The biosynthesis of quinolinate 3, the precursor to the pyridine ring of NAD, is still poorly understood. Two pathways have been identified, one involving the direct formation of quinolinic acid from aspartate and dihydroxyacetone phosphate, the other requiring a five-step degradation of tryptophan. The final step in this degradation is catalyzed by the non-heme Fe(II)-dependent enzyme 3-hydroxyanthranilate-3,4-dioxygenase (HAD). This enzyme catalyzes the oxidative ring opening of 3-hydroxyanthranilate (1) to 2-amino-3-carboxymuconic semialdehyde (ACMS, 2) which then cyclizes to quinolinate (3). In this communication, we demonstrate the following: (1) cyclization of ACMS to 3 is not HAD catalyzed, (2) the most stable form of ACMS in solution is an all trans isomer which undergoes facile cis to trans isomerization about the C2-C3 and C4-C5 double bonds via transient formation of its enol tautomer (6), (3) a model study on the ring opening of N,N-dimethylcarbamoylpyridinium with hydroxide and methoxide suggests that the cyclization of ACMS occurs by an electrocyclization reaction of its enol tautomer 6. Thus, the biosynthesis of quinolinic acid, by the tryptophan pathway, is likely to be a member of a growing family of natural products whose biosynthesis involves a pericyclic reaction.

ABSTRACT A new tryptophan catabolic pathway is characterized from Burkholderia cepacia J2315. In this pathway, tryptophan is converted to 2-amino-3-carboxymuconate semialdehyde, which is enzymatically degraded to pyruvate and acetate via the intermediates 2-aminomuconate and 4-oxalocrotonate. This pathway differs from the proposed mammalian pathway which converts 2-aminomuconate to 2-ketoadipate and, ultimately, glutaryl-coenzyme A.

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) catalyzes the oxidative ring opening of 3-hydroxyanthranilate in the final enzymatic step of the biosynthetic pathway from tryptophan to quinolinate, the universal de novo precursor to the pyridine ring of nicotinamide adenine dinucleotide. The enzyme requires Fe2+ as a cofactor and is inactivated by 4-chloro-3-hydroxyanthranilate. HAD from Ralstonia metallidurans was crystallized, and the structure was determined at 1.9 A resolution. The structures of HAD complexed with the inhibitor 4-chloro-3-hydroxyanthranilic acid and either molecular oxygen or nitric oxide were determined at 2.0 A resolution, and the structure of HAD complexed with 3-hydroxyanthranilate was determined at 3.2 A resolution. HAD is a homodimer with a subunit topology that is characteristic of the cupin barrel fold. Each monomer contains two iron binding sites. The catalytic iron is buried deep inside the beta-barrel with His51, Glu57, and His95 serving as ligands. The other iron site forms an FeS4 center close to the solvent surface in which the sulfur atoms are provided by Cys125, Cys128, Cys162, and Cys165. The two iron sites are separated by 24 A. On the basis of the crystal structures of HAD, mutagenesis studies were carried out in order to elucidate the enzyme mechanism. In addition, a new mechanism for the enzyme inactivation by 4-chloro-3-hydroxyanthranilate is proposed.

3-Hydroxyanthranilate-3,4-dioxygenase (HAD) is a non-heme Fe(II) dependent enzyme that catalyzes the oxidative ring-opening of 3-hydroxyanthranilate to 2-amino-3-carboxymuconic semialdehyde. The enzymatic product subsequently cyclizes to quinolinate, an intermediate in the biosynthesis of nicotinamide adenine dinucleotide. Quinolinate has also been implicated in important neurological disorders. Here, we describe the mechanism by which 4-chloro-3-hydroxyanthranilate inhibits the HAD catalyzed reaction. Using overexpressed and purified bacterial HAD, we demonstrate that 4-chloro-3-hydroxyanthranilate functions as a mechanism-based inactivating agent. The inactivation results in the consumption of 2 +/- 0.8 equiv of oxygen and the production of superoxide. EPR analysis of the inactivation reaction demonstrated that the inhibitor stimulated the oxidation of the active site Fe(II) to the catalytically inactive Fe(III) oxidation state. The inactivated enzyme can be reactivated by treatment with DTT and Fe(II). High resolution ESI-FTMS analysis of the inactivated enzyme demonstrated that the inhibitor did not form an adduct with the enzyme and that four conserved cysteines were oxidized to two disulfides (Cys125-Cys128 and Cys162-Cys165) during the inactivation reaction. These results are consistent with a mechanism in which the enzyme, complexed to the inhibitor and O2, generates superoxide which subsequently dissociates, leaving the inhibitor and the oxidized iron center at the active site.