Phosphoenolpyruvate Carboxylation and Aspartate Synthesis in Acetobacter suboxydans

Dialyzed extracts of Acetobacter suboxydans ATCC 621 catalyze (14)CO(2) assimilation in the presence of phosphoenolpyruvate and a divalent cation. The formation of (14)C-oxalacetate was demonstrated and found not to be dependent upon the presence of orthophosphate or diphosphonucleotides. Oxalacetate synthesis was stimulated by orthophosphate and inhibited by aspartate. All attempts to demonstrate a reversible carboxylation mechanism have failed. (14)C-aspartate was synthesized when phosphoenolpyruvate, H(14)Co(3) (-), pyridoxal phosphate, and glutamate were added to dialyzed extracts. Chromatographic and spectrophotometric analyses and chemical degradation further demonstrate the presence of a reversible aspartate aminotransferase. The function of oxalacetate synthesis in a bacterium that reportedly lacks an operative tricarboxylic acid cycle is discussed.     

Isocitrate Dehydrogenase and Glutamate Synthesis in Acetobacter suboxydans

Acetobacter suboxydans is an obligate aerobe for which an operative tricarboxylic acid cycle has not been demonstrated. Glutamate synthesis has been reported to occur by mechanisms other than those utilizing isocitrate dehydrogenase, a tricarboxylic acid cycle enzyme not previously detected in this organism. We have recovered alpha-ketoglutarate and glutamate from a system containing citrate, nicotinamide adenine dinucleotide (NAD), a divalent cation, pyridoxal phosphate, an amino donor, and dialyzed, cell-free extract. Aconitase activity was readily detected in these extracts, but isocitrate dehydrogenase activity, measured by NAD reduction, was masked by a cyanide-resistant, particulate, reduced NAD oxidase. Isocitrate dehydrogenase activity could be demonstrated after centrifuging the extracts at 150,000 x g for 3 hr and treating the supernatant fluid with 2-heptyl-4-hydroxyquinoline N-oxide. It is concluded that A. suboxydans can utilize the conventional tricarboxylic acid cycle enzymes to convert citrate to alpha-ketoglutarate which can then undergo a transamination to glutamate.     

Biosynthesis of alpha-isopropylmalic and citric acids in Acetobacter suboxydans

Cell-free extracts of Acetobacter suboxydans were prepared which were capable of condensing alpha-ketoisovalerate with (14)C-labeled acetyl-coenzyme A to yield (14)C-labeled alpha-isopropylmalate. The product of the reaction was isolated by paper and column chromatography and was characterized by recrystallization with synthetic alpha-isopropylmalic acid to constant specific radioactivity. The formation of alpha-isopropylmalate by extracts of A. suboxydans plus the ability of the organism to grow in a simple glucose-glycerol medium containing glutamic acid as the only amino acid indicate that the pathway for leucine biosynthesis shown to exist in yeast and Salmonella typhimurium also occurs in A. suboxydans. As a comparison, the condensation of oxalacetate and ((14)C) acetyl-coenzyme A to yield ((14)C) citric acid was shown, by similar means, to occur in A. suboxydans. This is of interest since the existence of this classical condensing enzyme has hitherto not been demonstrated in this organism. This reaction was further demonstrated in cell-free extracts of A. suboxydans by means of a spectrophotometric assay at 232 mmu which measured the cleavage of the carbon-sulfur bond of acetyl-coenzyme A in the presence of oxalacetate. Comparison of the specific activities of crude cell-free extracts indicated a much more extensive occurrence of this reaction in yeast than in A. suboxydans.