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PubMed2002

Influence of threonine exporters on threonine production in Escherichia coli.

Threonine production in Escherichia coli threonine producer strains is enhanced by overexpression of the E. coli rhtB and rhtC genes or by heterologous overexpression of the gene encoding the Corynebacterium glutamicum threonine excretion carrier, thrE. Both E. coli genes give rise to a threonine-resistant phenotype when overexpressed, and they decrease the accumulation of radioactive metabolites derived from [(14)C] L-threonine. The evidence presented supports the conclusion that both RhtB and RhtC catalyze efflux of L-threonine and other structurally related neutral amino acids, but that the specificities of these two carriers differ substantially.

Amino Acid Transport Systems, Neutral↗
PubMed1987

Specificity of the attenuation response of the threonine operon of Escherichia coli is determined by the threonine and isoleucine codons in the leader transcript.

Expression of the threonine (thr) operon enzymes of Escherichia coli is regulated by an attenuation mechanism. The regulatory portion of the operon contains a region coding for a leader peptide that contains consecutive threonine and isoleucine codons. It is thought that translation of the leader peptide controls the frequency of transcription termination at the attenuator site. Using oligonucleotide-directed site-specific mutagenesis we have altered the putative control codons of the leader peptide coding region. In two of the mutants the threonine and isoleucine codons were changed to produce peptides containing histidine and tyrosine codons. Both mutants showed loss of regulation by threonine and isoleucine. A hisT mutation, which leads to an undermodification of tRNA(His), increased thr operon expression in the mutants threefold but did not affect expression of the wild-type thr operon. Two other mutants were constructed that contained two histidine codons early in the leader peptide. Expression in both of these mutants was unaltered by the presence of the hisT allele or by the addition of threonine and isoleucine to the growth medium. In addition, a wild-type strain containing a temperature-sensitive threonyl-tRNA synthetase mutation showed increased thr operon expression at the non-permissive temperature, whereas none of the mutants showed any change. Taken together these data indicate that the specificity of the attenuation response is effected by specific control codons within the thr leader peptide coding region. We have also directly demonstrated thr leader peptide synthesis in vitro using a plasmid encoding the wild-type thr leader region to direct the synthesis of a peptide of the appropriate molecular weight when labeled with [3H]threonine but not with [3H]histidine or [3H]tyrosine. Conversely, when extracts were incubated with templates containing the mutated DNAs, peptides were labeled that showed patterns consistent with the expected amino acid compositions. These data indicate that the thr leader RNA is translated into the predicted leader peptide.

Codon↗
PubMed1978

Threonine inhibition of the aspartokinase--homoserine dehydrogenase I of Escherichia coli. Threonine binding studies.

Both activities of the aspartokinase--homoserine I (AK-HSD) of Escherichia coli are inhibited by threonine. Careful threonine binding studies have now been done which have allowed us to distinguish the various effects of threonine on the enzyme. The ultrafiltration technique of H. Paulus ((1969) Anal. Biochem. 32, 101) for measuring ligand binding was shown to be comparable with equilibrium dialysis techniques. Reduction in error by utilization of this procedure enabled us to obtain evidence for two different sets of threonine sites by direct binding studies. The binding data were mathematically consistent with two independent classes of threonine sites, each of which contained four sites per tetramer and had a Hill coefficient of about 2.3--2.5. KD for the second set of sites was five- to tenfold greater than the high affinity sites, depending upon conditions. The data now suggest that the sequential model for site--site interactions adequately describes the cooperativity of threonine binding to the high affinity set of sites.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1954

Enzymatic breakdown of threonine by threonine aldolase.

1. The enzyme which splits threonine to acetaldehyde and glycine has been partially purified from rat liver (five- to sixfold purification) and the name threonine aldolase proposed for it. 2. The general properties of threonine aldolase have been studied. The enzyme is unstable to a pH below 5. The pH optimum of the enzyme reaction is at 7.5-7.7. The initial rate of production of acetaldehyde is proportional to the enzyme concentration, and when the enzyme concentration is constant, the production of acetaldehyde is proportional to the time, provided that the substrate is in excess. The enzyme is inhibited by the carbonyl group reagent, hydroxylamine. Attempts to demonstrate that pyridoxal phosphate is a cofactor were unsuccessful. 3. The enzyme splits only L-allothreonine and L-threonine and is inactive against the D-forms of these amino acids. 4. The enzyme reaction on DL-allothreonine follows first order kinetics. From the first order velocity constants and the initial rates of the rates of the reaction at various substrate concentrations the Michaelis constant, Ks, for this substrate has been evaluated. Michaelis constants have also been determined for threonine. 5. The optimum temperature for the enzymatic breakdown of DL-allothreonine at pH 7.65 was found to be 50 degrees C. in phosphate buffer and 48 degrees C. in tris-maleate buffer. The rate of thermal inactivation of the enzyme threonine aldolase obeys a first order reaction. The heat of thermal inactivation was calculated by the aid of the van't Hoff-Arrhenius equation to be 43,000 cal. per mole for the temperature range 41.2-46.6 degrees C. 6. Equivalent amounts of acetaldehyde and glycine were formed from DL-allothreonine and the enzymatic breakdown of DL-allothreonine was found to be irreversible.

