOR SALE OR2 DISTRIBUTION

HO FIGURE 8.22 Examples of volatile natural products produced O from monolignols. Removal of the hydroxyl group from coniferyl H3C alcohol© generates Jones two & resonance- Bartlett stabilized Learning, carbocations. LLC © Jones & Bartlett Learning,Eugenol LLC ReductionNOT of theFOR allylic SALEcation generates OR DISTRIBUTION the methylene group NOT FOR SALE OR DISTRIBUTION in eugenol, the volatile compound in cloves. Reduction of the O CH propenyl cation and subsequent dehydration generates anethole, O 2 H3C OH the volatile found in many plants such as fennel. Removal of the H3C hydroxyl group of sinapyl alcohol and reduction of the resulting HO allylic cation to a methylene group initiates the formation of O myristicin, which also requires ring closure to form the methylene O © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O dioxy ring. Myristicin is the volatile that adds to the fragrance of H3C NOT FORnutmeg SALE seeds. OR DISTRIBUTION NOT FORSinapyl SALEalcohol OR DISTRIBUTION Myristicin

8.4 Natural Products Derived from the Phenylpropanoid Pathway 135

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. O OOH 8.5 Synthesis and Properties of © Jones & Bartlett Learning,C LLC ©C Jones & Bartlett Learning, LLC – – Polyketides NOT FOR SALE OR DISTRIBUTIONO NOTO FOR SALE OR DISTRIBUTION As described in the previous section, the phen- HO + H2O HO ylpropanoid pathway furnishes monolignols O O H3C H3C that are essential for lignin synthesis and, Hydroxyferulic acid therefore, can be considered part of the pri- © Jones & Bartlett Learning, LLC mary metabolism of© plants. Jones This & sameBartlett pathway Learning, LLC reverse aldol NOT FOR SALE OR DISTRIBUTION also provides precursorsNOT FOR for manySALE different OR DISTRIBUTION O aromatic compounds that are unique to a par- CH ticular plant, such as eugenol and myristicin O (Figure 8.22), and are generally regarded as secondary metabolites or natural products. In HO + © Jones & Bartlett Learning, LLC O– addition,© Jones coumaroyl-CoA & Bartlett from Learning, the phenylpro- LLC NOT FOR SALE OR DISTRIBUTIONO NOT FOR SALE OR DISTRIBUTION H3C panoid pathway is the starting material for the synthesis of polyketides such as the flavonoids, Vanillin FIGURE 8.23 Synthesis of vanillin. Vanillin is produced by which have traditionally been considered nat- a fermentation process that alters ferulic acid derivatives ural products. found in secondary cell walls. Hydration of the side chain of Many flavonoids are unique to a particu- © Jones & Bartlett Learning,hydroxyferulic LLCacid is followed by a reverse aldol reaction© Jones to & larBartlett family Learning,of plants. Both LLC their synthesis in a generate the aldehyde, vanillin. NOT FOR SALE OR DISTRIBUTION NOT FOR SALEspecific ORplant DISTRIBUTION and molecular details have been used to construct biochemically based phylog- enies. Most land plants and many aquatic spe- as fennel (Foeniculum vulgare). If the starting cies, however, contain some class of flavonoids that function to protect the organisms against material© Jonesis sinapyl & alcohol, Bartlett a similarLearning, reduction LLC © Jones & Bartlett Learning, LLC and subsequent formation of a methylene dioxy UV radiation, free-radical damage, pathogens, ring yieldsNOT myristicin FOR SALE (Figure OR 8.22), DISTRIBUTION the volatile and herbivores or toNOT attract FOR pollinators, SALE mycor-OR DISTRIBUTION compound found in nutmeg (Myristica fragrans). rhizal fungi, and symbiotic microorganisms. 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 their environment, flavonoids and other © Jonesin & FIGURE Bartlett 8.23, the Learning, double bond LLC in hydroxyferulic © Jones & Bartlett Learning, LLC acid is hydrated followed by a reverse aldol reac- polyketides are definitely of primary impor- NOT FORtion SALE that cleaves OR DISTRIBUTION the side chain to yield vanillin. tanceNOT in plantFOR metabolism. SALE OR DISTRIBUTION In some plants an alternate route uses the CoA Synthesis of Chalcones ester of ferulic acid as a precursor. Commercial vanilla extract from the pods of the vanilla Coumaroyl-CoA is a key intermediate in the orchid (Vanilla planiflora) is produced after a long phenylpropanoid pathway, and its synthesis © Jones & Bartlett Learning,fermentation LLC period. The pods are then© Jonesdried & fromBartlett phenylalanine Learning, is described LLC in Figures 8.11 NOT FOR SALE OR DISTRIBUTIONand extracted with ethanol. Real vanillaNOT extract FOR SALEand 8.12. OR It isDISTRIBUTION a substrate for the enzyme, chal- contains vanillin and a number of additional cone synthase (CHS). CHS is found in most compounds. Imitation vanilla extracts can be plants and provides the precursor for a large produced by fermentation of the ferulic acid number of flavonoids. A cysteine thiolate anion found in some woody stems. in the enzyme’s active site attacks the carbonyl © Jones & Bartlett Learning, LLC carbon of coumaroyl-CoA,© Jones displacing & Bartlett the CoA Learning, LLC SalicylicNOT Acid FORThe plantSALE hormone, OR DISTRIBUTION salicylic acid, and binding the coumaroylNOT FOR moiety SALE (FIGURES OR 8.24 DISTRIBUTION can be synthesized by a variety of pathways. It is and 8.26). The second substrate is malonyl- derived in some plants from coumaroyl-CoA. CoA, which is produced by an acyl-CoA carbox- In bacteria and other plants, such as Arabidopsis, ylase (ACC) complex found in the cytoplasm. salicylic acid can be produced from chorismic Unlike the plastid enzyme complex, the ACC © Jonesacid, & Bartlett an intermediate Learning, in the LLC shikimic acid path- complex© Jones in the & Bartlettcytoplasm Learning,is one large LLC fusion NOT FORway SALE (Figures OR 8.1 DISTRIBUTION and 8.7). Salicylic acid is a protein.NOT ItsFOR mechanism SALE ORof action DISTRIBUTION is the same as hormone produced after pathogen invasion. It the multisubunit ACC found in chloroplasts; it is transported to undamaged plant tissues where uses bicarbonate, ATP, and acetyl-CoA to pro- it mediates systemic acquired resistance (SAR). duce the three-carbon acid, malonyl-CoA (see It is also the active ingredient in aspirin (acetyl Chapter 6). © Jones & Bartlett Learning,salicylic acid), LLC although the modern commercial© Jones & BartlettMalonyl-CoA Learning, coordinates LLC with an aspara- NOT FOR SALE OR DISTRIBUTIONproduct is made by chemical synthesis. NOT FOR SALEgine (Asn) OR DISTRIBUTIONresidue in the CHS active site,

