Aromatic Amino Acids And Simple Benzoic Acids

The shikimate pathway begins with a coupling of phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate to give the seven-carbon 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP) (Figure 4.1). This reaction, shown here as an aldol-type condensation, is known to be mechanistically more complex in the enzyme-catalysed version; several of the other transformations in the pathway have also been found to be surprisingly

PEP CO2H

aldol-type reaction

D-erythrose 4-P

D-erythrose 4-P

formally an elimination; it actually involves oxidation of the hydroxyl adjacent to the proton lost and therefore requires NAD+ cofactor; the carbonyl is subsequently reduced back to an alcohol

NAD+

aldol-type reaction

CO2H

CO2H

CO2H

CO2H

HO X "*OH OH shikimic acid o x oh

3-dehydroshikimic acid

HO X "*OH OH shikimic acid

CO2H HO _CO2H

3-dehydroshikimic acid dehydration and enolization

protocatechuic acid

3-dehydroquinic acid

HO CO2H

3-dehydroquinic acid

- 2H oxidation and enolization

- 2H oxidation and enolization

protocatechuic acid

Figure 4.1

OH gallic acid

HO CO2H

OH quinic acid

OH quinic acid

Figure 4.1

complex. Elimination of phosphoric acid from DAHP followed by an intramolecular aldol reaction generates the first carbocyclic intermediate 3-dehydroquinic acid. However, this also represents an oversimplification. The elimination of phosphoric acid actually follows an NAD+-dependent oxidation of the central hydroxyl, and this is then re-formed in an NADH-dependent reduction reaction on the intermediate carbonyl compound prior to the aldol reaction occurring. All these changes occur in the presence of a single enzyme. Reduction of 3-dehydroquinic acid leads to quinic acid, a fairly common natural product found in the free form, as esters, or in combination with alkaloids such as quinine (see page 362). Shikimic acid itself is formed from 3-dehydroquinic acid via 3-dehydroshikimic acid by dehydration and reduction steps. The simple phenolic acids pro-tocatechuic acid (3,4-dihydroxybenzoic acid) and gallic acid (3,4,5-trihydroxybenzoic acid) can be formed by branchpoint reactions from 3-dehy-droshikimic acid, which involve dehydration and enolization, or, in the case of gallic acid, dehy-drogenation and enolization. Gallic acid features as a component of many tannin materials (gal-lotannins), e.g. pentagalloylglucose (Figure 4.2), found in plants, materials which have been used for thousands of years in the tanning of animal hides to make leather, due to their ability to crosslink protein molecules. Tannins also contribute to the astringency of foods and beverages, especially tea, coffee and wines (see also condensed tannins, page 151).

A very important branchpoint compound in the shikimate pathway is chorismic acid (Figure 4.3), which has incorporated a further molecule of PEP as an enol ether side-chain. PEP combines with shikimic acid 3-phosphate produced in a

Pentagalloylglucose

pentagalloylglucose

CO2H

co2h

P OH OH

glyphosate

Figure 4.2

simple ATP-dependent phosphorylation reaction. This combines with PEP via an addition - elimination reaction giving 3-enolpyruvylshikimic acid 3-phosphate (EPSP). This reaction is catalysed by the enzyme EPSP synthase. The synthetic N-(phosphonomethyl)glycine derivative glypho-sate (Figure 4.2) is a powerful inhibitor of this enzyme, and is believed to bind to the PEP binding site on the enzyme. Glyphosate finds considerable use as a broad spectrum herbicide, a plant's subsequent inability to synthesize aromatic amino acids causing its death. The transformation of EPSP to chorismic acid (Figure 4.3) involves a 1,4-elimination of phosphoric acid, though this is probably not a concerted elimination.

4-hydroxybenzoic acid (Figure 4.4) is produced in bacteria from chorismic acid by an elimination reaction, losing the recently introduced enolpyruvic acid side-chain. However, in plants, this phenolic acid is formed by a branch much further on in the pathway via side-chain degradation of cinnamic acids (see page 141). The three phenolic acids so far encountered, 4-hydroxybenzoic, protocatechuic, and gallic acids, demonstrate some of the hydroxylation patterns characteristic of shikimic acid-derived metabolites, i.e. a single hydroxy para to the side-chain function, dihydroxy groups arranged ortho to each other, typically 3,4- to the side-chain, and tri-hydroxy groups also ortho to each other and 3,4,5-to the side-chain. The single para-hydroxylation and the ortho-polyhydroxylation patterns contrast with the typical meta-hydroxylation patterns characteristic of phenols derived via the acetate pathway (see page 62), and in most cases allow the biosynthetic origin (acetate or shikimate) of an aromatic ring to be deduced by inspection.

