The L-aspartate oxidase reported to be present in higher plants is actually glutamic oxaloacetic transaminase.

We previously reported (Biochem. Biophys. Res. Commun. (1983) 111, 188-193) that cotton callus cells contain an L-aspartate oxidase which requires an unidentified cofactor with an apparent molecular weight of 1,050. Further study has revealed that this report was in error. The enzyme is actually glutamic oxaloacetic transaminase and the "cofactor" has been identified as alpha-ketoglutarate.

Higher plants contain L-asparate oxidase, the first enzyme of the Escherichia coli quinolinate synthetase system.

Cotton callus cells contain an L-aspartate oxidase which does not appear to be active with D-aspartate, L-glutamate or D- or L-alanine. The enzyme requires for activity a dialyzable cofactor with an apparent molecular weight of 1,050. Since L-aspartate oxidase is the first enzyme of the pathway for de novo synthesis of the pyridine ring in Escherichia coli, this finding suggests that higher plants may use the L-aspartate-dihydroxyacetone phosphate pathway for de novo pyridine nucleotide biosynthesis.

The mammalian enzyme which replaces B protein of E. coli quinolinate synthetase is D-aspartate oxidase.

In Escherichia coli quinolinic acid, a precursor of NAD+, is synthesized from L-aspartate and dihydroxyacetone phosphate by two enzymes, an FAD-containing 'B protein' and 'A protein'. An enzyme which can replace the B protein in the E. coli quinolinate synthetase system when D-aspartate replaces L-aspartate as a substrate has been purified 300-fold from bovine kidney. This enzyme is shown to be identical with the previously described D-aspartate oxidase (D-aspartate:oxygen oxidoreductase (deaminating), EC 1.4.3.1). The immediate reaction product of D-aspartate oxidase (iminoaspartate) is condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by E. coli quinolinate synthetase A protein. In the absence of A protein (or dihydroxyacetone phosphate) iminoaspartate is spontaneously hydrolyzed to form oxaloacetate with a half-life of about 2.5 min at 25 degrees C and pH 8.0.

L-Aspartate oxidase, a newly discovered enzyme of Escherichia coli, is the B protein of quinolinate synthetase.

In Escherichia coli, quinolinic acid, a precursor of NAD+, is synthesized from L-aspartate and dihydroxyacetone phosphate. This synthesis requires two enzymes, a FAD-containing "B protein" and an "A protein." The B protein has been purified 500-fold from E. coli cells. The enzyme behaves as an L-aspartate oxidase. In the absence of A protein, it converts L-aspartate to oxaloacetate. To our knowledge, no enzyme with this activity has been described previously. The enzyme displays some unusual properties. In its role as B protein in quinolinic acid synthetase, product formation (quinolinic acid) is linear with protein concentration; however, when it functions as an L-aspartate oxidase, product formation (oxaloacetate) is a parabolic function of protein concentration. The L-aspartate oxidase activity also shows marked substrate activation at substrate concentrations above 1.0 mM. The L-aspartate oxidase and B protein activities of the enzyme are inhibited by NAD+, which is competitive with FAD. The immediate reaction product of the enzyme has the same characteristics (rate of decay to oxaloacetate, and condensation with dihydroxyacetone phosphate to form quinolinate) as the unstable reaction product (iminoaspartate) formed from D-aspartate oxidase. A reaction mechanism for the A protein-catalyzed condensation of dihydroxyacetone phosphate and iminoaspartate to form quinolinate is presented.

Evidence for an intermediate in quinolinate biosynthesis in Escherichia coli.

Evidence for the formation of an unstable intermediate in the synthesis of quinolinate from aspartate and dihydroxyacetone phosphate by Escherichia coli was obtained using toluenized cells of nadA and nadB mutants of this organism and partially purified A and B proteins in dialysis and membrane cone experiments. The results of these experiments indicate that the nadB gene product forms an unstable compound from aspartate in the presence of flavine adenine dinucleotide, and that this compound is then condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by the nadA gene product.

The mode of condensation of aspartic acid and dihydroxyacetone phosphate in quinolinate synthesis in Escherichia coli.

