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.

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.

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.

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.