Cloning and nucleotide sequence of the aspartase gene of Pseudomonas fluorescens.
The aspartase gene (aspA) of Pseudomonas fluorescens was cloned and the nucleotide sequence of the 2,066-base-pair DNA fragment containing the aspA gene was determined. The amino acid sequence of the protein deduced from the nucleotide sequence was confirmed by N- and C-terminal sequence analysis of the purified enzyme protein. The deduced amino acid composition also fitted the previous amino acid analysis results well (Takagi et al. (1984) J. Biochem. 96, 545-552). These results indicate that aspartase of P. fluorescens consists of four identical subunits with a molecular weight of 50,859, composed of 472 amino acid residues. The coding sequence of the gene was preceded by a potential Shine-Dalgarno sequence and by a few promoter-like structures. Following the stop codon there was a structure which is reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 62.3%. Inspection of the codon usage for the aspA gene revealed as high as 80.0% preference for G or C at the third codon position. The deduced amino acid sequence was 56.3% homologous with that of the enzyme of E. coli W (Takagi et al. (1985) Nucl. Acids Res. 13, 2063-2074). Cys-140 and Cys-430 of the E. coli enzyme, which had been assigned as functionally essential (Ida & Tokushige (1985) J. Biochem. 98, 793-797), were substituted by Ala-140 and Ala-431, respectively, in the P. fluorescens enzyme.
D000595:Amino Acid Sequence; D000642:Ammonia-Lyases / Q000235:genetics*; D001220:Aspartate Ammonia-Lyase / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000032:analysis; D004269:DNA, Bacterial / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D005798:Genes, Bacterial; D011551:Pseudomonas fluorescens / Q000201:enzymology / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D004269:DNA, Bacterial; D000642:Ammonia-Lyases; D001220:Aspartate Ammonia-Lyase