Recombinant kynurenine-3-hydroxylase enzyme and process for its

Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase

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4352523, 4352542, 4353201, 435325, 435348, 536 232, 536 235, C12N 902, C12N 121, C12N 1553, C12N 119, C07H 2104

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061070696

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BRIEF SUMMARY
BACKGROUND OF THE INVENTION

The present invention concerns DNA sequences encoding for kynurenine-3-hydroxylase (kyn-3-OHase).
This enzyme is a flavin-containing monooxygenase which is localized in the outer mitochondrial membrane (Okamoto H., Yamamoto S., Nozaki M. and Hayaishi O. 1967. Biochem. Biophys. Res. Commun. 26: 309-314); it catalyses the 3 hydroxylation of L-kynurenine (L-kyn), an intermediate in the oxidative metabolism of tryptophan (DeCastro F. T., Price J. M. and Brown R. R., 1956. J. Am. Chem. Soc. 78: 2900-2904).
The kynurenine pathway (see the scheme below) is the major route of peripheral tryptophan metabolism in mammals: most of this metabolism takes place in the liver. The abbreviations used are the following: IDO, Indoleamine 2,3-dioxygenase; TDO, Tryptophan 2,3-dioxygenase; ED, 3-hydroxyanthranilate 3,4-dioxygenase; QPRT, Quinolinate phosphoribosyltransferase; ATP, Adenosine 5'-triphosphate; NAD, Nicotinamide adenine dinucleotide; NMN, Nicotinamide mononucleotide. ##STR1##
This pathway not only provides a route for total oxidation of tryptophan to acetyl-Co-A, but it is also responsible for the synthesis de novo of the nicotinamide nucleotide coenzymes NAD and NADP (Bender D. A. and McCreanor G. M. 1985. Biochem. Soc. Trans. 13:441-443).
Most of the current interest in this pathway arises from the observations that two intermediate metabolites, kynurenic acid (KYNA) and quinolinic acid (QUIN), seem to play a significant role in neurological diseases, the first acting as a neuroprotectant and the second as a neurotoxic agent. Kyn-3-OHase is the first enzyme in the route of production of QUIN.
The importance of QUIN as a neurotoxic agent was first evident from work by Lapin (1978. J. Neural. Trans. 42:37-43) who demonstrated that the administration of QUIN to rats caused convulsions. This was not sufficient to classify QUIN as a neurotoxin; its action in the central nervous system was better clarified when electrophysiological studies revealed that it was an agonist at the excitatory amino acid receptor sites normally activated by glutamate and aspartate (Stone T. W. and Perkins M. N., 1981. Eur J. Pharmacol. 72: 411-412).
Moreover, using the intrastriatal injection model, QUIN toxicity has been shown to be mediated through the N-methyl-D-aspartate (NMDA) receptors (Beal M., Kowall N., Swartz K. J., Ferrante R. J. and Martin J. B. 1989. J. Neurosci. 8: 3901-3908; Foster A. C., Gill R. and Woodruff G. N. 1988. J. Neurosci. 8: 4745-4754). Consistent with the involvment of NMDA receptors were the studies that showed reversal of QUIN-induced damage pathology by competitive NMDA antagonists (Foster A. C., Vezzani A., French E. D. and Schwarcz R. 1984. Neurosci. Lett. 48: 273-278; Leeson P. D., Baker R., Carling R. W., Curtis N. R., Moore K. W., Williams B. J. et al. 1991. J. Med. Chem. 34: 1243-1252).
It is becoming clear that some of the most important functions of the nervous system, such as synaptic plasticity and synapse formation, critically depend on the behavior of NMDA receptor channels and that neurological damages caused by a variety of pathological states can result from exaggerated activation of NMDA receptor channels (For a review see: Mori H. and Mishina M. 1995. Neuropharmacology 34: 1219-1237). Excessive activation of these receptors may play an important role in the neuronal injury associated with several disease states, including hypoxia-ischemia (Simon R. P., Swan J. H., Griffiths T. and Meldrum B. A. 1984. Science 226: 850-852), hypoglycemia (Wieloch T. 1985. Science 230: 681-683) and Huntington's disease (Schwarcz R., Whetsell W. O. Jr. and Mangano R. M. 1983. Science 219: 316-318. Koh J. Y., Peters S. and Choi D. W. 1986. Science 234: 73-76).
Assuming that QUIN is pathogenic for certain disorders, it is desirable to inhibit its formation. To accomplish this goal, knowledge must be gained about the enzymes that make QUIN and the sites at which the pathway is controlled.
In theory, QUIN could be formed in the brain in several ways (see the Kynurenine pathway above): from

REFERENCES:
Warren et al. (May 31, 1996) Genetica, vol. 98, pp. 249-262.
Sigel (1990) J. Membrane Biol., vol. 117, pp. 201-221.
Nishimoto et al. (1979) J. Chromatography, vol. 169, pp. 357-364 (abstract) .

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