136 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. His O © Jones & Bartlett Learning, LLC Cys © Jones & Bartlett Learning,O LLC HN CH OC O NOT FOR SALE OR DISTRIBUTION NOT FOR SALEHN ORCH DISTRIBUTIONOC CH 2 HN CH C O CH O 2 CH N 2 S – SH © JonesN & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC O NOT FOR SALE OR DISTRIBUTIONHO NOT FOR SALE OR DISTRIBUTION S-CoA

p O HO -Coumaroyl-CoA O C © Jones & Bartlett Learning, LLC CH © Jones & Bartlett Learning, LLC NOT FORAsn SALEO OR DISTRIBUTION HN NOT FOR SALE OR DISTRIBUTION O CH2 O C C O + CH N H CH2 HN C CH2 O H C S-CoA © Jones & Bartlett Learning, LLC O O © Jones & Bartlett Learning, LLC N H NOT FOR SALE OR DISTRIBUTIONC NOT FOR SALE OR DISTRIBUTION H O– S-CoA Malonyl-CoA – CoASH – CO © Jones & Bartlett Learning, LLC 2 © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION O O NOT FOR SALE OR DISTRIBUTION

S-CoA

HO © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC p NOT FOR SALE OR DISTRIBUTION -CoumaroylNOT diketide FOR SALE OR DISTRIBUTION FIGURE 8.24 Mechanism of action of chalcone synthase (CHS). All polyketide synthases have a conserved catalytic triad consisting of histidine (His), cysteine (Cys), and asparagine (Asn) residues in their active sites. The histidine imidazole-N 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-CoA and displaces the CoA. The second substrate, malonyl-CoA, binds near the asparagine residue. The Asn amide-NH2 forms an H bond with the carbonyl oxygen of malonyl-CoA and facilitates its decarboxylation. The resulting © Jones &acetyl Bartlett cation attacks Learning, the carbonyl LLCcarbon of the coumaroyl–cysteine complex© Jones and produces & Bartlett a diketide Learning,product. LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

which facilitates its decarboxylation. The (FIGURE 8.25). The overall reaction catalyzed by remaining two-carbon fragment, an acetyl- CHS is as follows: CoA cation, attacks the carbonyl carbon of Coumaroyl-CoA 3 Malonyl-CoA → the coumaroyl moiety© Jonesand displaces & Bartlett it from Learning, LLC © Jones & Bartlett Learning, LLC Chalcone 4 CoA-SH 3CO the cysteine active site.NOT The FOR product SALE is ORcou- DISTRIBUTION NOT FOR2 SALE OR DISTRIBUTION maroyl diketide (Figure 8.24). This diketide Chalcone synthases s are found in all vascu- intermediate is reattached to the cysteine lar plants and some bryophytes. They also occur thiolate in the active site of CHS and a second in fungi and certain classes of algae. Most CHSs equivalent of malonyl-CoA is coordinated have a monomer molecular weight of approxi- to the© Jonesasparagine & Bartlett residue, Learning, decarboxylated, LLC mately 40 kDa and© Jones function & Bartlettas dimers. Learning, The LLC and NOTadded FORto the SALE diketide. OR The DISTRIBUTION resulting tri- CHSs found in plantsNOT are FOR part SALEof a large OR family DISTRIBUTION ketide intermediate undergoes another round of enzymes that can catalyze the synthesis of an of binding to the active site cysteine and aromatic ring from fatty acid precursors, usually acetate addition to yield a tetraketide. This malonyl-CoA. The plant synthases are part of final ketide intermediate undergoes cycliza- a larger superfamily, the polyketide synthases. © Jones &tion Bartlett and aromatization Learning, whileLLC still bound to Polyketides© Jones are& Bartlettproduced Learning,by many organisms, LLC NOT FORCHS SALE to produce OR DISTRIBUTION the product called a chalcone includingNOT FOR bacteria SALE such OR asDISTRIBUTION Streptomyces, and

8.5 Synthesis and Properties of Polyketides 137

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. CoA-SH CO2 form the core structures of certain antibiotics, O © Jones & Bartlett Learning, LLC O O © Jones & suchBartlett as erythromycin. Learning, AlthoughLLC they catalyze NOT FOR SALE OR DISTRIBUTION NOT FOR SALEsimilar ORreactions, DISTRIBUTION the polyketide synthases of S-CoA + – C O S-CoA plants have very little amino acid sequence HO similarity to their bacterial counterparts. The plant enzymes probably evolved from fatty p-Coumaroyl-CoA Malonyl-CoA acid synthases (see Chapter 6). © Jones & Bartlett Learning, LLC The x-ray crystal© structure Jones &of Bartlettthe CHS from Learning, LLC O NOTO FOR SALE OR DISTRIBUTION alfalfa (Medicago sativaNOT) has FOR been SALEsolved to OR 1.5-Å DISTRIBUTION O O resolution (FIGURE 8.26A). The homodimer has two S- + C CoA – S-CoA structurally separate active sites. Coumaroyl- O CoA binds first in a cleft that excludes other HO phenolics due to its size and the charged residues – CoA-SH © Jones & Bartlett Learning, LLC in ©the Jones vicinity & of Bartlett the active Learning, site. As shown LLC in – CO2 NOT FOR SALE OR DISTRIBUTION FIGURENOT 8.26B FOR, the SALEactive siteOR consistsDISTRIBUTION of a cata- O OOO+ O lytic triad of histidine, cysteine, and asparagine C residues. The histidine acts as a general base and S-CoA – S-CoA O removes a proton from the cysteine sulfhydryl – CoA-SH HO group to produce an active thiolate anion that © Jones & Bartlett Learning, LLC– CO2 © Jones & bindsBartlett coumarate Learning, by displacing LLC the CoA. Malo- NOT FOR SALE OR DISTRIBUTION NOT FOR SALEnyl-CoA OR enters DISTRIBUTION the active site near the aspara- O OOO gine residue, which provides hydrogen bonds to ␥ S-CoA this substrate by way of its amide-NH2 ( -amino group). Decarboxylation of malonyl-CoA pro- – CoA-SH HO duces the cation that attacks the coumaroyl moi- © Jones & Bartlett Learning, LLC ety, producing a CoA-bound© Jones diketide. & Bartlett This cycle Learning, LLC NOT FORO SALEOH OR DISTRIBUTION is repeated two moreNOT times FOR to yield SALE a tetraketide OR DISTRIBUTION intermediate that then folds and is desaturated to produce an aromatic ring. Naringenin chalcone diffuses out of the active site, which is not large HO HO OH enough to allow further condensation to longer © Jones & BartlettChalcone Learning, LLC polyketides.© Jones The & Bartlett CHS from Learning, alfalfa has LLCa high FIGURE 8.25NOT Chalcone FOR synthesis SALE catalyzed OR DISTRIBUTION by chalcone synthase (CHS). CHS degreeNOT of FORsequence SALE similarity OR DISTRIBUTIONto other polyketide binds p-coumaroyl-CoA ﬁ rst and then adds a two-carbon fragment resulting from the synthases found in plants, including those that decarboxylation of malonyl-CoA (see Figure 8.24). Loss of CO2 drives the reaction 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. © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION (a) (b) FIGURE 8.26 X-ray crystal structure of CHS from alfalfa. (a) CHS 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 CHS 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 © Jones & Bartlettin yellow. TheLearning, ketide intermediates LLC are bound to this cysteine residue.© Jones Asparagine-336 & Bartlett in red sticks Learning, is the binding site LLC for malonyl-CoA. (Structure a from Protein Data Bank 1CHW. J.-L. Ferrer, J. Jez, M. E. Bowman, R. Dixon, and J. P. Noel, Nat. Struct. Biol. 6 [1999]: 775–784. Structure b NOT FOR SALEfrom ProteinOR DISTRIBUTIONData Bank 1CHGK. J.-L. Ferrer, J. Jez, M. E. Bowman,NOT R. Dixon, FOR and SALEJ. P. Noel, ORNat. Struct. DISTRIBUTION Biol. 6 [1999]: 775–784.)

