Mechanistic enzymology of quinolinic acid biosynthesis

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DESCRIPTION (provided by applicant): Nicotinamide adenine dinucleotide (NAD) and its phosphate (NADP) are essential redox cofactors in all living systems. Surprisingly, the biosynthesis of the key catalytic part of this cofactor, the quinolinic acid derived pyridine ring, is still poorly understood. We have recently discovered a bacterial system containing the enzymes required for the conversion of tryptophan to quinolinate. In dramatic contrast to the eukaryotic enzymes, all of the bacterial enzymes overexpress at a high level as soluble stable enzymes, opening up this system for detailed characterization. In this proposal, we describe mechanistic studies on tryptophan dioxygenase, formylkynurenine formamidase, kynurenine monooxygenase, kynureninase and hydroxyanthranilate dioxygenase. These studies will include structural studies, the characterization of the presteady state kinetics of each enzyme, the synthesis and testing of substrate analogs designed to trap reaction intermediates and ESR studies. The tryptophan to quinolinic acid pathway intermediates play an important role in several biological processes. Kynurenine is a precursor to kynurenic acid, an antagonist of the glutamate receptor. Both quinolinic acid and 3-hydroxykynurenine concentrations are elevated in patients with AIDS related dementia, Huntington's disease and hepatic encephalopathy. 3-Hydroxykynurenine induces apoptosis of neurons prepared from rat striatum. This intermediate also functions as a filter in the eye, protecting the retina from the damaging effects of UV-light. Crosslinking reactions mediated by 3-hydroxykynurenine may play a role in cataract formation. Mechanistic studies on the quinolinic acid biosynthetic enzymes will facilitate the identification of small molecule inhibitors to control these disease states.