Biosynthesis of α-Isopropylmalic and Citric Acids in Acetobacter suboxydans

Cell-free extracts of Acetobacter suboxydans were prepared which were capable of condensing α-ketoisovalerate with 14 C-labeled acetyl-coenzyme A to yield 14 C-labeled α-isopropylmalate. The product of the reaction was isolated by paper and column chromatography and was characterized by recrystallization with synthetic α-isopropylmalic acid to constant specific radioactivity. The formation of α-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 mμ 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 .