138 CHAPTER 8 Aromatic and Phenolic Compounds

chalcone isomerase O OH

NOT FOR SALE OR DISTRIBUTION5’ NOT FOR SALE OR DISTRIBUTION OH 6’ (b) 4’ 1 B 8 O HO 3’ 7 2 1’ A C 2’ © Jones & Bartlett Learning, LLC 3 © Jones & Bartlett Learning, LLC 6 NOT FOR SALE OR DISTRIBUTION5 4 NOT FOR SALE OR DISTRIBUTION

OH O

Naringenin flavanone © Jones &FIGURE Bartlett 8.27 Synthesis Learning, of naringenin LLC fl avanone. (a) A specifi c chalcone© Jonesisomerase &(CHI) Bartlett catalyzes formation Learning, of the pyran LLC ring NOT FORin SALEthe S-confi OR guration. DISTRIBUTION (b) The letter designations and numbering systemNOT for the FOR general SALE fl avonoid ORmolecule DISTRIBUTION 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.© FurtherJones ring& Bartlett closure toLearning, possible. LLC The acyl groups are added© to Jones an OH in& Bartlett Learning, LLC produce a pyran ring inNOT naringenin FOR SALEoccurs spon- OR DISTRIBUTIONone of the rings or to the sugar moietyNOT inFOR gly- SALE OR DISTRIBUTION taneously. Plants, however, have an isomer- cosylated flavonoids. Acylation can change the 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, (FIGURE 8.27). LLC The of the B ring of ©some Jones flavonoids. & Bartlett The sulfur Learning, LLC uncatalyzedNOT FOR reaction SALE results OR in DISTRIBUTION a racemic mix- NOT FOR SALE OR DISTRIBUTION 5 ture. The product of isomerization is classified ’ OH 6 ’ 4 as a flavanone and is the key intermediate in 1 B ’ 8 O the synthesis of other classes of flavonoids. HO 3’ 7 2 1’ The major differences in these compounds are A C 2 3 ’ © Jones &changes Bartlett in the Learning, oxidation stateLLC of the pyran (C) © Jones & Bartlett6 5 Learning,4 LLC NOT FORring SALE (FIGURE OR 8.28 DISTRIBUTION). NOT FOR SALE OR DISTRIBUTION OH O Many of the modifications shown in Figure 8.28 are catalyzed by oxygenases that add hydroxyl groups or a double bond to the C ring of naringenin flavanone. These enzymes O O OH O are associated with the© outerJones face & of Bartlett the endo- Learning, LLC © JonesO & Bartlett Learning, LLC 2 2 C C CC plasmic reticulum. AdditionalNOT FOR enzymatic SALE reac-OR DISTRIBUTION 3 NOT FOR SALE OR DISTRIBUTION 4 3 tions can also modify the A and B rings to OH produce a large array of derivatives that are B OH O O O collectively referred to as flavonoids. OH Most flavonoids are glycosylated for storage or transport.© Jones The & sugarBartlett attached Learning, to the flavo-LLC Flavanol Isoflavone© Jones & BartlettFlavone Learning,Dihydroflavonol LLC noid NOTis usually FOR glucose SALE but OR can alsoDISTRIBUTION be galactose, NOT FOR SALE OR DISTRIBUTION O O arabinose, xylose, or rhamnose. These reactions FIGURE 8.28 Structures of the various are catalyzed by glycosyl transferases that use a fl avonoid classes. Flavonoids are placed C C UDP-sugar substrate and make an ether bond in different classes, fl avonols, isofl avones, OH between the sugar and an OH group in the fl avones, and dihydrofl avonols, based on OH © Jones & Bartlett Learning, LLC the© modifi Jones cations & shown Bartlett in the C ring.Learning, O LLC OH flavonoid. Additional sugars are often added to Within each class, further additions are NOT FORthe SALE first one. OR DISTRIBUTION oftenNOT made FOR to the ASALE and B rings. OR DISTRIBUTIONFlavonol Leucoanthocyanidin

