The Shikimate Pathway: Aromatic Amino Acids and Phenylpropanoids
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Medicinal Natural Products. Paul M Dewick Copyright 2002 John Wiley & Sons, Ltd ISBNs: 0471496405 (Hardback); 0471496413 (paperback); 0470846275 (Electronic) 4 THE SHIKIMATE PATHWAY: AROMATIC AMINO ACIDS AND PHENYLPROPANOIDS Shikimic acid and its role in the formation of aromatic amino acids, benzoic acids, and cinnamic acids is described, along with further modifications leading to lignans and lignin, phenylpropenes, and coumarins. Combinations of the shikimate pathway and the acetate pathway are responsible for the biosynthesis of styrylpyrones, flavonoids and stilbenes, flavonolignans, and isoflavonoids. Terpenoid quinones are formed by a combination of the shikimate pathway with the terpenoid pathway. Monograph topics giving more detailed information on medicinal agents include folic acid, chloramphenicol, podophyllum, volatile oils, dicoumarol and warfarin, psoralens, kava, Silybum marianum, phyto-oestrogens, derris and lonchocarpus, vitamin E, and vitamin K. The shikimate pathway provides an alternative glycolysis) and D-erythrose 4-phosphate (from the route to aromatic compounds, particularly the aro- pentose phosphate cycle) to the aromatic amino matic amino acids L-phenylalanine,L-tyrosine acids was broadly outlined. Phenylalanine and and L-tryptophan. This pathway is employed by tyrosine form the basis of C6C3 phenylpropane microorganisms and plants, but not by animals, units found in many natural products, e.g. cin- and accordingly the aromatic amino acids fea- namic acids, coumarins, lignans, and flavonoids, ture among those essential amino acids for man and along with tryptophan are precursors of a wide which have to be obtained in the diet. A cen- range of alkaloid structures. In addition, it is found tral intermediate in the pathway is shikimic acid that many simple benzoic acid derivatives, e.g. (Figure 4.1), a compound which had been iso- gallic acid (Figure 4.1) and p-aminobenzoic acid lated from plants of Illicium species (Japanese (4-aminobenzoic acid) (Figure 4.4) are produced ‘shikimi’) many years before its role in metabolism via branchpoints in the shikimate pathway. had been discovered. Most of the intermediates in the pathway were identified by a careful study of AROMATIC AMINO ACIDS AND aseriesofEscherichia coli mutants prepared by SIMPLE BENZOIC ACIDS UV irradiation. Their nutritional requirements for growth, and any by-products formed, were then The shikimate pathway begins with a coupling characterized. A mutant strain capable of growth of phosphoenolpyruvate (PEP) and D-erythrose usually differs from its parent in only a single 4-phosphate to give the seven-carbon 3-deoxy- gene, and the usual effect is the impaired syn- D-arabino-heptulosonic acid 7-phosphate (DAHP) thesis of a single enzyme. Typically, a mutant (Figure 4.1). This reaction, shown here as an blocked in the transformation of compound A into aldol-type condensation, is known to be mecha- compound B will require B for growth whilst nistically more complex in the enzyme-catalysed accumulating A in its culture medium. In this version; several of the other transformations in the way, the pathway from phosphoenolpyruvate (from pathway have also been found to be surprisingly 122 THE SHIKIMATE PATHWAY formally an elimination; it actually involves oxidation of the hydroxyl adjacent to the proton lost and therefore requires NAD+ PEP CO H 2 aldol-type cofactor; the carbonyl is subsequently reduced back to an alcohol reaction CO H P O 2 CO2H H PO PO O H – HOP O H NAD+ HO O H HO OH HO OH OH OH OH D-erythrose 4-P DAHP aldol-type reaction CO2H CO2H HO CO2H HO CO2H NADPH – H2O NADH HO OH O OH O OH HO OH OH OH OH OH shikimic acid 3-dehydroshikimic 3-dehydroquinic quinic acid acid acid – 2H – H O oxidation and dehydration and 2 enolization enolization CO2H CO2H HO HO OH OH OH protocatechuic gallic acid acid Figure 4.1 complex. Elimination of phosphoric acid from gallic acid (3,4,5-trihydroxybenzoic acid) can be DAHP followed by an intramolecular aldol reac- formed by branchpoint reactions from 3-dehy- tion generates the first carbocyclic intermediate 3- droshikimic acid, which involve dehydration and dehydroquinic acid. However, this also represents enolization, or, in the case of gallic acid, dehy- an oversimplification. The elimination of phos- drogenation and enolization. Gallic acid features phoric acid actually follows an NAD+-dependent as a component of many tannin materials (gal- oxidation of the central hydroxyl, and this is then lotannins), e.g. pentagalloylglucose (Figure 4.2), re-formed in an NADH-dependent reduction reac- found in plants, materials which have been used tion on the intermediate carbonyl compound prior for thousands of years in the tanning of animal to the aldol reaction occurring. All these changes hides to make leather, due to their ability to cross- occur in the presence of a single enzyme. Reduc- link protein molecules. Tannins