Do Thiol Amino Acids Inhibit Threonine Deaminase Activity in E. coli? (Letter to the Editor).

Cysteine and homocysteine suppress the growth of Escherichia coli. It was explained by the inhibition of threonine deaminase (TD) (Harris in J Bacteriol 145(2):1031-1035, 1981). TD inhibition was detected by a decline in its product, 2-ketobutyrate (2-KB). We propose that cysteine or homocysteine may not inhibit TD activity. Instead, 2-KB binds to them forming stable cyclic adducts. This binding possibly leads to isoleucine limitation and growth inhibition.

Homocysteine toxicity in Escherichia coli is caused by a perturbation of branched-chain amino acid biosynthesis.

In Escherichia coli the sulfur-containing amino acid homocysteine (Hcy) is the last intermediate on the methionine biosynthetic pathway. Supplementation of a glucose-based minimal medium with Hcy at concentrations greater than 0.2 mM causes the growth of E. coli Frag1 to be inhibited. Supplementation of Hcy-treated cultures with combinations of branched-chain amino acids containing isoleucine or with isoleucine alone reversed the inhibitory effects of Hcy on growth. The last intermediate of the isoleucine biosynthetic pathway, alpha-keto-beta-methylvalerate, could also alleviate the growth inhibition caused by Hcy. Analysis of amino acid pools in Hcy-treated cells revealed that alanine, valine, and glutamate levels are depleted. Isoleucine could reverse the effects of Hcy on the cytoplasmic pools of valine and alanine. Supplementation of the culture medium with alanine gave partial relief from the inhibitory effects of Hcy. Enzyme assays revealed that the first step of the isoleucine biosynthetic pathway, catalyzed by threonine deaminase, was sensitive to inhibition by Hcy. The gene encoding threonine deaminase, ilvA, was found to be transcribed at higher levels in the presence of Hcy. Overexpression of the ilvA gene from a plasmid could overcome Hcy-mediated growth inhibition. Together, these data indicate that in E. coli Hcy toxicity is caused by a perturbation of branched-chain amino acid biosynthesis that is caused, at least in part, by the inhibition of threonine deaminase.

Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine.

Arabidopsis thaliana threonine deaminase (TD) is a tetramer composed of identical approximately 59600 Da subunits. TD activity has been shown to be inhibited by isoleucine. This effect is reversed by a large excess of valine. Nondenaturant gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry experiments demonstrated that binding of isoleucine on TD induces dimerization of the enzyme, whereas tetramerization is restored by addition of a high valine concentration. Nondenaturant gel filtration and electrospray ionization mass spectrometry of the enzyme in the presence of increasing amounts of isoleucine suggest a fast equilibrium between the tetramer and the dimer. Finally, study of TD mutants allowed us to focus on the specific role of each isoleucine-binding site.

The regulation of aminotransferase activity by carbamoyl-phosphate.

We studied the effect of carbamoylphosphate (CP) on L-aspartate aminotransferase (GOT) and L-alanine aminotransferase (GPT), compared to its effect on L-threonine deaminase (TD). GPT and GOT were slightly inhibited by CP, while TD was strongly inhibited. GPT and TD, but not GOT, were inactivated when preincubated with CP. Only GOT was enhanced by pyridoxal 5'-phosphate (PLP), but not when the coenzyme was preincubated with CP. When the enzymes were resolved by p-chloromercuribenzoate (PCMB) treatment to apoenzymes, only GOT retained 47% of the original activity. Reconstitution of the apoenzymes with PLP also followed different course; activities of GPT and TD were completely restored while GOT remained partially inactivated. Treatment of apoenzymes with CP resulted in impairment of their reconstitution except GPT, activity of which could be completely restored. When PLP was pre-treated with CP before reconstitution, however, even GPT was only partially restored. The data indicated that CP affect activities of these enzymes at different levels, holoenzymes, PLP and probably apoenzymes. Under a concentration of PLP, activity of GOT would be most enhanced, followed by TD then GPT. In the presence of CP, this effect would be eliminated.

The inhibition of rat liver threonine dehydratase by carbamoyl-phosphate. The formation of carbamoylpyridoxal 5'-phosphate.