8.5 Synthesis and Properties of Polyketides 139

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. donor is phosphoadenosine phosphosulfate formation of a double bond as in the fatty acid © Jones & Bartlett Learning,(PAPS). Sulfated LLC flavonoids are often© signal- Jones & desaturases.Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONing molecules used by plants such as legumesNOT FOR SALEAs ORillustrated DISTRIBUTION in FIGURE 8.29, the iron in to attract the appropriate species of symbiotic flavone synthase I is ligated to histidine and bacteria (see Chapter 5). aspartic acid residues in the active site. This Flavonoids can be prenylated, usually with Fe3 is reduced by ascorbic acid. The ferrous dimethylallyldiphosphate but occasionally form of the enzyme then binds oxogluta- with longer© Jones terpene & Bartlett precursors. Learning, This increases LLC rate and molecular© oxygen. Jones The& Bartlett oxoglutarate Learning, LLC the hydrophobicityNOT FOR SALE of the products. OR DISTRIBUTION Prenylated undergoes oxidativeNOT decarboxylation FOR SALE toOR suc- DISTRIBUTION flavonoids generally function as phytoalexins. cinate, which is released from the enzyme. This 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 flava- formation of a double bond in the substrate, © Jonesnone & Bartlett by addition Learning, of a double LLC bond to the C-ring naringenin© Jones flavanone. & Bartlett Additional Learning, reactions LLC in NOT FORbetween SALE C-2 OR and DISTRIBUTION C-3 (Figure 8.28). Two dif- flavonoidNOT FOR modification SALE areOR catalyzed DISTRIBUTION by ODDs, ferent flavone synthases (FNS) can catalyze and genomic analyses suggest that this type of this desaturation. In many angiosperms, fla- dioxygenase is more common than previously vone synthase II (FNS II) is a cytochrome thought. The Arabidopsis genome has approxi- P450 monooxygenase. In others, such as some mately 64 genes for putative ODDs, and some © Jones & Bartlett Learning,species in LLCthe Apiaceae (parsley, carrot),© Jones the & oxygenase-catalyzedBartlett Learning, reactions LLC may use this NOT FOR SALE OR DISTRIBUTIONsynthase (flavone synthase I [FNS I]) is NOTan oxo- FOR SALEtype of ORenzyme DISTRIBUTION rather than cytochrome P450 glutarate-dependent dioxygenase (ODD). The monooxygenases. ODDs are nonheme iron–containing enzymes Flavones are colorless compounds with that can bind O2 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© atomJones to &a Bartlettsubstrate Learning,or catalyze LLCthe in cells as 7-O-glycosides© Jones and & canBartlett be found Learning, LLC NOT FOR SALE OR DISTRIBUTION in all tissues of mostNOT plants, FOR exceptSALE forOR the DISTRIBUTION Brassicaceae, which do not produce flavones. ENZYME OH Flavones function to protect plant cells from Asp UV radiation and may also act as allelochemi- His His O O cals (i.e., compounds that inhibit growth of Fe3+ HO © Jones & Bartlett Learning, LLCAscorbate competing© Jones photosynthetic & Bartlett Learning, organisms). LLC They NOT FOR SALE OR DISTRIBUTIONHO OH mayNOT also FOR serve SALE to attract OR and DISTRIBUTION guide pollina- OH tors. As seen in FIGURE 8.30, colorless flavones O fluoresce under UV light, and the patterns can ENZYME O HO be detected by bees and some other potential Asp Dehydroascorbate His His pollinators. The flavones serve as a nectar guide 2 O O © Jones & Bartlett Learning,Fe + LLC © Jones & butBartlett also ensure Learning, that pollen LLC is transferred onto + O2 NOT FOR SALEO OR DISTRIBUTIONO O NOT FOR SALEthe insect. OR DISTRIBUTION – – – O O O – Synthesis and Properties of Anthocyanidins O + CO2 O O Anthocyanidins are the precursors of plant Succinate 2-Oxoglutarate pigments collectively referred to as anthocyanins. © Jones & Bartlett Learning, LLC Their colorless precursors,© Jones leuco-anthocyanidins, & Bartlett Learning, LLC ENZYMENOT FOR SALEO OR DISTRIBUTION are synthesized fromNOT dihydroflavonols, FOR SALE ORwhich DISTRIBUTION Asp 2 His His C are the products of another ODD that intro- + 3 duces an OH group at C-3 in the C ring of FeIV = O naringenin flavanone. The keto group in the O © Jones & Bartlett Learning,Flavanone LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION FIGURE 8.29 Mechanism of action of ﬂ avone synthase I (FNS I). FNS I belongs O to the 2-oxoglutarate–dependent nonheme iron family of dioxygenases (ODD). Iron 2 is liganded to histidine and aspartic acid residues in the protein. The ferric iron is ENZYME C reduced by ascorbate. The Fe2 form of the enzyme binds oxoglutarate and catalyzes Asp 3 IV His His an oxidative-decarboxylation to succinate. The resulting Fe O form of the enzyme © Jones & Bartlett Learning,Fe3+ LLC then catalyzes© Jones formation & ofBartlett a double bond Learning, in the ﬂ avanone LLC substrate between C-2 and O C-3 in the C ring. In a more generalized reaction, an ODD transfers the Fe-bound NOT FOR SALE OR DISTRIBUTION Flavone oxygen NOTto a second FOR substrate SALE to generate OR DISTRIBUTIONa hydroxylated product.

140 CHAPTER 8 Aromatic and Phenolic Compounds

(a) © Jones & Bartlett Learning, LLC (b) © Jones & Bartlett Learning, LLC FIGURENOT 8.30 ColorlessFOR SALE fl avones areOR visible DISTRIBUTION under UV light. Flower (Coreopsis leavenworthiiNOT, top; Bidens FOR mitis SALE, bottom) OR images DISTRIBUTION 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 fl owers. 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 &C Bartlettring is then Learning, reduced, LLCand a second ODD, Changes© Jones in cell & Bartlettshape influence Learning, stacking LLC of the NOT FORanthocyanidin SALE OR DISTRIBUTIONsynthase, introduces two double aromaticNOT FOR rings SALE of the ORanthocyanins DISTRIBUTION and lead bonds into the ring to produce the anthocyani- to variations in the perceived colors. The pres- din flavylium cation (FIGURE 8.31). The final steps ence of cations, mainly Mg2 and Al3, can also in anthocyanin synthesis include glycosylation influence the final color of flower pigments. A of the OH group in the C ring and further modi- particularly dramatic example are the colors of fications of the A and© B Jonesrings. & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC The unmodified NOTanthocyanidin FOR SALE flavylium OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION cation can be generated in vitro and exhibits a FIGURE 8.31 Examples of anthocyanidins. R2 reddish hue at neutral pH. The color is influ- The product of anthocyanidin synthase is OH the fl avylium cation shown at the top that is enced both by the type of solvent and pH. In further modifi ed to produce different colored O flower petals the color of the anthocyanins can HO products. The hydroxyl (OH) group at R1 R vary ©depending Jones &on Bartlett the chemical Learning, modifications, LLC in most anthocyanidins© is glycosylatedJones & to Bartlett Learning, LLC 3 includingNOT further FOR SALEglycosylation, OR DISTRIBUTION acylation, and stabilize the ring structure.NOT Hydroxyl FOR groups SALE OR DISTRIBUTIONR in the A and B rings are also often glycosyl- 1 prenylation. Anthocyanins are often stored in ated. The colors shown above can be OH 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: R = OH, R = H, R = H depend on a number of physiological 1 2 3 rise to a blue color. Changes in the vacuolar pH, Luteolinidin: R = H, R = OH, R = H © Jones & Bartlett Learning, LLC parameters© Jones such & as BartlettpH of the compartment Learning, LLC 1 2 3 such as raising the pH to 7 or higher, can shift in which the pigments are stored and Delphinidin: R1 = OH, R2 = OH, R3 = OH NOT FORthe SALE color ORtoward DISTRIBUTION the red end of the spectrum. presenceNOT ofFOR metal ionsSALE and copigments. OR DISTRIBUTIONCyanidin: R1 = OH, R2 = OH, R3 = H 8.5 Synthesis and Properties of Polyketides 141

