BIOSYNTHESIS OF AMINO-ACIDS BY OXALOBACTER-FORMIGENES - ANALYSIS USING C-13-NMR

The gram-negative anaerobe Oxalobacter formigenes, grows on oxalate as the principal carbon and energy source, but a small amount of acetate is also required for growth. Experiments were conducted to determine the distribution and the position of label in cellular amino acids fro m cells grown on [C-13]oxalate, [C-13]acetate (1-C-13, 2-C-13 and U-C- 13), and (CO3)-C-13. The labeling pattern (determined with NMR spectro scopy) of amino acids was consistent with their formation through comm on biosynthetic pathways. The majority of the carbons in the amino aci ds that are usually derived from pyruvate, oxaloacetate, alpha-ketoglu tarate, 3-phosphoglycerate, and carbon in the aromatic amino acids wer e labeled by oxalate. Carbon from (CO3)-C-13 was assimilated primarily into amino acids expected to be derived from oxaloacetate and alpha-k etoglutarate. Approximately 60% of the acetate that was assimilated in to amino acids was incorporated as a C-2 unit into proline, arginine, glutamate, and leucine. The pattern of labeling from acetate in glutam ate, arginine, and proline was consistent with acetate incorporation v ia citrate (si)-synthase and subsequent formation of alpha-ketoglutara te via the first third of the tricarboxylic acid pathway. Acetate was also assimilated into amino acids derived from pyruvate and oxaloaceta te, but results indicated that this incorporation was as single carbon atoms. Based on these findings, cell-free extracts were assayed for s everal key biosynthetic enzymes. Enzymatic activities found included g lutamate dehydrogenase, phosphoenolpyruvate carboxylase, and pyruvate carboxylase. These findings are consistent with proposed biosynthetic mechanisms.