Salmonella typhimurium Mutants with Alternate Requirements for Vitamin B 6 or Isoleucine

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.

supporting

confidence: 72%

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

Feldner¹,

Grimminger²

1976

“…Data supporting this hypothesis were not obtained because of the failure of pyridoxal-5-phosphate to restore transaminase C activity to cell extracts of the mutant strain. Such behavior has been observed for other pyridoxine-amino acid auxotrophs (7).…”

supporting

confidence: 62%

Escherichia coli K-12 mutant with alternate requirements for vitamin B6 or branched-chain amino acids and lacking transaminase C activity

Falkinham¹

1977

“…Firstly, the enzymatic defects in most pyridoxineless mutants have not been defined and therefore there cannot be at present a foolproof test that mutants classified phenotypically as Pdx are genotypically pdx. See, for example, the studies on "Km" mutants recently reported (10,11,13). Accordingly, the experiments were done with mutants from the three genetic classes which represent the majority of pdx mutants in E. coli B to establish the generality of the results.…”

Section: Resultsmentioning

confidence: 99%

Isoleucine and Threonine Can Prolong Protein and Ribonucleic Acid Synthesis in Pyridoxine-Starved Mutants of Escherichia coli B

Dempsey

Sims

1972

Pyridoxineless mutants of Escherichia coli B stopped incorporation of nucleosides into trichloroacetic acid-insoluble material about 40 to 60 min after pyridoxine starvation was initiated, whereas incorporation of amino acids (measured the same way) slowed but did not stop for several hours. Both these incorporations and cell density were increased most effectively by the presence of either threonine or isoleucine. Arginine, glutamate, histidine, methionine, tryptophan, and tyrosine also caused significant but less dramatic increases. Inducibility of j3-galactosidase continued beyond the point where nucleic acids appeared to stop their synthesis, suggesting that messenger ribonucleic acid synthesis continued beyond ribosomal ribonucleic acid synthesis. This inducibility was also increased by isoleucine and threonine. The overall results suggest that the threonine-isoleucine biosynthetic pathway is the most sensitive to starvation for pyridoxine.