Acetaldehyde↗
PubMed1982

Threonine imbalance and the threonine requirement of the chicken.

Experiments were conducted to determine the influence of dietary amino acids on the threonine requirement of chicks. A diet limiting in threonine and containing the equivalent of 20.6% crude protein was imbalanced by supplements of 0.9 to 1.5% L-tryptophan, 3.0% L-serine, a mixture of 2% each, leucine, isoleucine and valine or a 6.0% mixture of all essential amino acids but threonine. All amino acid supplements decreased the growth of chicks, and decreased food intake and/or efficiency of food utilization (P less than 0.05). Supplemental threonine prevented these effects in all cases except for 1.5% tryptophan in which the adverse effect of tryptophan was not prevented completely. The threonine requirement of Leghorn chicks was 0.69 to 0.72 percent of the diet when the diet did not contain the imbalancing amino acid supplements. We concluded that the threonine requirement of the chick is higher than previously reported, and is influenced by the amino acid content of the diet.

Amino Acids↗
PubMed1991

Insulin-stimulated serine and threonine phosphorylation of the human insulin receptor. An assessment of the role of serines 1305/1306 and threonine 1348 by their replacement with neutral or negatively charged amino acids.

Insulin promotes insulin receptor beta-subunit phosphorylation on tyrosine, serine, and threonine residues in a variety of cells, including simian COS cells which transiently express human insulin receptors following transfection with a cDNA encoding the wild-type receptor protein. To examine the potential roles of serines 1305 and 1306 and threonine 1348 as sites of insulin-stimulated phosphorylation in these cells, these residues (i.e. either serines 1305 and 1306, or threonine 1348) were replaced with neutral (alanine) or negatively charged (aspartate) amino acids. Following transient expression of each of these mutant receptors in COS cells, two-dimensional phosphopeptide mapping reveals that threonine 1348 is the major, if not the only, insulin-stimulated threonine phosphorylation site. In contrast, while serines 1305 and/or 1306 are phosphorylated in an insulin-dependent manner, these sites comprise only a minor proportion of insulin receptor serine phosphorylation in these cells. Substitution of either serines 1305 and 1306 or threonine 1348 with neutral or negatively charged amino acids has no effect on insulin-stimulated tyrosine autophosphorylation of these mutant receptors in intact cells. Furthermore, insulin-stimulated exogenous protein-tyrosine kinase activity of the mutant receptors is unaffected, as assessed following either phosphorylation of receptors in intact cells or following immunopurification of receptors and their autophosphorylation in vitro.

Amino Acid Sequence↗
PubMed2001

Metabolic redirection of carbon flow toward isoleucine by expressing a catabolic threonine dehydratase in a threonine-overproducing Corynebacterium glutamicum.

Carbon destined for lysine synthesis in Corynebacterium glutamicum ATCC 21799 can be diverted toward threonine by overexpression of genes encoding a feedback-insensitive homoserine dehydrogenase (hom(dr)) and homoserine kinase (thrB). We studied the effects of introducing two different threonine dehydratase genes into this threonine-producing system to gauge their effects on isoleucine production. Co-expression of hom(dr), thrB, and ilvA, which encodes a native threonine dehydratase, caused isoleucine to accumulate to a final concentration of 2.2+/-0.2 g l(-1), five-fold more than accumulates in the wild-type strain, and approximately twice as much as accumulates in the strain expressing only hom(dr) and thrB. Comparing these data with previous results, we found that overexpression of the three genes, hom(dr), thrB, and ilvA, in C. glutamicum ATCC 21799 is no better in terms of isoleucine production than the expression of a single gene, tdcB, encoding a catabolic threonine dehydratase from Escherichia coli. Co-expression of hom(dr), thrB, and tdcB, however, caused the concentration of isoleucine to increase 20-fold compared to the wild-type strain, about four times more than the corresponding ilvA-expressing strain. In this system, the apparent yield of isoleucine production was multiplied by a factor of two [2.1 mmol (g dry cell weight)(-1)]. While the balance of excreted metabolites showed that the carbon flow in this strain was completely redirected from the lysine pathway into the isoleucine pathway, it also showed that more pyruvate was diverted into amino acid synthesis.

