Purification of threonine deaminase from <i>Escherichia coli</i>

Grimminger, H.; Rahimi-Laridjani, I.; Koerner, K.; Lingens, Franz

doi:10.1016/0014-5793(73)80302-3

Cited by 9 publications

(7 citation statements)

References 8 publications

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“…The protein-protein interaction network only showed two clusters: (1) the respiration chain including hydrogenase (hyaA, hyaC, hyaE, hyaF) and ubiquinol oxidase (cbdA); (2) flagellar biosynthesis and mobility including fliP, fliS, motB, cheW, and tap, which was found also in H 2 O 2 -resistance genes. [41]. rho Transcription termination factor; required for one of the two major types of termination of RNA transcription [42].…”

Section: Genome-wide Screening Of Resistance Genes Against Hoclmentioning

confidence: 99%

Genome-Wide Screening of Oxidizing Agent Resistance Genes in Escherichia coli

et al. 2021

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The use of oxidizing agents is one of the most favorable approaches to kill bacteria in daily life. However, bacteria have been evolving to survive in the presence of different oxidizing agents. In this study, we aimed to obtain a comprehensive list of genes whose expression can make Escherichiacoli cells resistant to different oxidizing agents. For this purpose, we utilized the ASKA library and performed a genome-wide screening of ~4200 E. coli genes. Hydrogen peroxide (H2O2) and hypochlorite (HOCl) were tested as representative oxidizing agents in this study. To further validate our screening results, we used different E. coli strains as host cells to express or inactivate selected resistance genes individually. More than 100 genes obtained in this screening were not known to associate with oxidative stress responses before. Thus, this study is expected to facilitate both basic studies on oxidative stress and the development of antibacterial agents.

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Section: Genome-wide Screening Of Resistance Genes Against Hoclmentioning

confidence: 99%

Genome-Wide Screening of Oxidizing Agent Resistance Genes in Escherichia coli

et al. 2021

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“…The mobility of the mutant enzyme corresponds to a subunit molecular weight of 43,000. These data show the threonine deaminase from mutant IP 7 to be a dimer, whereas the wild-type enzyme is a tetramer (1,5).…”

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confidence: 92%

“…The enzyme appears to be pure as judged by acrylamide gel electrophoresis (2), sodium dodecyl sulfate gel electrophoresis (9), and analytical ultracentrifugation. The specific activity of pure preparations is 0.32 U/mg of protein, whereas the specific activity of pure wild-type threonine deaminase is 230 U/mg of protein (5).…”

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confidence: 94%

“…Meniscus depletion sedimentation equilibrium experiments (10) show linear plots of log c versus r2 corresponding to a molecular weight of approximately 85,000 (6 C; 21,000 and 25,000 rpm, respectively; 0.4 mg of protein per ml of 0.1 M phosphate buffer [pH 7.4]; assumed partial specific volume, 0.74). The wild-type enzyme shows a molecular weight of 200,000 (1,5).…”

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Threonine Deaminase from a Mutant Requiring Isoleucine or Pyridoxine: Inability to Form a Tetrameric State

Grimminger

Feldner

1974

J Bacteriol

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“…The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al, 1973;Grimminger et al, 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments.…”

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Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity

Feldner¹,

Grimminger²

1976

J Bacteriol

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The mutant IP7 of Escherichia coli B requires isoleucine or pyridoxine for growth as a consequence of a mutation in the gene coding for biosynthetic threonine deaminase. The mutation of IP7 was shown to be of the nonsense type by the following data: (1) reversion to isoleucine prototrophy involves the formation of external suppression at a high frequency, as shown by transduction experiments; and (ii) the isoleucine requirement is suppressed by lysogenization with a phage carrying the amber suppressor su-3. Cell extracts of the mutant strain contain a low activity of threonine deaminase. The possibility that this activity is biodegradative was ruled out by kinetic experiments. The mutant threonine deaminase was purified to homogeneity by conventional procedures. The enzyme is a dimer of identical subunits of an approximate molecular weight of 43,000 (Grimminger and Feldner, 1974), whereas the wild-type enzyme is a tetramer of 50,000-dalton subunits (Calhoun et al., 1973; Grimminger et al., 1973). The mutant enzyme is not inhibited by isoleucine and does not bind isoleucine, as shown by equilibrium dialysis experiments. Pyridoxal phosphate enhances the maximum catalytic activity of the mutant enzyme by a factor of five, whereas the wild-type enzyme is not affected. In wild-type and mutant threonine deaminase the ratio of protein subunits and bound pyridoxal phosphate is 2:1. The activation of threonine deaminase from strain IP7 is due to a second coenzyme binding site, as shown by (i) spectrophotometric titration of the enzyme with pyridoxal phosphate and by (ii) measurement the pyridoxal phosphate content of the enzyme after sodium borohydride reduction of the protein. The observation of one pyridoxal phosphate binding site per peptide dimer in the wild-type enzyme and of two binding sites per dimer in the mutant strongly suggests that one of the potential sites in the wild-type enzyme is masked by allosteric effects. The factors responsible for the half-of-the-sites reactivity of the coenzyme sites appear to be nonoperative in the mutant protein.

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Purification of threonine deaminase from Escherichia coli

Cited by 9 publications

References 8 publications

Genome-Wide Screening of Oxidizing Agent Resistance Genes in Escherichia coli

Genome-Wide Screening of Oxidizing Agent Resistance Genes in Escherichia coli

Threonine Deaminase from a Mutant Requiring Isoleucine or Pyridoxine: Inability to Form a Tetrameric State

Threonine deaminase from a nonsense mutant of Escherichia coli requiring isoleucine or pyridoxine: evidence for half-of-the-sites reactivity

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