Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti

Abstract

Aspartate aminotransferase (AAT) is an important enzyme in aspartate catabolism and biosynthesis and, by converting tricarboxylic acid cycle intermediates to amino acids, AAT is also significant in linking carbon 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 complementation of an Escherichia coli auxotroph that lacks three aminotransferases. pJA10 contained a gene, aatB, that coded for a previously undescribed AAT, AatB. pJA30 encoded an aromatic aminotransferase, TatA, that had significant AAT activity, and pJA20 encoded a branched-chain aminotransferase designated BatA. Genes for the latter two enzymes, WttA and batA, were previously isolated from R. meliloti. aatB is distinct from but hybridizes to aaL4, which codes for AatA, a protein required for symbiotic nitrogen fixation. The DNA sequence of aatB contained 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 sequence 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 Km for aspartate of 5.3 mM and a Km for 2-oxoglutarate of 0.87 mM. Its pH optimum is between 8.0 and 8.5. Mutations were constructed in aatB and hztA and transferred to the genome ofR. melioti 104A14. Both mutants were prototrophs and were able to carry out symbiotic nitrogen fixation.Bacteria of the genus Rhizobium fix nitrogen in a symbiotic association with leguminous plants. Carbon compounds produced by the plant are fed to the bacteria and metabolized to supply the energy and reductant needed for nitrogen fixation. Identifying which carbon compounds are transferred to the bacteria and how these compounds are metabolized is important in understanding the physiology of the symbiosis. Although sucrose is the major carbon source transported from the shoot to the nodule (44), most data suggest that it is not an important source of reductant for bacteroids (58) and that dicarboxylic acids are more likely to be their main carbon and energy sources (8,13,56). Succinate and malate are found in high concentrations in nodules (20,49,57) and bacteroids (15,29,45,51)

Genetics of the serine cycle in Methylobacterium extorquens AM1: identification of sgaA and mtdA and sequences of sgaA, hprA, and mtdA

“…However, within the group of aminotransferases many sequences are available, and similarity has been found for primary structures of tyrosine, histidinol-phosphate, and aspartate aminotransferases (33). For all of these enzymes, a few invariant residues that play an important role in structure and function of aminotransferases were found (1,33,60). However, enzymes belonging to the serine:pyruvate aminotransferase family, including SGAT from M. extorquens AM1, do not show any similarity with these enzymes in their primary structure, and most of the invariant residues were not found in corresponding locations of these sequences.…”

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

“…However, our analysis and the most recent study (Graindorge et al, 2014) revealed that aminotransferases having PPA-AT activity are present not only in plants but also in a broader range of microbes. Plant PPA-ATs belong to the AspAT Ib class (Graindorge et al, 2010;Dal Cin et al, 2011;Maeda et al, 2011), which falls within subfamily Ig (Mehta et al, 1993;Jensen and Gu, 1996) and is distantly related to class-Ia AspATs (subfamily Ia) commonly present in all organisms (Alfano and Kahn, 1993;Okamoto et al, 1996;Nobe et al, 1998). In addition to plants, AspAT Ib-type enzymes having strong PPA-AT activity were found in Chlorobi bacteria (C. tepidum; Figure 3, Table 1) and Kinetic parameters were obtained for prephenate using aspartate amino donor (20 mM) in 10-min reactions using 0.5 and 2 mg/mL recombinant enzymes for wild type and mutants of Arabidopsis PPA-AT enzymes, respectively.…”

A Thermostable Aspartate Aminotransferase from Aeropyrum pernix K1

“…These findings were similar to AAT of Rhizobium meliloti (pH 8.0 -8.5). 12 At the same time, acidic or neutral ranges of optimum pH have been observed for AATs from Sulfolobus solfataricus (pH 5.8) and Bacillus YM-2 (pH 7.2). (Fig.…”

“…12 We then determined the optimum pH and temperature for ApAAT with the transamination reaction toward L-aspartate and α-ketoglutarate. The optimum pH for ApAAT was 8.0 (Fig.…”