Base Sequence↗
PubMed1977

Role of threonine dehydrogenase in Escherichia coli threonine degradation.

Threonine was used as nitrogen source by Escherichia coli K-12 through a pathway beginning with the enzyme threonine dehydrogenase. The 2-amino-3-ketobutyrate formed was converted to glycine, and the glycine was converted to serine, which acted as the actual nitrogen donor. The enzyme formed under anaerobic conditions and known as threonine deaminase (biodegradative) is less widespread than threonine dehydrogenase and may be involved in energy metabolism rather than in threonine degradation per se.

Adenylyl Cyclases↗
PubMed1998

Restoration of rat liver L-threonine dehydratase activity by pyridoxamine 5'-phosphate: the half-transaminating activity of L-threonine dehydratase and its regulatory role.

When a highly purified preparation of rat liver l-threonine deaminase (l-TDH, EC 4.2.1.16) was 99% inactivated by dialysis, removing bound pyridoxal 5'-phosphate (PLP), the apoenzyme was reactivated not only by PLP but also by pyridoxamine 5'-phosphate (PMP). When purified by HPLC, the commercial PMP used in the incubation mixture was found to contain only extremely small amounts of PLP, which could not account for restoration of l-threonine dehydratase activity. HPLC analysis of the assay mixtures showed that during incubation, sufficient PLP had been formed for reactivation of the apoenzyme. The apoenzyme evidently bound PMP and triggered transamination between PMP and the keto acids, which either contaminated, or were formed by the minimal amount of PLP-holoenzyme always present even in the dialyzed preparation. When sufficient PLP was formed, the PLP-holoenzyme and the original 'true' l-threonine dehydratase activity were restored. When PMP was incubated with the apoenzyme in the presence of small quantities of keto acids (pyruvate or 2-oxobutyrate) small amounts of l-alanine or l-aminobutyrate were formed. The reaction was not reversible; l-alanine and l-aminobutyrate did not react with the PLP-holoenzyme. No transaminating activity occurred with other amino acids. These results show that l-threonine dehydratase exists in two forms: the well known stable apoenzyme-PLP (hydrolase deaminating) and the transient apoenzyme-PMP (non-reversible half-transaminating). Half-transamination has the biological role of keeping the activity of the 'true' l-TDH constant and of regulating intracellular levels of pyruvate, alanine, oxobutyric acid, l-aminobutyric acid, l-threonine and l-serine.

Acetoacetates↗
PubMed1993

The expression of Escherichia coli threonine synthase and the production of threonine from homoserine in mouse 3T3 cells.

We have subcloned the coding sequence for the Escherichia coli threonine synthase gene into a eukaryotic expression vector based on the simian-virus-40 early promoter. When mouse 3T3 cells which already expressed homoserine kinase were transfected with the new plasmid, the cells were able to incorporate radioactivity from [14C]homoserine into their cell proteins. Stable cell lines were established by co-transfecting 3T3 cells with the plasmid coding for threonine synthase and another coding for homoserine kinase and G-418 (Geneticin) resistance. Cells were selected for G-418 resistance and then screened for an ability to synthesize threonine from homoserine and incorporate it into the cell protein. A cell line which expressed both the homoserine kinase and threonine synthase genes was capable of growth in a threonine-deficient medium containing homoserine.

3T3 Cells↗
PubMedDate not supplied

Contribution of erythrocytes and plasma in threonine and lysine transfer across the portal drained viscera and the liver in pigs. Effect of threonine and lysine dietary supply.

Contributions of erythrocytes and plasma to threonine and lysine transport across the PDV and the liver were determined in growing pigs successively fed a threonine deficient diet and a control well-balanced diet (experiment 1) or a lysine deficient or a well-balanced diet (experiment 2). The animals were surgically prepared for insertion of chronic catheters in the mesenteric vein (MV), the portal vein (PV), a hepatic vein (HV) and the carotid artery (CA). Plasma and whole blood AA concentrations in PV, HV and CA and PV and HV blood flows were determined during 6 hours of para-aminohippuric acid constant infusion. During this period the pigs were continuously fed (1 meal per hour). The contribution of plasma to lysine and threonine transport was higher in pigs fed the well balanced diets. More than 50% of threonine and lysine appearing in the PV and in the HV are transported by the plasma. Our results suggest that erythrocytes are probably little involved in lysine and threonine transfer across the liver and digestive tract of pig continuously fed.