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. hydrangea flowers, which are generally pink The attenuation of oxygen evolution in pho- © Jones & Bartlett Learning,when grown LLC in alkaline soils but can© change Jones & tosystemBartlett II Learning, by anthocyanins LLC helps prevent the NOT FOR SALE OR DISTRIBUTIONto blue after application of an acidic mixtureNOT ofFOR SALEformation OR of DISTRIBUTION oxygen free radicals. The antho- soluble aluminum salts to the soil (see photo cyanins themselves are also very effective on page 119). The presence of other flavonoids, antioxidants. They can readily dissipate reac- such as the colorless flavones, act as copig- tive oxygen species such as the hydroperoxy ments and can also modulate color. Anthocya- radical and other radical species produced in nins in© flowers Jones can & obviouslyBartlett haveLearning, a variety LLC of peroxisomes (see Appendix© Jones 5). & Bartlett Learning, LLC colors NOTand are FOR a major SALE factor OR in DISTRIBUTION the attraction AnthocyanidinsNOT can FORbe polymerized SALE OR into DISTRIBUTION of pollinators. Genetic engineering techniques oligomers called proanthocyanidins or con- have been used to try to alter flower colors for densed tannins. The precursors accumulate 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 © Jonesthis & Bartlettis not readily Learning, accomplished LLC by modifica- linear© Jones polymers & Bartlettwith a deep Learning, purple to LLCbrown NOT FORtions SALE of a few OR genes. DISTRIBUTION colorNOT characteristic FOR SALE of someOR DISTRIBUTIONseeds, fruits, and In contrast to flower petals, anthocyanins in tree bark. These compounds inhibit proteo- leaves are usually a red color and are responsi- lytic enzymes in herbivores and also slow the ble for the bright red of autumn leaves, partic- growth of bacterial and fungal pathogens. ularly in some maple species. Anthocyanins in © Jones & Bartlett Learning,photosynthetic LLC tissues are stored in vacuoles© Jones & BartlettSynthesis Learning, and Properties LLC of Isoﬂ avonoids NOT FOR SALE OR DISTRIBUTIONand protect cells against the damagingNOT effects FOR SALEIsoflavonoids OR DISTRIBUTION are synthesized from narin- of intense sunlight. Their synthesis can be genin flavanone by a cytochrome P450 mono- increased during periods of high light inten- oxygenase. Isoflavone synthase (IFS) catalyzes sity and low temperatures, such as early spring the cleavage of the C–H bond at C-3 in the C ring in the northern hemisphere. Under these con- and generates a radical intermediate. The B ring ditions,© theJones light &absorption Bartlett byLearning, anthocyanins LLC then migrates to C-3© Jonesand the &enzyme Bartlett adds Learning, an LLC reducesNOT the FORrate of SALE the light OR absorptionDISTRIBUTION and OH group to C-2 (FIGURENOT 8.32 FOR). A dehydratase SALE OR can DISTRIBUTION transfer in photosystem II. In cool conditions, remove H2O from this initial product to yield a CO2 fixation is limiting and cannot recycle specific isoflavone such as genistein. Isoflavone the cofactors required in the light reaction. synthase can also act on deoxyflavonones that lack the hydroxyl group on C-5 of ring A to © Jones & Bartlett Learning, LLC produce© Jones an additional & Bartlett class Learning, of isoflavonoids. LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION OH OH As in the other flavonoids, modifying groups OH such as sugars can then be added to the basic HO O HO O isoflavonoid ring system. Isoflavonoids are not widespread among O2 NADPH plants but are commonly found in legumes. © Jones & Bartlett Learning,H LLC ©+ Jones H O & IsoflavonoidBartlett Learning, synthesis LLCis inducible, and the NADP 2 NOT FOR SALEOH ORO DISTRIBUTION OH O NOT FOR SALEproducts OR act DISTRIBUTION as phytoalexins in leaf tissues to protect against pathogens. Isoflavonoids are excreted from legume roots and serve as attrac- tants for nitrogen-fixing symbiotic bacteria (see Chapter 5). © JonesHO & BartlettO Learning,OH LLC The isoflavone ©ring Jones system & bears Bartlett a resem- Learning, LLC NOT FOR SALE OR DISTRIBUTION blance to the mammalianNOT FOR sex SALE hormones, OR DISTRIBUTION the estradiols (FIGURE 8.33). Isoflavonoids are able to bind weakly to mammalian estrogen OH O OH

© Jones & Bartlett Learning,−H2 OLLCdehydratase © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION HO O FIGURE 8.32 Synthesis of genistein isofl avone. 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 H atom at C-3 in the C ring, producing a radical intermediate. Migration of the aromatic B ring to C-3 and OH O © Jones & Bartlett Learning, LLC © JonesOH hydroxylation & Bartlett of C-2 Learning, yield an isofl avone LLC intermediate. In soybean (Glycine max), a dehydratase removes H2O from the C ring, NOT FOR SALE OR DISTRIBUTION Genistein NOT FORproducing SALE the ORisofl avone, DISTRIBUTION genistein.

142 CHAPTER 8 Aromatic and Phenolic Compounds

R O OH Daidzein: R = H Genistein: R = OH © JonesIsoflavones & Bartlett in soy Learning,HO LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

HO O

Coumestrol Isoflavone in clover, alfalfa sprouts FIGURE 8.33 Some isoﬂ avonoids 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 © Jones &between Bartlett the hydroxyl Learning, groups. LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

receptors and can function as phytoestrogens. Curcumin is produced mainly in the roots of The phytoestrogen in clover, coumestrol, is the plant as a phytoalexin. Humans use the known to have negative© Jones effects &on Bartlett the repro- Learning,roots toLLC provide a pungent taste© and Jones yellow & Bartlett Learning, LLC ductive cycles of ruminantsNOT FORsuch asSALE sheep. OR The DISTRIBUTIONcolor to foods. Curcumin also inhibitsNOT the FOR secre- SALE OR DISTRIBUTION phytoestrogens in soy-rich foods, such as tofu, tion of mucus in mammalian lungs and may be may have beneficial effects in humans. Clini- useful in treatment of the symptoms of asthma cal trials have shown that a soy-rich diet can and cystic fibrosis. reduce blood cholesterol levels and the risk of heart© Jones attacks & in Bartlett postmenopausal Learning, women. LLC Pyrone Synthases ©The Jones pyrone & Bartlettsynthases Learning, are LLC The NOTU.S. Food FOR and SALE Drug OR Administration DISTRIBUTION rec- another type ofNOT polyketide FOR SALEsynthase OR with DISTRIBUTION ommends an intake of 25 g per day of soy restricted distribution among plants. The protein to diminish the effects of menopause basic pyrone synthase uses acetyl-CoA as its and reduce cholesterol levels. The effects are initial substrate and adds malonyl-CoA in a variable and may depend on the activity of gut condensation reminiscent of fatty acid syn- © Jones &flora Bartlett that can Learning, modify the LLC phytoestrogens and thesis© Jones (FIGURE & 8.35 Bartlett). Pyrone Learning, synthases LLCcatalyze NOT FORpromote SALE uptakeOR DISTRIBUTION from the intestines. theNOT production FOR SALE of a ORtriketide DISTRIBUTION intermediate and its subsequent cyclization to 6-methyl- Examples of Other Plant Polyketide Synthases 4-hydroxy-pyrone. CHS is ubiquitous in plants and provides Like the CHSs, the pyrone synthases are the naringenin precursor for a wide variety dimers with two active sites. The chalcone and of flavonoids. Other ©types Jones of polyketide & Bartlett syn- Learning,basic pyroneLLC synthases exhibit 30%© Jones to 50% & Bartlett Learning, LLC thases have been describedNOT FOR that SALE are oftenOR DISTRIBUTIONamino acid similarity and the same catalyticNOT FOR triad SALE OR DISTRIBUTION species specific, leading to the production of of histidine, cysteine, and asparagine. A com- unique natural products. Only a few of these parison of the x-ray crystal structures of CHS 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 FIGURE 8.36. The variety© Jonesof different & Bartlett products. Learning, One example LLC is structural data clearly© Jones illustrate & Bartlettthat production Learning, LLC the synthesisNOT FOR of curcuminSALE OR in CurcumaDISTRIBUTION longa, a of different productsNOT is a FOR result SALEof minor OR residue DISTRIBUTION plant in the Zingiberaceae (ginger) family. mutations that affect the size and hydrophobic- In C. longa, a unique diketide synthase adds ity of the ketide binding cavity. malonyl-CoA to the CoA ester of ferulic acid (FIGURE 8.34). A second feruloyl-CoA is added Stilbene Synthase An especially perplexing © Jones &to Bartlettthe diketide Learning, product LLCto yield curcumin, example© Jones of polyketide & Bartlett synthesis Learning, is the LLCproduc- NOT FORthe SALE main OR ingredient DISTRIBUTION in the spice, turmeric. tionNOT of stilbenes.FOR SALE OR DISTRIBUTION

