ISOLATION AND CHARACTERIZATION OF A GENE CODING FOR A NOVEL ASPARTATE-AMINOTRANSFERASE FROM RHIZOBIUM-MELILOTI

Aspartate aminotransferase (AAT) is an important enzyme in aspartate c atabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carb on metabolism with nitrogen metabolism. To examine the role of AAT in symbiotic nitrogen fixation further, plasmids encoding three different aminotransferases from Rhizobium meliloti 104A14 were isolated by com plementation of an Escherichia coli auxotroph that lacks three aminotr ansferases. pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase, Tat A, that had significant AAT activity, and pJA20 encoded a branched-cha in aminotransferase designated BatA. Genes for the latter two enzymes, tatA and batA, were previously isolated from R. meliloti. aatB is dis tinct from but hybridizes to aatA, which codes for AatA, a protein req uired for symbiotic nitrogen fixation. The DNA sequence of aatB contai ned an open reading frame that could encode a protein 410 amino acids long and with a monomer molecular mass of 45,100 Da. The amino acid se quence of aatB is unusual, and AatB appears to be a member of a newly described class of AATs. AatB expressed in E. coli has a K(m) for aspa rtate of 5.3 mM and a K(m) for 2-oxoglutarate of 0.87 mM. Its pH optim um is between 8.0 and 8.5. Mutations were constructed in aatB and tatA and transferred to the genome of R. meliloti 104A14. Both mutants wer e prototrophs and were able to carry out symbiotic nitrogen fixation.