Folic acid (vitamin B9) (Figure 4.6) is a conjugate of a pteridine unit, p-aminobenzoic acid, and glutamic acid. It is found in yeast, liver, and green vegetables, though cooking may destroy up to 90% of the vitamin. Deficiency gives rise to anaemia, and supplementation is often necessary during pregnancy. Otherwise, deficiency is not normally encountered unless there is malabsorption, or chronic disease. Folic acid used for supplementation is usually synthetic, and it becomes sequentially reduced in the body by the enzyme dihydrofolate reductase to give dihydrofolic acid and then tetrahydrofolic acid (Figure 4.6). Tetrahydrofolic acid then functions as a carrier of one-carbon groups, which may be in the form of methyl, methylene, methenyl, or formyl groups, by the reactions outlined in Figure 4.7. These groups are involved in amino acid and nucleotide metabolism. Thus a methyl group is transferred in the regeneration of methionine from homocysteine, purine biosynthesis involves methenyl and formyl transfer, and pyrimidine biosynthesis utilizes methylene transfer. Tetrahydrofolate derivatives also serve as acceptors of one-carbon units in degradative pathways.
Mammals must obtain their tetrahydrofolate requirements from their diet, but microorganisms are able to synthesize this material. This offers scope for selective action and led to the use of sulphanilamide and other antibacterial sulpha drugs, compounds which competitively inhibit dihydropteroate synthase, the biosynthetic enzyme incorporating p-aminobenzoic acid into the structure. These sulpha drugs thus act as antimetabolites of p-aminobenzoate. Specific dihydrofolate reductase inhibitors have also become especially useful as antibacterials,
OH H HN1
W5-formyl-FH4 ATP (folinic acid)
methotrexate methotrexate co2h o co2h
FH2 Figure 4.9
e.g. trimethoprim (Figure 4.8), and antimalarial drugs, e.g. pyrimethamine, relying on the differences in susceptibility between the enzymes in humans and in the infective organism. Anticancer agents based on folic acid, e.g. methotrexate (Figure 4.8), primarily block pyrimidine biosynthesis, but are less selective than the antimicrobial agents, and rely on a stronger binding to the enzyme than the natural substrate has. Regeneration of tetrahydrofolate from dihydrofolate is vital for DNA synthesis in rapidly proliferating cells. The methylation of deoxyuridylate (dUMP) to deoxythymidylate (dTMP) requires N5,N70-methylenetetrahydrofolate as the methyl donor, which is thereby transformed into dihydrofolate (Figure 4.9). N5-Formyl-tetrahydrofolic acid (folinic acid, leucovorin) (Figure 4.7) is used to counteract the folate-antagonist action of anticancer agents like methotrexate. The natural 6S isomer is termed levofolinic acid (levoleucovorin); folinic acid in drug use is usually a mixture of the 6R and 6S isomers.
In a sequence of complex reactions, which will not be considered in detail, the indole ring system is formed by incorporating two carbons from phosphoribosyl diphosphate, with loss of the original anthranilate carboxyl. The remaining ribosyl carbons are then removed by a reverse aldol reaction, to be replaced on a bound form of indole by those from L-serine, which then becomes the side-chain of L-tryptophan. Although a precursor of L-tryptophan, anthranilic acid may also be produced by metabolism of tryptophan. Both compounds feature as building blocks for a variety of alkaloid structures (see Chapter 6).
Returning to the main course of the shikimate pathway, a singular rearrangement process occurs transforming chorismic acid into prephenic acid phosphoribosyl PP
phosphoribosyl anthranilic acid
L-Trp phosphoribosyl PP
phosphoribosyl anthranilic acid
L-Ser co2h reverse aldol reaction reverse aldol reaction
imine-enamine tautomerism co2h
chorismic acid (pseudoequatorial conformer)
chorismic acid (pseudoaxial conformer)
prephenic acid co2h chorismic acid (pseudoequatorial conformer)
chorismic acid (pseudoaxial conformer)
(Figure 4.11). This reaction, a Claisen rearrangement, transfers the PEP-derived side-chain so that it becomes directly bonded to the carbocycle, and so builds up the basic carbon skeleton of phenylalanine and tyrosine. The reaction is catalysed in nature by the enzyme chorismate mutase, and, although it can also occur thermally, the rate increases some 106-fold in the presence of the enzyme. The enzyme achieves this by binding the pseudoaxial conformer of chorismic acid, allowing a transition state with chairlike geometry to develop.