8.5 Synthesis and Properties of Polyketides 143

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. O © Jones & Bartlett Learning, LLC O © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION S-CoA S-CoA HO O2 HO O NADPH SAM CH Feruloyl-CoA p-Coumaroyl-CoA 3 © Jones & Bartlett Learning, LLC © JonesO O& Bartlett Learning, LLC NADP SAH diketide NOT FOR SALE OR DISTRIBUTION + NOT FOR SALE OR DISTRIBUTION synthase HO S-CoA Malonyl-CoA

O O

© Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC HO OH NOT FOR SALEO OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION CH3 O CH3 Curcumin FIGURE 8.34 Synthesis of curcumin. A unique diketide synthase binds the CoA ester of ferulic acid (see Figure 8.14) and catalyzes the addition of malonyl-CoA. The feruloyl-CoA diketide intermediate is further modiﬁ ed by a speciﬁ c polyketide synthase © Jonesthat & addsBartlett a second Learning, feruloyl moiety toLLC the side chain to yield curcumin. This© Jonesparticular polyketide& Bartlett synthase Learning, is unique to some LLC NOT FORplants SALE of the Zingiberaceae OR DISTRIBUTION (ginger) family. NOT FOR SALE OR DISTRIBUTION

O O O OOO Stilbene synthases utilize coumaroyl-CoA © Jones & Bartlett Learning, LLC © Jones & asBartlett their initial Learning, substrate LLCand add three units of CoA-S CH3 CoA-S CH CoA-S NOT FOR SALE OR DISTRIBUTION3 NOT FORCH3 SALEmalonyl-CoA OR DISTRIBUTION to produce the same tetraketide Acetyl-CoA –CO2 –CO2 intermediate that is found in chalcone synthesis. The stilbene synthases, however, catalyze a + CoA-SH O O Malonyl-CoA CoA-SH subsequent decarboxylation of the intermediate and a C-2 to C-7 cyclization instead of the CoA-S O– © Jones & Bartlett Learning, LLC C-1 to C-6 cyclization© Jones in chalcone & Bartlett synthases Learning, LLC Malonyl-CoA NOT FOR SALE OR DISTRIBUTION (FIGURE 8.37). Both NOTtypes FORof synthases SALE exhibitOR DISTRIBUTION over 50% amino acid similarity and have iden- HO CH3 tical residues in the active site and surrounding substrate entrance cavity. Yet they consistently O catalyze the formation of different products © Jones & Bartlett Learning, LLC from© Jonesthe same & tetraketide Bartlett intermediate.Learning, LLC Subtle NOT FOR SALE OR DISTRIBUTIONO differencesNOT FOR in the SALE active OR site DISTRIBUTION region apparently 6-Methyl-4-hydroxy-2-pyrone favor the formation of a larger molecule by FIGURE 8.35 Activity of pyrone synthase. The enzyme CHSs and decarboxylation of the tetraketide catalyzes the sequential condensation of two molecules of and synthesis of a smaller product by stilbene malonyl-CoA with an acetyl-CoA starter unit instead of the synthases. © Jones & Bartlett Learning,p-coumaroyl-CoA LLC starter in CHS. A triketide intermediate© Jones is & Bartlett Learning, LLC produced in this reaction and then cyclized into an unsatu- As in the case of the flavonoids, the stilbenes NOT FOR SALE OR DISTRIBUTIONrated ␦-lactone called a pyrone. NOT FOR SALEcan be furtherOR DISTRIBUTION modified, mainly by glycosylation.

144 CHAPTER 8 Aromatic and Phenolic Compounds

FIGURE 8.36 Comparison of the active site structures of 2-pyrone synthase (2-PS) (left) and chalcone synthase (CHS) (right). The major difference between these homologous, dimeric enzymes is the size of the binding cavity, which is considerably smaller in the case of 2-PS. In addition, 2-PS has an active site entrance that is constricted compared with CHS and lined with several hydrophobic residues. Relatively small molecules have access to the active site cavity and result in the condensation and exit of © Jones &relatively Bartlett hydrophobic Learning, products. This LLC is in contrast to the more open and© hydrophilicJones cavity& Bartlett of CHS (see Learning, also Figure 8.26). LLC NOT FOR(Reproduced SALE OR from :DISTRIBUTION J. M. Jez, M. B. Austin, J. Ferrer, M. E. Bowman, J.NOT Schröder, FOR and J.SALE P. Noel, ChemistryOR DISTRIBUTION & Biology 7 [2000]: 919–930, Figure 6. Courtesy of Joseph P. Noel, Howard Howard Hughes Medical Institute.)

