A complete pathway for Azorhizobium caulinodans nicotinate catabolism has been determined from mutant phenotype analyses, isolation of metabolic intermediates, and structural studies. Nicotinate serves as a respiratory electron donor to 02 via a membrane-bound hydroxylase and a specific c-type cytochrome oxidase. The resulting oxidized product, 6-hydroxynicotinate, is next reduced to 1,4,5,6-tetrahydro-6-oxonicotinate. Hydrolytic ring breakage follows, with release of pyridine N as ammonium. Decarboxylation then releases the nicotinate C-7 carboxyl group as C02, and the remaining C skeleton is then oxidized to yield glutarate. Transthioesterification with succinyl coenzyme A (succinyl-CoA) yields glutaryl-CoA, which is then oxidatively decarboxylated to yield crotonyl-CoA. As with general acyl x oxidation, L-P-hydroxybutyryl-CoA, acetoacetylCoA, and finally two molecules of acetyl-CoA are produced. In sum, nicotinate is catabolized to yield two CO2 molecules, two acetyl-CoA molecules, and ammonium. Nicotinate catabolism stimulates Azorhizobium N2 fixation rates in culture. Nicotinate catabolism mutants still able to liberate pyridine N as ammonium retain this capability, whereas mutants so blocked do not. From, mutant analyses and additional physiological tests, N2 fixation stimulation is indirect. In N-limited culture, nicotinate catabolism augments anabolic N pools and, as a consequence, yields N2-fixing cells with higher dinitrogenase content.Azorhizobium caulinodans invades both stems and roots of the host legume Sesbania rostrata and yields symbiotic nodules that fix N2 at very high rates. All wild Azorhizobium isolates are phenotypically indistinguishable and are auxotrophic for pyridine nucleotide cofactor (NAD+) biosynthesis; culture in defined medium requires nicotinate supplementation (10). When so cultured, A. caulinodans also uses nicotinate as the sole N source for growth (22). Whereas all other studied rhizobia exclusively fix N2 in symbiosis, A. caulinodans avidly fixes N2 in culture (10). Paradoxically, when fixing N2 in culture under optimal conditions, A. caulinodans exhaustively catabolizes nicotinate, and its growth becomes NAD+ limited. Relative to a submicromolar nicotinate vitamin requirement, high-level (0.3 mM) nicotinate supplementation strongly stimulates N2 fixation in culture (4, 11). We have sought to understand how nicotinate stimulates N2 fixation.Although nicotinate is a good N source, it is a poor C source for Azorhizobium growth (18). However, stoichiometric measurements of nicotinate-stimulated 02 consumption indicate that A. caulinodans completely utilizes the nicotinate C skeleton (18). Cyclic intermediates in this pathway were previously isolated and characterized, and a mechanism of pyridine ring N release was proposed (18) (Fig. 1). Nicotinate is first oxidized to yield 6-hydroxynicotinate by a membrane-bound hydroxylase, which uses 02 as an electron acceptor. The nicotinate-specific respiratory chain to 02 terminates at a c-type cytochrome oxidase (17). The reaction prod...
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