Elucidation of the complete Azorhizobium nicotinate catabolism pathway

Abstract

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...

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group

“…There is, however, a precedent for the involvement of c-type cytochromes in aromatic hydroxylation. The catabolism of nicotinic acid by Pseudomonas jluorescens, Pseudornonas putidu and Azorhizobiurn caulinodans proceeds via an initial hydroxylation reaction that is not NADPH dependent but is instead associated with membrane-associated electron transport to oxygen (Hunt, 1959;Jones and Hughes, 1972;Kitts et al, 1989) involving b-type cytochrome(s), ctype cytochrome(s) and an o-type cytochrome oxidase all of which are reducible by nicotinate but not by NADH or succinate, thus constituting a dedicated electron transport chain (Jones and Hughes, 1972) ; c-type cytochrome-deficient mutants of A. caulinodans are reportedly unable to catalyse nicotinate hydroxylation (unpublished results cited in Kitts et al, 1992). Our failure to observe quinone staining of the accumulated methylamine dehydrogenase L subunit in the cytochrome-c-deficient mutants suggests that a c-type cytochrome-linked hydroxylase similar to that described in f!…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Since it is known that the alphaproteobacterium Azorhizobium caulinodans is able to catabolize nicotinate aerobically via 1,4,5,6-tetrahydro-6-oxonicotinate and 2-formylglutarate (187), it could be expected that the first steps of this aerobic pathway might be encoded by ndh, hnr, and ena orthologs. The further catabolism of 2-formylglutarate in the nine identified proteobacteria probably proceeds via the glutarate pathway described for A. caulinodans (187), thus explaining the absence of hgd, hmd, mgm, mii, dmdAB, and dml homologs in such aerobic gene clusters (4).…”

Nicotinate catabolism is dispensable and nicotinate anabolism is crucial in Azorhizobium caulinodans growing in batch culture and chemostat culture on N2 as The N source

“…Subsequently, the complex growth interdependence of N 2 -fixing A. caulinodans cultures and nicotinate was investigated in depth (3, 10-12). Importantly, A. caulinodans fails to synthesize pyridine nucleotide cofactors de novo and thus requires nicotinate (vitamin) supplementation; A. caulinodans is thus auxotrophic for NAD ϩ biosynthesis (11).Apart from nicotinate's fate as an anabolic substrate, nicotinate catabolism seems to somehow stimulate N 2 fixation in culture (3,(10)(11)(12). Nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase (11).…”

“…When these vitamin requirements were reexamined by measuring the maximal amount of biomass (by the absorbance at 570 nm [A 570 ]) produced in batch cultures, only biotin and pantothenate were needed in (sub)micromolar quantities, whereas nicotinate was required at relatively high (0.3 mM) levels (7). Subsequently, the complex growth interdependence of N 2 -fixing A. caulinodans cultures and nicotinate was investigated in depth (3,(10)(11)(12). Importantly, A. caulinodans fails to synthesize pyridine nucleotide cofactors de novo and thus requires nicotinate (vitamin) supplementation; A. caulinodans is thus auxotrophic for NAD ϩ biosynthesis (11).…”

“…Apart from nicotinate's fate as an anabolic substrate, nicotinate catabolism seems to somehow stimulate N 2 fixation in culture (3,(10)(11)(12). Nicotinate is first oxidized to 6-OH-nicotinate by a membrane-bound nicotinate hydroxylase (11).…”