Dihydroxy [3-14C]acetone phosphate was prepared enzymatically from [1-14C]glucose and use as a substrate in a partially purified quinolinate synthetase system prepared from Escherichia coli mutants. Carbon-by-carbon degradation of the resulting [14C]quinolinate showed that 96% of the 14C was located in carbon-4, indicating that carbon-3 of dihydroxyacetone phosphate condenses with carbon-3 of aspartate in quinolinate synthesis in E. coli.

Metabolism of 6-aminonicotinic acid in Escherichia coli.

A late-log-phase culture of an Escherichia coli nadB pncA double mutant took up 6-[7-14C]aminonicotinic acid and excreted 6-[14C]aminonicotinamide. This mutant also accumulated intracellularly several radioactive compounds which have been tentatively identified as 6-amino analogs of compounds in the pyridine nucleotide cycle. It is concluded that 6-aminonicotinamide and 6-aminonicotinic acid probably exert at least a portion of their bacteriostatic effects by being metabolized, by the enzymes of the pyridine nucleotide cycle, to 6-aminonicotinamide adenine dinucleotide and 6-aminonicotinamide adenine dinucleotide phosphate. These compounds are not electron acceptors and are known inhibitors of some pyridine nucleotide-linked dehydrogenases.

Chemical Basis for Greenbug Resistance in Small Grains: II. Identification of the Major Neutral Metabolite of Benzyl Alcohol in Barley.

((14)C)Benzyl alcohol was administered either by uptake through the roots or by injection directly into the stems of wheat (Triticum aestivum L. em Thell), sorghum (Sorghum bicolor. L Moench) and two strains of barley (Hordeum vulgare L.). One strain of barley was susceptible to the greenbug (Schizaphis granium Rondani), and the other was greenbug-resistant. In all four plants, several radioactive metabolites were formed. The major neutral metabolite has been identified as benzyl-beta-d-glucopyranoside. This glucoside was found to have no biological activity against the greenbug under conditions in which the parent compound, benzyl alcohol, inhibits the reproduction of this insect pest.

Studies on the de novo biosynthesis of NAD in Escherichia coli. The separation of the nadB gene product from the nadA gene product and its purification.

Quinolinic acid (pyridine 2,3-dicarboxylic acid) which is an immediate precursor of the pyridine nucleotides, is synthesised from L-asparate and dihydroxyacetone phosphate in Escherichia coli. Extracts from certain nadB mutants complement the extracts prepared from all nadA mutants for the enzymic synthesis of quinolinate. Using the complementation assay, the quinolinate synthetase B protein has been purified more than 300-fold. The quinolinate synthetase B protein exists in all nadA and nadC mutants examined. The quinolinate synthetase A protein was present in all nadC mutants and most (but not all) nadB mutants. The facile separation of the wild-type quinolinate synthetase A and B proteins out of a nadC mutant suggests that quinolinate synthetase does not exists as a tightly bound complex. The partially purified quinolinate synthetase is inhibited by physiological concetrations of NAD and NADH but not by NADP or NADPH.

De novo biosynthesis of nicotinamide adenine dinucleotide in Escherichia coli: excretion of quinolinic acid by mutants lacking quinolinate phosphoribosyl transferase.

The excretion of quinolinic acid was studied in growing and resting cells of Escherichia coli K-12 nadC(13). Under optimal conditions, this organism could synthesize quinolinic acid in several-fold excess of the amount which would be required for normal growth. The excretion of quinolinic acid was controlled by the concentration of nicotinamide adenine dinucleotide (NAD) precursors available to the organism either during growth or during incubation in dense cell suspensions. These observations suggest that biosynthesis of NAD de novo is regulated by both repression and feedback inhibition. Analogues of niacin which inhibit bacterial growth also inhibited and repressed the synthesis (excretion) of quinolinic acid. The pH optimum for quinolinic acid excretion agreed favorably with the optimum observed for its synthesis in vitro. The rate of quinolinic acid excretion was strongly influenced by the concentration of ribose or glycerol in the medium.