The effects exerted by carbamoyl phosphate (CP) and cyanate (KCNO) on rat liver L-threonine deaminase have been studied. The two compounds showed that same effects, inhibiting through a competitive mechanism both the holoenzyme and the dialyzed enzyme; inhibition was more evident for the latter. Ki values, both for L-threonine and pyridoxal 5'-phosphate (PLP), were lower for the apoenzyme and the inhibitors also affected the Km of the apoenzyme for PLP. The effects of CP and KCNO are mainly due to an interference in the association reaction apoenzyme + PLP in equilibrium holoenzyme This was clearly demonstrated by the fact that, when PLP was incubated with CP or KCNO, it failed to enhance the activity of the holoenzyme nor did it reactivate the resolved apoenzyme. Such interference of CP and KCNO in the L-threonine deaminase activity was mainly due to a specific mechanism, the formation of a new derivative of PLP. The reaction of PLP with either CP or KCNO occurred readily, at low concentrations, under physiological conditions. The new compound was identified as 3,4-dihydro-2H-pyrido[3,4-e]1,3-oxazin-2-one derivative by ultraviolet-visible spectra, elemental analysis, infrared, NMR and MS spectra. In this paper we formulate the hypothesis that this compound is involved in the regulation of the CP and PLP intracellular content and in the control of PLP dependent enzymes.

Cloning, expression, purification, and characterization of biosynthetic threonine deaminase from Escherichia coli.

Feedback inhibition of the regulatory enzyme threonine deaminase by isoleucine provides an important level of enzymic control over branched chain amino acid biosynthesis in Escherichia coli. Cloning ilvA, the structural gene for threonine deaminase, under control of the trc promoter results in expression of active enzyme upon induction by isopropyl 1-thio-beta-D-galactoside to levels of approximately 20% of the soluble protein in cell extracts. High level expression of threonine deaminase has facilitated the development of a rapid and efficient protocol for the purification of gram quantities of enzyme with a specific activity 3-fold greater than previous preparations. The catalytic activity of threonine deaminase is absolutely dependent on the presence of pyridoxal phosphate, and the tetrameric molecule is isolated containing 1 mol of cofactor/56,000-Da chain. Wild-type threonine deaminase demonstrates a sigmoidal dependence of initial velocity on threonine concentration in the absence of isoleucine, consistent with a substrate-promoted conversion of the enzyme from a low activity to a high activity conformation. The enzymic dehydration of threonine to alpha-ketobutyrate measured by steady-state kinetics, performed at 20 degrees C in 0.05 M potassium phosphate, pH 7.5, is described by a Hill coefficient, nH, of 2.3 and a K0.5 of 8.0 mM. The negative allosteric effector L-isoleucine strongly inhibits the enzyme, yielding a value for nH of 3.9 and K0.5 of 74 mM whereas enzyme activity is greatly increased by L-valine, which yields nearly hyperbolic kinetics characterized by a value for nH of 1.0 and a K0.5 of 5.7 mM. Thus, these effectors promote dramatic and opposing effects on the transition from the low activity to the high activity conformation of the tetrameric enzyme.

In vitro regulation of rat liver L-threonine deaminase by different effectors.

The regulation of liver L-threonine deaminase by different effectors--bile acids, bile pigments and monocarbon molecules--was investigated. Total inhibition of the enzyme was observed with physiological concentrations of bile acids and biliverdin. Purely competitive inhibition of the holoenzyme by several monocarbon molecules was demonstrated; the mechanism was partially competitive for bicarbonate. Inhibition was more pronounced in the case of the dialyzed enzyme. From the higher Km values for pyridoxal-5'-phosphate (PLP), obtained in the presence of the inhibitors, the results are explained on the basis of interference in the association reaction: apoprotein + PLP----holoenzyme. The various effects determined by bicarbonate may play a specific role in vivo since they occur at physiological concentrations of this compound.

Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli.

Incubation of purified Escherichia coli biodegradative threonine dehydratase with glyoxylate resulted in covalent binding of 1 mol of glyoxylate per mol of protein with concomitant loss of enzyme activity. The glyoxylate-binding site was identified as a heptapeptide representing amino acid residues Ser-33-Asn-Tyr-Phe-Ser-Glu-Arg-39 in the protein primary structure. Addition of glyoxylate to a culture of E. coli cells led to time-dependent enzyme inactivation. Immunoprecipitation with anti-dehydratase antibody of extract from [14C]glyoxylate-treated cells revealed labeled dehydratase polypeptide. These results are interpreted to mean that enzyme inactivation by glyoxylate in E. coli cells is associated with covalent protein modification.