nucleophilic attack on to protonated double bond of PEP h^

co2h co2h co2h

HO Y OH OH shikimic acid

po^XO2H

HO Y OH OH shikimic acid nucleophilic attack on to protonated double bond of PEP h^

co2h

PO it OH OH

shikimic acid 3-P

CO2H

PO it OH OH

shikimic acid 3-P

CO2H

L-Phe L-Tyr prephenic acid

chorismic acid

EPSP synthase

1,4-elimination of phosphoric acid

CO2H

1,2-elimination of phosphoric acid

*O co2h

chorismic acid

OH EPSP

OH EPSP

elimination of pyruvic acid (formally as enolpyruvic acid) generates aromatic ring isomerization via elimination of pyruvic acid (formally as enolpyruvic acid) generates aromatic ring co2h

isomerization via elimination of pyruvic acid (formally as enolpyruvic acid) generates aromatic ring

H © chorismic acid

"O CO2H isochorismic acid salicylic acid

4-hydroxybenzoic acid

H © chorismic acid

"O CO2H isochorismic acid salicylic acid

L-Gln / \ L-Gln amination using ammonia (generated CO2H from glutamine) as CO H

I nucleophile nh2

4-amino-4-deoxy-chorismic acid

| elimination of pyruvic acid

CO2H

p-aminobenzoic acid (PABA)

hydrolysis of enol ether side-chain

2-amino-2-deoxy-isochorismic acid

CO2H

CO2H

anthranilic acid

anthranilic acid hydrolysis of enol ether side-chain

CO2H

2-amino-2-deoxy-isochorismic acid oxidation of 3-hydroxyl to ketone, then enolization

CO2H

CO2H

CO2H

2,3-dihydroxybenzoic acid

L-Trp

Figure 4.4

2,3-dihydroxybenzoic acid, and salicylic acid

(2-hydroxybenzoic acid) (in microorganisms, but not in plants, see page 141), are derived from chorismic acid via its isomer isochorismic acid (Figure 4.4). The isomerization involves an Sn2'-type of reaction, an incoming water nucleophile attacking the diene system and displacing the hydroxyl. Salicyclic acid arises by an elimination reaction analogous to that producing 4-hydroxybenzoic acid from chorismic acid. In the formation of 2,3-dihydroxybenzoic acid, the side-chain of isochorismic acid is first lost by hydrolysis, then dehydrogenation of the 3-hydroxy to a 3-keto allows enolization and formation of the aromatic ring. 2,3-Dihydroxybenzoic acid is a component of the powerful iron chelator (siderophore) enterobactin (Figure 4.5) found in Escherichia coli and many other Gram-negative bacteria. Such compounds play an important role in bacterial growth by making available sufficient concentrations of essential iron. Enterobactin comprises three molecules of 2,3-dihydroxybenzoic acid and three of the amino acid L-serine, in cyclic triester form.

Simple amino analogues of the phenolic acids are produced from chorismic acid by related transformations in which ammonia, generated from glutamine, acts as a nucleophile (Figure 4.4). Chorismic acid can be aminated at C-4 to give 4-amino-4-deoxychorismic acid and then p-aminobenzoic (4-aminobenzoic) acid, or at C-2 to give the isochorismic acid analogue which will yield 2-aminobenzoic (anthranilic) acid. Amination at C-4 has been found to occur with retention of configuration, so perhaps a double inversion mechanism is involved. p-Aminobenzoic acid (PABA) forms part of the structure of folic acid (vitamin B9)* (Figure 4.6). The folic acid structure is built up (Figure 4.6) from a dihydropterin diphosphate which reacts with p-aminobenzoic

activation to AMP derivative, compare peptide formation, Figure 7.15 CO2H COAMP L_Ser

2,3-dihydroxy-benzoic acid

activation to AMP derivative, compare peptide formation, Figure 7.15 CO2H COAMP L_Ser

Sulphanilamide

enterobactin as iron chelator enterobactin

Figure 4.5

enterobactin

Figure 4.5

OH Ho"

enterobactin as iron chelator

CO2H

PABA

hydroxymethyl-dihydropterin

SO2NH2 sulphanilamide

(acts as antimetabolite of PABA and is enzyme inhibitor)

I Sn2 reaction |

OH OH

co2h

hydroxymethyl-dihydropterin PP

dihydropteroic acid

reduction

L-Glu ATP

NADPH

tetrahydrofolic acid (FH4)

O CO2H

CO2H

the pteridine system is sometimes drawn as the tautomeric amide form:

NADPH

h2n n n dihydrofolic acid (FH2)

dihydrofolate reductase (DHFR)

O CO2H

reduction

a pteridine

O CO2H

CO2H

p-amino-benzoic acid (PABA)

L-Glu folic acid acid to give dihydropteroic acid, an enzymic step for which the sulphonamide antibiotics are inhibitors. Dihydrofolic acid is produced from the dihydropteroic acid by incorporating glutamic acid, and reduction yields tetrahydrofolic acid. This reduction step is also necessary for the continual regeneration of tetrahydrofolic acid, and forms an important site of action for some antibacterial, anti-malarial, and anticancer drugs.

Anthranilic acid (Figure 4.4) is an intermediate in the biosynthetic pathway to the indole-containing aromatic amino acid L-tryptophan (Figure 4.10).

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