Pathways to the aromatic amino acids L-pheny-lalanine and L-tyrosine via prephenic acid may vary according to the organism, and often more than one route may operate in a particular species according to the enzyme activities that are available (Figure 4.12). In essence, only three reactions are involved, decarboxylative aromatization, transamination, and in the case of tyrosine biosynthesis an oxidation, but the order in which these reactions occur differentiates the routes. Decarboxylative aromatization of prephenic acid yields phenylpyruvic acid, and PLP-dependent transamination leads to L-phenylalanine. In the presence of an NAD+-dependent dehydrogenase enzyme, decarboxylative aromatization occurs with retention of the hydroxyl function, though as yet there is no evidence that any intermediate carbonyl analogue of prephenic acid is involved. Transamination of the resultant 4-hydroxyphenylpyruvic acid subsequently gives L-tyrosine. L-Arogenic acid is the result of transamination of prephenic acid occurring prior to the decarboxylative aromatization, and can be transformed into both L-phenylalanine and L-tyrosine depending on the absence or presence of a suitable enzymic dehydrogenase activity. In some organisms, broad activity enzymes are known to be capable of accepting both prephenic acid and arogenic acid as substrates. In microorganisms and plants, L-phenylalanine and L-tyrosine tend to be synthesized separately as in Figure 4.12, but in animals, which lack the shikimate pathway, direct hydroxylation of L-phenylalanine to L-tyrosine, and of L-tyrosine to L-DOPA (dihydroxyphenylalanine), may be achieved (Figure 4.13). These reactions are catalysed by tetrahydropterin-dependent hydroxylase enzymes, the hydroxyl oxygen being derived from molecular oxygen. L-DOPA is a precursor of the catecholamines, e.g. the neurotransmitter noradrenaline and the hormone adrenaline (see page 316). Tyrosine and DOPA are also converted by oxidation reactions into a heterogeneous polymer melanin, the main pigment in mammalian skin, hair, and eyes. In this material, the indole system is not formed from tryptophan, but arises from DOPA by cyclization of DOPAquinone, the nitrogen of the side-chain then attacking the ortho-quinone (Figure 4.13).
Some organisms are capable of synthesizing an unusual variant of L-phenylalanine, the aminated derivative L-p-aminophenylalanine (L-PAPA) (Figure 4.14). This is known to occur by a series of reactions paralleling those in Figure 4.12, but utilizing the PABA precursor 4-amino-4-deoxychori-smic acid (Figure 4.4) instead of chorismic acid. Thus, amino derivatives of prephenic acid and py-ruvic acid are elaborated. One important metabolite known to be formed from L-PAPA is the antibiotic phenylpyruvic acid transamination: keto acid ^^ amino acid co2h co2h
phenylpyruvic acid decarboxylation, aromatization and loss of leaving group
chorismic acid an additional oxidation step (of alcohol to ketone) means OH is retained on decarboxylation and aromatization; no discrete ketone intermediate is formed h©Coh prephenic acid NAD+
co2h o h©Coh prephenic acid NAD+
4-hydroxyphenyl-pyruvic acid oh
4-hydroxyphenyl-pyruvic acid co2h t
L-Phe decarboxylation, aromatization and loss of leaving group
L-arogenic acid co2h nh2
L-Tyr oxidation means OH is retained on decarboxylation and aromatization; no discrete CO2H ketone intermediate is formed
TT tetrahydro- „ teteatydro- CO H
C°2H biopteri^ biopterin CATECHOLAMINES
L-Phe L-Tyr L-DOPA
DOPAchrome DOPAquinone Figure 4.13
chloramphenicol*, produced by cultures of Strep-tomyces venezuelae. The late stages of the pathway (Figure 4.14) have been formulated to involve hydroxylation and N-acylation in the side-chain, the latter reaction probably requiring a coenzyme A ester of dichloroacetic acid. Following reduction of the carboxyl group, the final reaction is oxidation of the 4-amino group to a nitro, a fairly rare substituent in natural product structures.
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