Tryptophan-kynurenine-xanthurenic acid pathway

Quinolobactin, 8-hydroxy-4-methoxy-2-quinoline carboxylic acid, is an alkaloid produced by Pseudomonas fluorescens ATCC17400. The biosynthesis of quinolobactin involves the tryptophan-kynurenine-xanthurenic acid pathway (Figure 6.13). 

Tryptophan catabolism is also associated with several dead-end pathways, for example the formation of kynurenic and xanthurenic acids. Normal urine contains small amounts of hydroxykynurenine, kynurenine, kynurenic acid, and xanthurenic add. When large amounts of tryptophan are fed to animals, the excretion of these compounds increases. Xanthurenic acid is excreted in massive quantities in vitamin B6 deficiency. 

Figure 8.4. Pathways of tryptophan metaholism. Tryptophan dioxygenase, EC 1.13.11.11 formylkynurenine formamidase, EC 3.5.1.9 kynurenine hydroxylase, EC 1.14.13.9 kynureninase, EC 3.7.1.3 3-hydroxyanthranilate oxidase, EC 1.10.3.5 picolinate carboxylase, EC 4.1.1.45 kynurenine oxoglutarate aminotransferase, EC 2.6.1.7 kynurenine glyoxylate aminotransferase, 2.6.1.63 tryptophan hydroxylase, EC 1.14.16.4 and 5-hydroxytryptophan decarboxylase, EC 4.1.1.26. Relative molecular masses (Mr) tryptophan, 204.2 serotonin, 176.2 kynurenine, 208.2 3-hydroxykynurenine, 223.2 kynurenic acid, 189.2 xanthurenic acid, 205.2 and quinolinic acid 167.1. CoA, coenzyme A.

Under normal conditions, the rate-limiting enzyme of the pathway is tryptophan dioxygenase (Section 8.3.2), and there is hide accumulation of intermediates. Kynurenine transaminase, the enzyme which catalyzes the transamination and ring closure of kynurenine to kynurenic acid, and of hydroxykynurenine to xanthurenic acid, has a high relative to the normal steady-state concentrations of its substrates in the liver. Kynureninase and kynurenine hydroxylase have lower values of K, so that there is normally litde accumuladon of kynurenine or hydroxykynurenine. 

Alloxan-diabetic animals, in which the conversion of tryptophan to methylnicotinamide was greatly impaired, excreted increased amounts of methylnicotinamide when very large doses of tryptophan were given, indicating that the defect may be due to changes in the kynurenine pathway of tryptophan (M5). Moreover, diabetic rats excreted much more xanthurenic acid and less anthranilic and 3-hydroxyanthranilic acids than did nondiabetics, following large doses (200-400 mg) of tryptophan. 

More recently, Olson et al. (05) reported that in 34 patients with chronic alcoholism the excretion of 5-hydroxyindoleacetic acid was significantly lower than in normal controls, after oral ingestion of 10 g DL-tryptophan. Such lowered excretion was unaffected by improved nutritional status or abstinence from ethyl alcohol. The urinary excr on of xanthurenic acid was similar in the alcoholic and control groups. The conclusions indicate that in chronic alcoholism the conversion of tryptophan to 5-hydroxyindoleacetic acid is preferentially depressed, while the kynurenine pathway is normal (05). 

Interest then moved to animals. Both isotopic and nutritional experiments showed that the pathway established in microorganisms applied equally to mammals. Thus hydroxyanthranilic acid was converted to nicotinic acid (9, 604), which it could replace as a growth factor (944), whereas there was no similar conversion of anthranilic acid (343). An outstanding series of isotopic experiments, especially by Heidelberger and co-workers, showed that the 8-carbon atom of the tryptophan side chain became the 8-carbon atom of the kynurenine side chain and that the side chain was lost in conversion of kynurenine to nicotinic acid (369, 371, 427). Moreover the carbon in the 3-position of the indole nucleus became the carboxyl carbon of nicotinic acid (370 this experiment proved conclusively the reality of the tryptophan-nicotinic acid conversion) and the indole nitrogen appeared with only slight dilution in kynurenine, kynurenic acid, and xanthurenic acid (759). All these relations are those to be expected for the pathway tryptophan —+ kynurenine —> hydroxykynurenine (or its phosphate) —> hydroxyanthranilic acid (or its phosphate) — nicotinic acid, illustrated in diagrams 17 and 18. 

If the dietary levels of niacin and tryptophan are insufficient, the condition known as pellagra results. The symptoms of pellagra are dermatitis, diarrhea, dementia, and, finally, death. In addition, abnormal metabolism of tryptophan occurs in a vitamin B6 deficiency. Kynurenine intermediates in tryptophan degradation cannot be cleaved because kynureninase requires PLP derived from vitamin B6. Consequently, these intermediates enter a minor pathway for tryptophan metabolism that produces xanthurenic acid, which is excreted in the urine. 