Double inhibition of L-threonine dehydratase by aminothiols.

The inhibition of highly purified rat liver L-threonine dehydratase (L-threonine hydro-lyase (deaminating), EC 4.2.1.16) by aminothiols (L-cysteine, D-cysteine, cysteamine) has been studied. Single inhibition experiments evaluated by Lineweaver-Burk and Dixon plots showed, in a given concentration range, partially (parabolic) competitive inhibitions, indicating two binding sites for each inhibitor. Double inhibition experiments revealed that the inhibition was antagonistic, the two inhibitors weakening each other's effect. Formation of EI1 and EI2 binary complexes, and ESI1, ESI2 and EI1I2 ternary complexes was demonstrated, while formation of the quaternary complex ESI1I2 was ruled out. It is assumed that one inhibitor-binding site coincides with the substrate-binding center while the second inhibitor-binding (allosteric, regulatory) site may comprise the pyridoxal-phosphate-binding SH group(s). The comparison between Km and Ki values and the evaluation of intracellular concentrations of L-threonine, L-cysteine and cysteamine suggest a possible physiological role of the inhibition.

[Genetic mapping of the ilv7434 mutation providing threonine deaminase resistance to isoleucine inhibition in Escherichia coli]. / Geneticheskoe kartirovanie mutatsii ilv7434, obespechivaiushchei ustoichivost' treonindezaminazy k ingibirovaniiu izoleitsinom u Escherichia coli.

Location of previously isolated ilv7434 mutation was determined by use of transductional shortening of the F'14 episome. The ilv7434 mutation causes resistance of threonine deaminase (coded for by ilvA gene) to feed-back inhibition by isoleucine. Another phenotype characteristics of the ilv7434 mutant is the ability to feed a lawn of isoleucine auxotrophs in the cross-streak test. The F'14 strain AB1206 carrying ilv7434 mutation was used as a donor for making transductionally shortened episomes in recA recipient. These shortened F'14 episomes containing variable segments of the ilv cluster were then tested for their ability to transfer ilv7434 phenotype by complementation with ilv recA recipients. The data of complementation test suggest that ilv7434 is situated between ilvD and ilvC genes. One of 20 tested shortened episomes carrying, as shown by complementation test, incomplete ilvA gene was found to transfer ilv7434 phenotype by recombination with ilvA527 recA+ recipient. These data allow to conclude that ilv7434 mutation is located within the ilvA gene.

Rat liver L-threonine deaminase: properties and purification.

A new method of purification of rat liver L-threonine deaminase has been developed, and the results obtained are compared with values obtained by other authors. Some properties of this enzyme (pH optimum, temperature optimum, thermal stability, specificity, etc.) have been examined and we found that the enzyme is inhibited by carbonate ions, that L-cysteine (a competitive inhibitor) is also an inactivator of the enzyme and that it is bound to the enzyme in a ratio of 0.25 mole of cysteine per mole of enzyme, supporting the hypothesis that the enzyme consists of 4 subunits.

Escherichia coli K-12 mutation that inactivates biodegradative threonine dehydratase by transposon Tn5 insertion.

From a collection of kanamycin-resistant mutants of Escherichia coli K-12 isolated by transposon Tn5 mutagenesis, we have identified a mutant that lacks functional biodegradative threonine dehydratase (EC 4.2.1.16) by direct enzyme assay and by the loss of cross-reacting material with affinity-purified antibodies against the purified enzyme. Aerobic and anaerobic growth of this strain on various carbon sources failed to reveal a phenotype. Evidence for the insertional inactivation of threonine dehydratase by Tn5 was obtained by cloning the DNA segments flanking the Tn5 insertion site into pBR322 and hybridizing the cloned DNA to a synthetic oligodeoxynucleotide probe complementary to the DNA segment coding for a unique hexapeptide at the amino terminus end of the enzyme; the region of homology to the synthetic cDNA sequence appears to be located within about 500 nucleotides from one end of Tn5. Genetic analysis with the transposon element that caused insertional inactivation located the tdc gene at min 67 on the E. coli chromosome.

