RESUMO
To elucidate the role of sulfhydryl groups in the enzymatic reaction of the aspartase from Escherichia coli, we used site-directed mutagenesis which showed that the enzyme was activated by replacement of Cys-430 with a tryptophan. This mutation produced functional alterations without appreciable structural change: The kcat values became 3-fold at pH 6.0; the Hill coefficient values became higher under both pH conditions; the dependence of enzyme activity on divalent metal ions increased; and hydroxylamine, a good substrate for the wild-type enzyme, proved a poor substrate for the mutant.
Assuntos
Aspartato Amônia-Liase/metabolismo , Escherichia coli/enzimologia , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Aspartato Amônia-Liase/genética , Dicroísmo Circular , Cisteína , Ativação Enzimática , Escherichia coli/genética , Cinética , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , TriptofanoRESUMO
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.
Assuntos
Amônia-Liases/genética , Aspartato Amônia-Liase/genética , DNA Bacteriano/análise , Genes Bacterianos , Pseudomonas fluorescens/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Códon/análise , Escherichia coli/enzimologia , Pseudomonas fluorescens/enzimologia , Homologia de Sequência do Ácido NucleicoRESUMO
Based on our recent determinations of the nucleotide sequences of the L-aspartate ammonia-lyase genes from Escherichia coli and Pseudomonas fluorescens, primary structures of the two L-aspartate ammonia-lyases and fumarate hydratases from Bacillus subtilis and E. coli (N-terminal partial sequence) were compared by computer analysis. These four enzymes exhibited a significant homology of at least 37%, implying that L-aspartate ammonia-lyase and fumarate hydratase share a common evolutionary origin. To authors' knowledge, this feature appears to be the first example showing that two kinds of enzymes catalyzing different types of reactions, albeit similar, share such a high degree of sequence homology.
Assuntos
Amônia-Liases/genética , Aspartato Amônia-Liase/genética , Bacillus subtilis/genética , Escherichia coli/genética , Fumarato Hidratase/genética , Genes Bacterianos , Genes , Pseudomonas fluorescens/genética , Sequência de Aminoácidos , Bacillus subtilis/enzimologia , Sequência de Bases , Escherichia coli/enzimologia , Pseudomonas fluorescens/enzimologia , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
The aspA gene of Escherichia coli W which encodes aspartase was cloned into the plasmid vector pBR322. The nucleotide sequences of aspA and its flanking regions were determined. The aspA gene encodes a protein with a molecular weight of 52,224 consisted of 477 amino acid residues. The amino acid sequence of the protein predicted from the nucleotide sequence was consistent with those of the NH2- and COOH-terminal regions and also with the amino acid composition of the purified aspartase determined previously. Potential promoter and terminator sequences for aspA were also found in the determined sequence.
Assuntos
Amônia-Liases/genética , Aspartato Amônia-Liase/genética , Escherichia coli/genética , Genes Bacterianos , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Enzimas de Restrição do DNA , DNA Bacteriano/genética , Genes Reguladores , Ligação Genética , Regiões Promotoras GenéticasRESUMO
Aspartase [L-aspartate ammonia-lyase, EC 4.3.1.1] of Pseudomonas fluorescens was highly purified to homogeneity and crystallized. The purified enzyme sedimented as a monodisperse entity upon ultracentrifugation with a s0(20),w value of 8.6S. Upon polyacrylamide gel electrophoresis (PAGE), the enzyme migrated as a single band. The molecular weight of the native enzyme was 173,000 +/- 3,000, as determined by sedimentation equilibrium analysis, and that of the enzyme subunit was determined to be 50,000 +/- 1,500 by sodium dodecyl sulfate (SDS)-PAGE. Cross-linking experiments using dimethyl suberimidate followed by SDS-PAGE indicated that the native enzyme was composed of four subunits with identical molecular weight. The amino acid composition of the enzyme was determined.