Animals↗
PubMed1978

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Carboxymethylation of the enzyme: threonine binding and inhibition are functionally dissociable.

The inactivation of the aspartokinase I-homoserine dehydrogenase I by iodoacetic acid and the effect on the sensitivity to its inhibitor, L-threonine, were examined. Both aspartokinase and homoserine dehydrogenase inactivation, as well as the dehydrogenase desensitization toward L-threonine occur as a pseudo-first order process. During its inactivation, the aspartokinase remains sensitive to L-threonine. At 50% inactivation, the inhibition curve of the aspartokinase by L-threonine displays homotropic cooperative effects. This alkylated protein retains eight binding sites for L-threonine. During the carboxymethylation, the protein remains in the tetrameric form until half of the kinase activity is lost. At the end of the inactivation aggregate forms and dimers appear.

Alkylation↗
PubMed1980

Synthesis, pharmacological, conformational, and dynamic studies of the potent hormone antagonists [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin. Conformational and dynamic considerations in the design of antagonists.

The solid phase synthesis of [1-penicillamine, 4-threonine]-oxytocin and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin is reported. The two compounds have no in vitro milk ejecting activity and no in vivo or in vitro oxytocic activity, but both are potent antagonists in these three assay systems. In the in vitro oxytocic assay, [1-penicillamine, 4-threonine]- and [1-penicillamine, 2-phenylalanine, 4-threonine]-oxytocin have pA2 values of 7.55 +/- 0.04 and 7.67 +/- 0.02, respectively, and both inhibit the uterine contractile response to oxytocin in nonpregnant and pregnant rats. [1-Penicillamine, 2-phenylalanine, 4-threonine]-oxytocin has a weak antipressor activity and at high doses, consistently caused a weak and transient fall in blood pressure in the rat. Carbon-13 nuclear magnetic resonance chemical shift parameters and spin-lattice relaxation times (T1) indicate that these two new oxytocin antagonists have very similar conformation and dynamic properties to oxytocin inhibitors which have previously been examined. These results are discussed in terms of conformational and dynamic models of oxytocin antagonism at the uterus. It is suggested that conformational restrictions at the 2- and 4-positions of penicillamine-1 analogues of oxytocin are important to antagonist activity and potency.

Amino Acid Sequence↗
PubMed1992

Serine and threonine catabolism in Saccharomyces cerevisiae: the CHA1 polypeptide is homologous with other serine and threonine dehydratases.

The catabolic L-serine (L-threonine) dehydratase of Saccharomyces cerevisiae allows the yeast to grow on media with L-serine or L-threonine as sole nitrogen source. Previously we have cloned the CHA1 gene by complementation of a mutant, cha1, lacking the dehydratase activity. Here we present the DNA sequence of a 1,766-bp fragment of the CHA1 region encompassing an open reading frame of 1080 bp. Comparison of the predicted amino acid sequence of the CHA1 polypeptide with that of other serine/threonine dehydratases revealed several blocks of sequence homology. Thus, the amino acid sequence of rat liver serine dehydratase (SDH2) and the CHA1 polypeptide are 44% homologous allowing for conservative substitutions, while 36% similarity is found between the catabolic threonine dehydratase (tdcB) of Escherichia coli and the CHA1 protein. This strongly suggests that CHA1 is the structural gene for the yeast catabolic serine (threonine) dehydratase. S1-nuclease mapping of the CHA1 mRNA ends showed a major transcription initiation site corresponding to an untranslated leader of about 19 nucleotides, while a major polyadenylation site was located about 86 nucleotides downstream from the open reading frame. Furthermore, we have mapped the chromosomal position of the CHA1 gene to less than 0.5 kb centromere proximal to HML on the left arm of chromosome III.

Amino Acid Sequence↗
PubMed1982

Attenuation in the threonine operon: effects of amino acids present in the presumed leader peptide in addition to threonine and isoleucine.

We have studied in vivo the contribution of amino acids corresponding to codons of the leader sequence other than the so-called "regulatory codons" (for threonine and isoleucine) in the expression of the threonine operon. In the presence of threonine and isoleucine, addition of each amino acid encoded in the proximal part of the leader sequence resulted in a significant decrease of the expression of the operon, over and above that decrease observed in the sole presence of threonine and isoleucine. These effects were cumulative. No such effect was found with the amino acids encoded by the distal part of the leader sequence. These findings are discussed in the light of the current model of attenuation.

