Kynurenine hydroxylation

Another major pathway of kynurenine metabolism (step/, Fig. 25-11) is hydroxylation to 3-hydrox-... 

In view of the current interest in hydroxylation reactions in vivo it is to be hoped that this stage of tryptophan metabolism will receive more detailed attention from enzymologists. At present it can be stated definitely only that kynurenine (or possibly a derivative such as A -acetylkynurenine 170, 173) is converted either to hydroxykynurenine or to some simple derivative at the same level of oxidation. It is of interest that both kynurenine and hydroxykynurenine are formed on photooxidation of tryptophan, especially in the presence of ferrous iron (972). 

Tryptophan is an essential amino acid involved in synthesis of several important compounds. Nicotinic acid (amide), a vitamin required in the synthesis of NAD+ and NADP+, can be synthesized from tryptophan (Figure 17-24). About 60 mg of tryptophan can give rise to 1 mg of nicotinamide. The synthesis begins with conversion of tryptophan to N-formylkynurenine by tryptophan pyrrolase, an inducible iron-porphyrin enzyme of liver. N-Formylkynurenine is converted to kynurenine by removal of formate, which enters the one-carbon pool. Kynurenine is hydroxylated to 3-hydroxykynurenine, which is converted to 3-hydroxyanthranilate, catalyzed by kynureninase, a pyridoxal phosphate-dependent enzyme. 3-Hydroxyanthranilate is then converted by a series of reactions to nicotinamide ribotide, the immedi-... 

As described above, KMO catalyzes the hydroxylation at the third position of kynurenine. The KMO enzyme is thus at a key position of the pathway as its activity determines the level of flux through the two arms of the pathway. KMO inhibition is expected to be beneficial in neurodegenerative disease as this would increase the availability of KYN to KATII, and thus achieves a shift away from QUIN and 3-HK production to an increase in KYNA production. Thus, KMO has been considered the most relevant target for therapeutic intervention in the KP for CNS disease [1]. 

Little of the tryptophan that enters the tryptophan-niacin pathway is actually used to form nicotinie acid ribonucleotide, and 60 mg of tryptophan results in the formation of only about 1 mg of nicotinic add (C8). Evidence recently reviewed (C8) indicates that this ratio is not a fixed one and shows considerable variation depending upon the amount of tryptophan and preformed nicotinic acid available to the organism and also the amount of PLP present. It is also of interest that nicotinie acid in the form of NADPH is required in one enzymic step in the kynurenine pathway, the hydroxylation of kymurenine to 3-hydroxy k murenine. 

L-Kynurenine obtained from for the degradation of tryptophan is hydroxylated by a KMO homolog encoded by qbsG. The 3-hydroxy-kynurenine could be transam-inated into xanthurenic acid by the QbsB protein. The bifunctional protein QbsL activates xanthurenic acid via its N-terminal AMP (adenosine monophosphate) lig-ase domain, whereas the C-terminal domain of QbsL is responsible for the addition of the methyl group. QbsCDE proteins transfer sulfur from an unknown sulfur donor molecule. The participation and exact role of QbsK, a putative oxidoreductase, was not clear. Quinolobactin was proposed to result from the spontaneous hydrolysis of 8-hydroxy-4-methoxy-2-quinoline thiocarboxylic acid 17 [18]. 

The precursors of kynurenic acid and its derivatives are L-kynurenine and hydroxylated L-kynurenines, like 3-hydroxy-L-kynurenine (D 21), 5-hydroxy-L-kynurenine (derived from 5-hydroxy-L-tryptophan, D 21), 3,4-dihydroxy-L-kynurenine, etc. Quinaldic acid and related substances not hydroxylated in position 4 are derived from the hydroxylated compounds in animals. The reduction is probably performed by the microorganisms living in the intestinal tract. 4-Hydroxyquinoline and related substances devoid of the carboxy group in position 2 are derived from kynuramine. 

Kynurenine formamidase catalyzes the hydrolysis of formylkynurenine. A variety of aromatic forma-mides will react, but the enzyme is more active with its natural than with the synthetic substrates. The product of the reaction, kynurenine, may then either be hydroxylated or decarboxylated. 

Kynurenine is hydroxylated to hydroxykynurenine by an enzyme (kynurenine-3-hydroxylase) found in rat liver mitochondria. The reaction requires NADPH and molecular oxygen. In the presence of pyridoxal phosphate, hydroxykynurenine is hydrolyzed by an enzyme (kynurenase) found in liver and kidney. The product of this reaction is 3-hydroxyanthranilic acid. The same enzyme catalyzes the cleavage of the side chain of kynurenine to yield alanine and anthranilic acid. Studies made with labeled 3-hydroxyanthranilic acid demonstrated its role as an intermediate of the biosynthesis of nicotinic acid. These studies established that the label of the carbon 3 of 3-hydroxyanthranilic acid is transferred to the a-carbon of quinolinic acid and is lost as C14O2 during the conversion of quinolinic to nicotinic acid. The details of the metabolic conversion of 3-hydroxyanthranilic acid to nicotinic acid are known. 