also contribute to tion of 3-dehydroquinic acid leads to quinic acid, the astringency of foods and beverages, especially a fairly common natural product found in the tea, coffee and wines (see also condensed tannins, free form, as esters, or in combination with alka- page 151). loids such as quinine (see page 362). Shikimic A very important branchpoint compound in the acid itself is formed from 3-dehydroquinic acid shikimate pathway is chorismic acid (Figure 4.3), via 3-dehydroshikimic acid by dehydration and which has incorporated a further molecule of reduction steps. The simple phenolic acids pro- PEP as an enol ether side-chain. PEP combines tocatechuic acid (3,4-dihydroxybenzoic acid) and with shikimic acid 3-phosphate produced in a AROMATIC AMINO ACIDS AND SIMPLE BENZOIC ACIDS 123 OH enzyme, and is believed to bind to the PEP bind- OH OH ing site on the enzyme. Glyphosate finds con- HO OH siderable use as a broad spectrum herbicide, a O OH plant’s subsequent inability to synthesize aromatic OH O amino acids causing its death. The transformation OH of EPSP to chorismic acid (Figure 4.3) involves a O O O O O O 1,4-elimination of phosphoric acid, though this is OO OH probably not a concerted elimination. O 4-hydroxybenzoic acid (Figure 4.4) is pro- duced in bacteria from chorismic acid by HO OH HO OH an elimination reaction, losing the recently OH introduced enolpyruvic acid side-chain. However, OH pentagalloylglucose in plants, this phenolic acid is formed by a branch much further on in the pathway via side-chain degradation of cinnamic acids (see CO H CO H 2 2 page 141). The three phenolic acids so far encoun- HN O O tered, 4-hydroxybenzoic, protocatechuic, and gallic O P P OH OH acids, demonstrate some of the hydroxylation OH OH patterns characteristic of shikimic acid-derived glyphosate PEP metabolites, i.e. a single hydroxy para to the side- chain function, dihydroxy groups arranged ortho to Figure 4.2 each other, typically 3,4- to the side-chain, and tri- hydroxy groups also ortho to each other and 3,4,5- simple ATP-dependent phosphorylation reaction. to the side-chain. The single para-hydroxylation This combines with PEP via an addition–elimi- and the ortho-polyhydroxylation patterns contrast nation reaction giving 3-enolpyruvylshikimic acid with the typical meta-hydroxylation patterns char- 3-phosphate (EPSP). This reaction is catalysed acteristic of phenols derived via the acetate path- by the enzyme EPSP synthase. The synthetic way (see page 62), and in most cases allow the N-(phosphonomethyl)glycine derivative glypho- biosynthetic origin (acetate or shikimate) of an aro- sate (Figure 4.2) is a powerful inhibitor of this matic ring to be deduced by inspection. nucleophilic attack on to protonated double bond of PEP H PEP CO H CO2H CO2H 2 H PO CO2H HH ATP HO OH PO OH EPSP synthase PO OCO2H OH OH OH OP shikimic acid shikimic acid 3-P 1,2-elimination of – HOP phosphoric acid 1,4-elimination of phosphoric acid CO2H CO2H L-Phe – HOP prephenic acid L-Tyr OCO2H PO OCO2H OH OH chorismic acid EPSP Figure 4.3 124 THE SHIKIMATE PATHWAY elimination of pyruvic acid elimination of pyruvic acid (formally as enolpyruvic acid) (formally as enolpyruvic acid) generates aromatic ring isomerization via generates aromatic ring CO H S 2′ reaction 2 CO2H N CO2H CO2H OH2 OH OH OCO2H OCO2H OH OH isochorismic acid salicylic acid 4-hydroxybenzoic H chorismic acid acid hydrolysis of L-Gln L-Gln enol ether amination using side-chain oxidation of 3-hydroxyl to ammonia (generated ketone, then enolization from glutamine) as CO2H CO H CO2H nucleophile 2 CO2H NH2 OH NAD+ OH OCOH 2 OCO2H OH OH NH 2 2,3-dihydroxybenzoic 2-amino-2-deoxy- 4-amino-4-deoxy- acid chorismic acid isochorismic acid elimination of pyruvic acid CO2H CO2H NH2 L-Trp NH2 anthranilic acid p-aminobenzoic acid (PABA) Figure 4.4 2,3-dihydroxybenzoic acid,andsalicylic acid available sufficient concentrations of essential (2-hydroxybenzoic acid) (in microorganisms, but iron. Enterobactin comprises three molecules of not in plants, see page 141), are derived from 2,3-dihydroxybenzoic acid and three of the amino chorismic acid via its isomer isochorismic acid L-serine, in cyclic triester form. acid (Figure 4.4). The isomerization involves Simple amino analogues of the phenolic an SN2 -type of reaction, an incoming water acids are produced from chorismic acid by nucleophile attacking the diene system and related transformations in which ammonia, gen- displacing the hydroxyl. Salicyclic acid arises by erated from glutamine, acts as a nucleophile an elimination reaction analogous to that producing (Figure 4.4). Chorismic acid can be aminated at 4-hydroxybenzoic acid from chorismic acid. In C-4 to give 4-amino-4-deoxychorismic acid and the formation of 2,3-dihydroxybenzoic acid, the then p-aminobenzoic (4-aminobenzoic) acid, or at side-chain of isochorismic acid is first lost by C-2 to give the isochorismic acid analogue which hydrolysis, then dehydrogenation of the 3-hydroxy will yield 2-aminobenzoic (anthranilic) acid.