They are widespread© but Jones not universally & Bartlett dis- Learning, LLC O © Jones O& BartlettO Learning, LLC tributed in floweringNOT plants. FOR They SALE have anti-OR DISTRIBUTION S-CoA NOT FOR SALE OR DISTRIBUTION bacterial and antifungal properties and act as + 3 HO S-CoA phytoalexins. Resveratrol, the stilbene shown HO in Figure 8.37, is abundant in grapes. Based on – 3 CO2 experiments in vitro and experiments in ani- – 3 CoA-SH mals,© it Jonesacts as an& Bartlettantioxidant Learning, and can inhibit LLC © Jones & Bartlett Learning, LLC bloodNOT platelet FOR aggregation SALE OR (clotting) DISTRIBUTION in mam- NOT FOR SALE OR DISTRIBUTION mals. Resveratrol is the compound in red wine OOOO that is presumably responsible for the “French 9 7 5 3 1 S-CoA paradox,” that is, how to consume a diet rich in 8 6 4 2 saturated fats and cholesterol and still maintain HO © Jones &a lowBartlett risk for Learning, atherosclerosis LLC and heart attack. © Jones & Bartlett Learning, LLC Experiments in mice have recently shown that – CoA-SH – COOH, – CoA-SH NOT FOR SALE OR DISTRIBUTION NOT1–6 FOR condensation SALE OR DISTRIBUTION2–7 condensation consumption of resveratrol protects mice from chalcone synthase stilbene synthase the adverse effects of a high-calorie diet even though the animals were relatively obese com- HO HO pared with controls. Larger quantities of resveratrol than are present in a normal diet, however, 4 © Jones & Bartlett Learning, LLC 9 HO OH © Jones & Bartlett9 Learning, LLC must be consumed to obtain these health ben- 3 NOT FOR SALE OR DISTRIBUTION 5 NOT FOR SALE OR DISTRIBUTION efits. Resveratrol is available in pure form and 6 2 8 7 1 2 8 OH 7 clinical trials are currently being done in humans 3 suffering from type 2 diabetes. O OH 6 5 4

Chalcone OH Synthesis© Jones and & Activity Bartlett of Coumarins Learning, LLC (Naringenin)© Jones & Bartlett Learning, LLC Stilbene TheNOT coumarins, FOR SALE like otherOR DISTRIBUTIONproducts of the NOT FOR SALE OR DISTRIBUTION(Resveratrol) phenylpropanoid pathway, start with the synthe- FIGURE 8.37 Comparison of the activity of chalcone and stilbene synthases. sis of the key intermediate, coumaroyl-CoA. Cou- Both enzymes catalyze the condensation of three molecules of malonyl-CoA with marin synthesis requires a specific hydroxylase a p-coumaroyl-CoA staring unit to generate the same tetraketide intermediate. (cytochrome P450 monooxygenase) that adds In chalcone synthase CHS, the intermediate undergoes a C-1 to C-6 cyclization © Jones & Bartlett Learning, LLC and© aromatizationJones & to Bartlett produce naringenin Learning, chalcone. LLC In stilbene synthase, the an OH to the aromatic ring at the ortho position. tetraketide intermediate undergoes a decarboxylation before cyclization of C-2 to NOT FORThe SALE next stepOR is DISTRIBUTION a nonenzymatic isomerization of C-7NOT to produce FOR the SALE second aromatic OR DISTRIBUTION ring to yield resveratrol.

8.5 Synthesis and Properties of Polyketides 145

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. O O OH OH © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC S CoA NOT FOR SALE OR DISTRIBUTION- NOTOH FOR SALE OR DISTRIBUTION O2 HO HO OH O O O O NADPH + CoA-SH NADP Dicoumarol visible light © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

HO O O OH O Umbelliferone © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC 5 4 6 3 NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OROO DISTRIBUTION 7 2 8 1 O O Warfarin FIGURE 8.39 Structures of two coumarin derivatives. Coumarin Dicoumarol is produced in damaged clover plants by C7 — usually substituted with OH dimerization of coumarol. It is a powerful anticoagulant in © Jones & Bartlett Learning,C6 and C8 — H,LLC OH, OCH3 © Jones & mammals.Bartlett W arfarinLearning, is a synthetic LLC coumarin with structural NOT FOR SALEFIGURE OR 8.38 DISTRIBUTION Synthesis of umbelliferone. Coumarins are synthesizedNOT FOR SALEsimilarity toOR dicoumarol DISTRIBUTION and is used therapeutically to prevent from p-coumaroyl-CoA, the key phenylpropanoid pathway intermediate. internal clot formation. A specifi c cytochrome P450 monooxygenase hydroxylates the ring at the ortho position with respect to the side chain and removes the CoA. A light-catalyzed isomerization of the double bond in the side chain is followed by spontaneous formation of the ␦-lactone. Coumarins can be further modifi ed by hydroxylation© Jones and& Bartlettmethylation. Learning,The inset shows LLCthe © Jones & Bartlett Learning, LLC numbering system and substitution patterns of coumarins. NOT FOR SALE OR DISTRIBUTION 6 NOT FOR SALE OR DISTRIBUTION + HO O O O˗PP the double bond in the side chain catalyzed by – PP visible light, followed by spontaneous formation © Jonesof & a Bartlett␦-lactone. FIGURELearning, 8.38 illustrates LLC the synthesis of © Jones & Bartlett Learning, LLC NOT FORumbelliferone SALE OR and DISTRIBUTION the general structural designa- NOT FOR SALE OR6 DISTRIBUTION tions for coumarins. HO Coumarins have a restricted distribution OO among angiosperms and are found in some NADPH O2 members of the Fabaceae, Asteraceae, and NADP © Jones & Bartlett Learning,Apiaceae. The LLC sweet smell of new-mown© hayJones is & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTIONdue to the release of coumarins from damagedNOT FOR SALE OR DISTRIBUTIONHO clover (Melilotus officinalis). Coumarins can be dimerized to dicoumarol, which is a highly O O O effective anticoagulant (FIGURE 8.39). Consump- tion of large amounts of sweet clover can cause NADPH O2 internal© bleedingJones in& ruminants.Bartlett Learning, Synthetic anti- LLC © Jones &NADP Bartlett Learning, LLC coagulants,NOT such FOR as SALE warfarin, OR are DISTRIBUTION based on the NOT FOR SALE OR DISTRIBUTION dicoumarol structure. Once widely used as a rat poison, warfarin is now prescribed as an O O O anticlotting agent for the treatment of humans with atherosclerosis. Psoralen © Jones & CoumarinsBartlett Learning, are often modified LLC by the addi- FIGURE© Jones 8.40 Synthesis & Bartlett of the furanocoumarin, Learning, psoralen. LLC NOT FORtion SALE of other OR chemical DISTRIBUTION groups. The C-7 posi- UmbelliferoneNOT FOR (7-hydroxycoumarin) SALE OR isDISTRIBUTION prenylated at C-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 C-6. The pre- second P450 monooxygenase catalyzes removal of the side nylated coumarin is then further modified chain and formation of a double bond in the furan ring to © Jones & Bartlett Learning, LLC © Jones & formBartlett psoralen. Learning, The ring may be LLC modifi ed by glycosylation. by two monooxygenases to yield a linear The linear furanocoumarins are phytophotosensitizers that NOT FOR SALE OR DISTRIBUTIONfurano derivative called a psoralen (FIGURENOT 8.40 FOR). SALEcause severe OR dermatitis DISTRIBUTION in humans.