Cysteine and growth inhibition of Escherichia coli: threonine deaminase as the target enzyme.

Cysteine has been shown to inhibit growth in Escherichia coli strains C6 and HfrH 72, but not M108A. Growth inhibition was overcome by inclusion of isoleucine, leucine, and valine in the medium. Isoleucine biosynthesis was apparently affected, since addition of this amino acid alone could alter the inhibitory effects of cysteine. Homocysteine, mercaptoethylamine, and mercaptoethanol inhibited growth to varying degrees in some strains, these effects also being prevented by addition of branched-chain amino acids. Cysteine, mercaptoethylamine, and homocysteine were inhibitors of threonine deaminase but not transaminase B, two enzymes of the ilvEDA operon. Cysteine inhibition of threonine deaminase was reversed by threonine, although the pattern of inhibition was mixed. These results suggest a relationship between the growth-inhibitory effects of cysteine and other sulfur compounds and the inhibition of isoleucine synthesis at the level of threonine deaminase.

Comparative studies on threonine and serine dehydratases in rat liver.

1. Two isoenzymes of serine (threonine) dehydratase can be separated by DEAE cellulose chromatography. 2. Each of these isoenzymes possesses different S:T (serine dehydratase activity: threonine dehydratase activity) ratio and kinetic properties. 3. In early postnatal development, the S:T ratio shows marked variation. 4. The hepatic S:T ratio also changes with dietary manipulations. Furthermore L-alanine inhibits enzymic activities towards serine and threonine to different extent. 5. The results are consistent with the hypothesis postulating the presence of two distinct proteins each having a different activity ratio towards serine and threonine.

Inhibition of Escherichia coli biodegradative threonine dehydratase by pyruvate.

Pyruvate inhibits Escherichia coli K-12 biodegradative threonine dehydratase activity by a mechanism distinct from product inhibition by alpha-ketobutyrate and catabolite inactivation by intermediary metabolites.

[Kinetics and thermodynamics of heat inactivation of L-threonine-L-serine dehydratase from human liver]. / Kinetika i termodinamika teplovoi inaktivatsii 8treonin-L-serindegidratazy iz pecheni cheloveka.

It has been shown that heat inactivation of L-threonine- and L-serine dehydratase activities at 37; 45; 50 and 55 degrees C in human liver extracts (the liver was ectracted with buffer containing 1.10(-5) M of pyridoxal 5'-phosphate) in course of time is practically identical, and characterizes by the same of values of activation energy of heat inactivation process, activation enthalpy of this process, activation free energy of that and activation enthalpy of the heat inactivation process. A rise of pyridoxal 5'-phosphate concentration (to 2.10(-4) M) in the buffer used for the liver extraction and hence in the medium in which the heat inactivation process was carried out stabilises L-threonine- and L-serine dehydratase activities against the inactivation at 55 degrees C. It has been concluded thatL-threonine- and L-serine dehydratase activities in human liver belong to the single protein or to two proteins having very like physico-chemical properties, and that pyridoxal 5'-phosphate is essential for this enzyme not only as coenzyme but also it is necessary to support active and stable conformation of this oligomeric protein.

Enhanced allosteric regulation of threonine deaminase and acetohydroxy acid synthase from Escherichia coli in a permeabilized-cell assay system.

A permeabilized-cell technique for rapid assay of enzyme activity has revealed enhanced allosteric regulation of both threonine deaminase (L-threonine hydrolyase (deaminating), EC 4.2.1.16) and acethohydroxy acid synthease (acetolactate pyruvate-lyase (carboxylating), EC 4.1.3.18) in Escherichia col K-12. In the permeabilized cell assay threonine deaminase exhibited a higher Hill coefficient for inhibition by L-isoleucine, and acetohydroxy acid synthase exhibited a hypersensensitivity to allosteric inhibition by L-valine when compared to studies on crude extracts. We propose that these effects reflect the in situ microenvironments of both enzymes. Preliminary evidence further indicates that acetohydroxy acid synthase may loosely associate with the cell membrane.