Tryptophan is also metabolized along the kynurenine pathway en route to nicotinic acid following its initial oxidation by tryptophan pyrro-lase. Several metabolites along this pathway are electroactive and include w-formylkynurenine, kynurenine, kynurenic acid, anthranilic acid, 3-hy-droxykynurenine, and xanthurenic acid. 

Research on tryptophan metabolism can be divided into three stages a first in which the end products of tryptophan metabolism were studied by investigating the composition of urine a second in which the intermediates of the various metabolic pathways were studied by following the urinary excretion of injected radioisotopes and a third in which an attempt was made to identify the enzymes involved in tryptophan metabolism. In the middle of the 19th century, it was demonstrated that kynurenine is excreted in dogs urine. Fifty years later, it was shown that kynurenic acid excretion is stimulated by the administration of tryptophan. A variety of other compounds were also found in the urine, including xanthurenic acid and quinolinic-2-carboxylic acid. 

Of the parasitic diseases the most devastating one is malaria with a worldwide death toll of more than a million people. It is transmitted by the mosquito. To combat this, the life cycle and metabolism of the parasite Plasmodium falciparum is affected through a reduction in the synthesis of xanthurenic acid (92). Xanthurenic acid, required for gametogenesis and fertility of the parasite, is synthesized in the tryptophan degradation pathway through the PLP-dependent enzyme kynurenine aminotransferase. Hence, this enzyme is a target for the development of antimalarial drugs. 

This pathway occurs in a incomplete form in the vertebrate organism. In certain bacteria it is an important pathway for the total oxidation of tryptophan. The quinolinic pathway arises by transamination of kynurenine and hydroxykynurenine. The resulting products qiontaneously cyclize to form kynurenic and xanthurenic acids. 

The experiments discussed above on the biosynthesis of a pseudan have also shown that kynurenic acid is not the precursor of the 2-alkylquinolin-4(lH)-ones. Based on the putative function of the genes of the qbs operons (176), a pathway was proposed for the biosynthesis of kynurenic acid (I), xanthurenic acid [10(8)], and quinolobactin [IO(8) j in Pseudomonas fluorescens ATCC 17400 (Scheme 5). The first step, the oxidation of tryptophan to N-formylkynurenine, is likely to be catalyzed by the enzyme tryptophan 2,3-dioxygenase (TDO) (QbsF), which is a heme-dependent enzyme. The second step, the deformylation of N-formyl-kynurenine to L-kynurenine is catalyzed by kynurenine formamidase (KFA). The product of qbsH, a metal-dependent hydrolase found also in other bacterial genomes, is the likely candidate. 

The oxidative pathway of tryptophan metabolism is shown in Figure 3. Kynureninase is a pyridoxal phosphate-dependent enzyme, and in deficiency its activity is lower than that of tryptophan dioxygenase, so that there is an accumulation of hydroxy-kynurenine and kynurenine, resulting in greater metabolic flux through kynurenine transaminase and increased formation of kynurenic and xanthurenic acids. Kynureninase is exquisitely sensitive to vitamin Bg deficiency because it undergoes a slow inactivation as a result of catalysing the half-reaction of transamination instead of its normal reaction. The resultant enzyme with pyridoxamine phosphate at the catalytic site is catalytically inactive and can only be reactivated if there is an adequate concentration of pyridoxal phosphate to displace the pyridoxamine phosphate. 

As discussed in Section 8.3.3, estrogen metabolites inhibit kynureninase and reduce the activity of kynurenine hydroxylase to such an extent that, even without induction of tryptophan dioxygenase (Section 9.5.4.1), the activity of these enzymes is lower than is needed for the rate of flux through the pathway, thus leading to increased formation of xanthurenic and kynurenic acids. 

Xanthurenic and kynurenic acids, and kynurenine and hydroxykynurenine, are easy to measure in urine, so the tryptophan load test (the ability to metabolize a test dose of 2—5 g of tryptophan) has been widely adopted as a convenient and very sensitive index of vitamin nutritional status. However, because glucocorticoid hormones increase tryptophan dioxygenase activity, abnormal results of the tryptophan load test must be regarded with caution, and cannot necessarily be interpreted as indicating vitamin B deficiency. Increased entry of tryptophan into the pathway will overwhelm the capacity of kynureninase, leading to increased formation of xanthurenic and kynurenic acids. Similarly, oestrogen metabolites inhibit kynureninase, leading to results that have been misinterpreted as vitamin B deficiency. 

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