146 CHAPTER 8 Aromatic and Phenolic Compounds

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. The furanocoumarins are produced in some ing of the skin. In the United States, cow © Jones crop& Bartlett plants such Learning, as celery LLC in response to fun- parsnip© Jones or hogweed & Bartlett (Heracleum Learning, sphondylium LLC ) NOT FORgal SALE pathogens. OR DISTRIBUTION These coumarin derivatives isNOT an invasive FOR SALEplant that OR produces DISTRIBUTION furanocou- 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 dermati- come in contact with the furanocoumarin; tis-causing properties present a major barrier subsequent exposure of the skin to sunlight to removal and eradication of this invasive causes dermatitis followed© Jones by severe& Bartlett blister- Learning,species. LLC © Jones & Bartlett Learning, LLC NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION

CHAPTER SUMMARY © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC A general outline describing the synthesis of aromatic the central product of the shikimic acid pathway; compoundsNOT FOR in plants SALE is illustratedOR DISTRIBUTION in FIGURE 8.41. Syn- it is the substrateNOT FOR for phenylalanineSALE OR DISTRIBUTION ammonia lyase thesis begins in plastids with two simple intermediates (PAL), a cytoplasmic enzyme and the gateway to 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 deriv- © Jones &shikimic Bartlett acid Learning, pathway to LLC produce chorismic acid and© Jonesative, &most Bartlett often Learning,coumaroyl-CoA. LLC The synthesis of the essential aromatic amino acids, tyrosine, phenyl- primary products such as lignin draws from the pool NOT FORalanine, SALE andOR tryptophan. DISTRIBUTION Intermediates in the shikimicNOT of FOR coumaroyl-CoA. SALE OR DISTRIBUTIONNatural products such as flavo- acid pathway can provide the building blocks for addi- noids also draw from the same precursor pool and can tional aromatics such as the gallotannins and the plant be considered primary metabolites because of their hormone, salicylic acid (Figure 8.1). Phenylalanine is essential functions in plant–environmental interac- © Jones & Bartlett Learning, LLCtions. Many polyketide synthases© Jones use &coumaroyl-CoA Bartlett Learning, LLC as a starting unit and produce unique natural products NOT FOR SALE OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION + such as curcumin (Figure 8.34). Phosphoenol pyruvate Erythrose-4-phosphate Additional aromatic compounds are synthesized by branches from the phenylpropanoid pathway, and only Phenolics O O a few of these have been covered in this chapter such as O + the coumarins. Aromatic metabolism in plants comprises + © CJonesO– & Bartlett Learning, LLC CH C O– © Jones & Bartlett Learning, LLC H N CH + H3N 3 CH C – a rich assortment of chemical reactions and products. NOT FOR SALEH3N OR DISTRIBUTIONO CH NOT FOR SALE OR DISTRIBUTION CH2 2 The structures and syntheses of many new compounds H C 2 are still not fully resolved and others are waiting to be HN discovered. The aromatic amino acids are also precursors OH in other unique synthetic pathways, such as the gluco- Tryptophan © Jones & Bartlett Learning, LLC Tyrosine © Jonessinolates & Bartlettcovered in Learning, Chapter 5. TheLLC alkaloids are a rich Phenylalanine assortment of additional natural products that are also NOT FOR SALE OR DISTRIBUTION NOTderived FOR SALEfrom amino OR DISTRIBUTION acids, particularly the aromatic phenylalanine ammonia ayase (PAL) amino acids. A small number of these are described in H O Chapter 9. CC C O– © Jones &H Bartlett Learning, LLC © Jones & Bartlett Learning, LLC NOTtrans-Cinnamic FOR SALE Acid OR DISTRIBUTION NOT FOR SALE OR DISTRIBUTION O coumarate synthase 2 FIGURE 8.41 Summary of general aromatic metabolism in H O plants. The synthesis of aromatic compounds starts in plastids with the shikimic acid pathway and results in the production of the three HO C C C O– aromatic amino acids, tyrosine, phenylalanine, and tryptophan. © Jones & Bartlett Learning,H LLC Phenylalanine occupies© Jones a key position& Bartlett as the precursor Learning, of the LLC p-Coumaric Acid phenylpropanoid pathway. The cytoplasmic enzyme, PAL, removes NOT FOR SALE OR DISTRIBUTIONCoumarins the amino groupNOT from phenylalanine FOR SALE and yields OR trans DISTRIBUTION-cinnamic Polyketides, acid, which is a substrate for a cytochrome P450 monooxygenase. flavonoids, stilbenes, etc. The product is p-coumaric acid, which can be further modifi ed, usually by esterifi cation to coenzyme A. The resulting pool of Monolignols coumaroyl-CoA in the cytoplasm is a source for the synthesis of Volatiles monolignols and lignin, which are primary products, and a large © Jones & Bartlett Learning, LLC © Jonesnumber of & natural Bartlett products, Learning, including the vital LLC fl avonoids and their NOT FOR SALE OR DISTRIBUTIONLignin NOTderivatives FOR andSALE defensive OR compounds DISTRIBUTION such as coumarins.

8.5 Synthesis and Properties of Polyketides 147

© Jones & Bartlett Learning, LLC. NOT FOR SALE OR DISTRIBUTION. REFERENCES FOR FURTHER INFORMATION © Jones & BartlettBoerjan, Learning,W., Ralph, J., LLC & Baucher, M. (2003). Lignin© JonesKoes, & Bartlett R., Verweij, Learning, W., & Quattrocchio, LLC F. (2005). NOT FORbiosynthesis. SALE OR Annu. DISTRIBUTION Rev. Plant Biol. 54:519–546. NOTFlavonoids: FOR SALE a colorfulOR DISTRIBUTION model for the regulation of Grotewold, E. (2006). Genetics and biochemistry of evolution of biochemical pathways. Trends Plant Sci. ﬂ oral pigments. Annu. Rev. Plant Biol. 57:761–780. 10:236–242. Herrmann, K.M. (1995). The shikimate pathway: Tanaka, Y., Sasaki, N., & Ohmiya, A. (2008). early steps in the biosynthesis of aromatic compounds. Biosynthesis of plant pigments: anthocyanins, betalains Plant Cell 7:907–919.© Jones & Bartlett Learning, LLCand carotenoids. Plant J. 54:©733–749. Jones & Bartlett Learning, LLC Humphreys, J.M.NOT & Chapple, FOR SALE C. (2002). OR Rewriting DISTRIBUTION NOT FOR SALE OR DISTRIBUTION the lignin road map. Curr. Opin. Plant Biol. 5:224–229.

CH 3 © Jones & Bartlett Learning, LLC © Jones & Bartlett Learning, LLC N O N NOTPLANT FOR BIOCHEMISTRYSALE OR DISTRIBUTION on the web NOT FOR SALE OR DISTRIBUTION N N CH3 H C 3 O Visit the Plant Biochemistry student companion Web site at http://biology.jbpub.com/gleason/plantbiochemistry/ for additional online study resources on these topics.

148 CHAPTER 8 Aromatic and Phenolic Compounds