Kynurenine Hydroxylase. Kynurenine is hydroxylated by an enzyme prepared from mitochondria of animal livers. The properties of this enzyme are very similar to those described from the hydroxylation of other aromatic amines, steroids, and phenylalanine molecular oxygen is consumed and an equivalent of TPNH is oxidized simultaneously with kynurenine oxidation (VIII). 

It has been observed that the metabolism of tryptophan is also greatly influenced by riboflavin deficiency. In this deficiency there is an increased excretion of metabolic products of tryptophan such as N -acetylkynurenine, N -acetyl-3-hydroxy-kynurenine, kynurenic acid, and xanthurenic acid. In a search for the specific metabolic defect Charconnet-Harding, Dalgliesh, and Neuberger Biochem. J. London) 63, 513, 1953) concluded that riboflavin might be concerned with an unknown phosphorylation step but is not concerned with the oxidative hydroxyl-ation of kynurenine to hydroxykynurenine or anthranilic acid to hydroxyanthranilic acid. The authors also point out that riboflavin may have no specific metabolic role in tryptophan metabolism. 

Several alternative pathways of L-tryptophan metabolism diverge from kynurenine (24). In mammals the quantitatively major fate of the benzene ring of the amino acid appears to be its oxidation to carbon dioxide via 3-hydroxyanthranilic acid (25), Figure 4.5. Kynurenine is first hydroxylated by a typical mixed function oxidase and the side chain is then removed, under the... 

The enzyme is specific in requiring L-kynurenine as the substrate and hydroxylating only at position 3. 

The enzymic hydroxylation of kynurenine has been reported by several groups of workers (62,63). The enzyme is present in rat liver mitochondria and can be solubilized by sonic oscillation or digitonin treatment (63a). Reduced triphosphopyridine nucleotide (TPNH) is cifically required for the hydroxylation and there is a stoichiometric relationship between the TPNH oxidized and the hydro kynureiiine formed (62). Molecularo gen is also required for the hydroxylation, as indicated by 0 studies ( ). The... 

Treatment of the antibiotic with carboxypeptidase Y showed the absence of a C-terminal amino acid, while L-kynurenine was released when the antibiotic was pretreated with base to open the lactone ring. Therefore the lactone ring involved the carboxyl group of kynurenine as well as the hydroxyl group of threonine. 

In the course of the metabolic transformation of trsrptophan to 3-hydroxyanthranilic acid and xanthurenic acid (Fig. 4), 3-hydroxy-kynurenine is formed from kynurenine (182,538). An enzymic system which catalyzes this reaction has been observed in the livers of rats and cats (198). This 3-hydroxylase occms in mitochondria. It appears to be specific for L-kynurenine because n-kynurenine, A -acetyl-L-kynurenine, AT-acetyl-D-kynurenine, kynurenic acid, and anthranilic acid are not attacked. Only the 3-position of kynurenine is hydroxylated. 

Functional end products of the essential amino acid tryptophan arise mainly through two distinctive pathways. The major pathway is degradation of tryptophan by oxidation, which fuels the kynurenine pathway (See 02011). The second and quantitatively minor pathway is hydroxylation of tryptophan and its subsequent decarboxylation to the indoleamine 5-hydroxytryptamine (serotonin) and subsequently melatonin. The metabolites of the kynurenine pathway, indicated as kynurenines, include quinolic acid and kynurenic acid. Quinolinic acid is an agonist of the NMDA receptor (see also section on glutamic acid), while kynurenic acid is a nonselective NMDA-receptor antagonist with a high affinity for the glycine site of the NMDA receptor (see also section on... 

The oxidation of tryptophan by various strains of Pseudomonas has been shown to proceed in all cases via kynurine. One sequence of reactions, the aromatic pathway, continues by eliminating the alanine side chain through the action of kynureninase, and subsequently utilizes oxygen for the formation of catechol and the pyrocatechase reaction already discussed. Another pathway retains the side chain of kynurenine and forms kynurenic acid through the action of kynurenine transaminase. A sequence of reactions has been indicated by recent work of Hayaishi and his associates (Kuno et al., 1961) this sequence appears to include three oxygenase reactions one hydroxylation and two phenolytic oxygenations (Fig. 18). 

Most of the kynurenine formed is, however, hydroxylated to xanthurenic acid and 8-hydroxyquinaldic acid. The enzyme kynureninase yields from 3-hydroxykynurenine 3-hydroxyanthranilic acid, which is then degraded via the glutarate pathway. 

Studies have been made on the effect on tryptophan of a system (ferrous sulfate, ascorbic acid, EDTA, pH 6.7, 37° C) which supposedly effects hydroxylation of aromatic compounds in a manner closely analogous to in vivo hydroxylations. Dalgliesh (94) detected 5- and possibly 7-hydroxy-tryptophan using this oxidizing system, while kynurenine gave 3- and 5-hydroxy-kynurenine as principal products. However, Pfaender et al. (301), using a similar system, obtained principally oxindolylalanine (13%) together with low yields of 5- and... 

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