Amino Acid Sequence↗
PubMed1993

Production of threonine by Brevibacterium flavum containing threonine biosynthesis genes from Escherichia coli.

Genes of the threonine operon of Escherichia coli were used for the construction of a Brevibacterium flavum strain excreting threonine. Using the shuttle vector pCEM300 and a newly constructed shuttle vector pEC71 (7.1 kb, Kmr/Nmr), various plasmids carrying E. coli thr genes were prepared. Mutants resistant to the threonine analog 2-amino-3-hydroxyvaleric acid (AHV) were isolated after the ethyl methanesulfonate treatment of B. flavum carrying these recombinant plasmids. A mutant of B. flavum CCM 351 carrying the cloned genes thrA and thrB accumulated 12 g/L of threonine after 48 h of cultivation.

Brevibacterium↗
PubMed1977

Synthesis and some pharmacological properties of [4-threonine, 7-glycine]oxytocin, [1-(L-2-hydroxy-3-mercaptopropanoic acid), 4-threonine, 7-glycine]oxytocin (hydroxy[Thr4, Gly7]oxytocin), and [7-Glycine]oxytocin, peptides with high oxytocic-antidiuretic selectivity.

[4-Threonine, 7-glycine]oxytocin and [1-(L-2-hydroxy-3-mercaptopropanoic acid), 4-threonine, 7-glycine]oxytocin (hydroxy[Thr4, Gly7]oxytocin) were synthesized by a combination of solid-phase and classical methods of peptide synthesis. A protected octapeptide was synthesized by the solid-phase method and following ammonolysis and purification 1 + 8 couplings in solution were employed to furnish the required key nonapeptide and acyl octapeptide intermediates, respectively. [7-Glycine]oxytocin was prepared from a sample of the protected nonapeptide intermediate used in the original synthesis of this peptide. [7-Glycine]oxytocin has an oxytocic potency (O) of 93 +/- 4 units/mg and an antidiuretic potency (A) of 0.0056 +/- 0.0003 units/mg. It has an O/A ratio of 16 000. [4-Threonine, 7-glycine]oxytocin has an oxytocic potency of 166 +/- 4 units/mg and an antidiuretic potency of 0.002 +/- 0.0004 units/mg. Its O/A ratio is 83 000. Threonine substitution has thus brought about a substantial enhancement in oxytocic activity and a fivefold enhancement in O/A selectivity. Hydroxy [Thr4, Gly7]oxytocin has an oxytocic potency of 218 +/- 8 units/mg and antidiuretic potency of 0.0040 +/- 0.0005 units/mg. Its O/A ratio is thus 54 500. All three 7-glycine-substituted analogues exhibit a marked sensitivity to Mg2+ on the rat uterus assay ststem and in the presence of 0.5 mM Mg2+ had oxytocic potencies in the range of 900-1000 units/mg. Should these peptides exhibit enhanced oxytocic selectivity in humans, they might offer a greater margin of safety than oxytocin in those clinical stiuations in which the latter is currently employed.

Amino Acids, Sulfur↗
PubMed1997

Conversion of DL-threonine, D-threonine and 2-oxobutyrate into propionate and 2-hydroxybutyrate by Fusobacterium species.

The present investigation examined DL-threonine, D-threonine and 2-oxobutyrate conversion into propionate and 2-hydroxybutyrate by various type strains and clinical isolates of Fusobacterium. Except for Fus. naviforme, the type strains were able to produce varying degrees of propionate and/or 2-hydroxybutyrate from DL-threonine. Additionally, D-threonine was converted into an equimolar amount of propionate by Fus. necrophorum subsp. necrophorum, Fus. nucleatum subsp. nucleatum and Fus. varium, and to a lower but significant amount by Fus. mortiferum and Fus. perfoetens. However, the level of propionate remained unchanged for Fus. nucleatum subsp. fusiforme, Fus. nucleatum subsp. vincentii, Fus. naviforme, Fus. necrophorum subsp. funduliforme, Fus. gonidiaforme and Fus. russii. 2-Oxobutyrate was fermented to propionate by all type strains, although Fus. russii reduced it mainly to 2-hydroxybutyrate. Thus, an attempt was made to make use of these features in order to identify clinical isolates.

Fusobacterium↗