Toxic accumulation of alpha-ketobutyrate caused by inhibition of the branched-chain amino acid biosynthetic enzyme acetolactate synthase in Salmonella typhimurium.

Biochemical and genetic analyses of the bacterium Salmonella typhimurium suggest that accumulation of alpha-ketobutyrate partially mediates the herbicidal activity of acetolactate synthase inhibitors. Growth inhibition of wild-type bacteria by the herbicide sulfometuron methyl was prevented by supplementing the medium with isoleucine, an allosteric inhibitor of threonine deaminase-catalyzed synthesis of alpha-ketobutyrate. In contrast, isoleucine did not rescue the growth of a mutant containing a threonine deaminase unresponsive to isoleucine. Moreover, the hypersensitivity of seven Tn10 insertion mutants to growth inhibition by sulfometuron methyl and alpha-ketobutyrate correlated with their inability to convert alpha-ketobutyrate to less noxious metabolites. We propose that alpha-ketobutyrate accumulation is an important component of sulfonylurea and imidazolinone herbicide action.     

Isoleucine and valine metabolism in Escherichia coli. XIX. Inhibition of isoleucine biosynthesis by glycyl-leucine

The inhibition of growth of the K-12 strain of Escherichia coli by glycyl-l-leucine observed originally by Simmonds and co-workers was investigated. The inhibition was reversed by isoleucine and those precursors of isoleucine beyond threonine in the biosynthetic pathway. Threonine reversed the inhibition poorly. With heavy cell suspensions, the inhibition was transient: the onset of growth followed the disappearance of the dipeptide from the medium and the appearance of glycine and leucine. Glycyl-leucine was shown to be an inhibitor of threonine deaminase (EC 4.2.1.16 l-threonine hydro-lyase [deaminating]). One kind of glycyl-leucine-resistant mutant had a threonine deaminase that was resistant to isoleucine and glycyl-leucine inhibition. The pattern of glycyl-leucine inhibition is compared with those of inhibition by isoleucine and by the weaker inhibitors leucine and valine.     

Biosynthesis of branched-chain amino acids in Schizosaccharomyces pombe: regulatory properties of threonine deaminase

Biosynthetic threonine deaminase (TD) from Schizosaccharomyces pombe has been partially purified from crude extracts by treatment with protamine sulfate, ammonium sulfate precipitation, and gel filtration through Sephadex G-25. In both crude extracts and purified preparations, TD showed marked stimulation by pyridoxal phosphate. A pH optimum for activity was found at pH 9.0, whereas the inhibition caused by the natural feedback inhibitor, l-isoleucine, was maximal at pH 7.4. l-Threonine exhibits homotropic cooperative effects at low pH (7.0-8.0), which are eliminated at pH 9.0, and the affinity for substrate (in terms of K(m)) increased with increasing pH. Enzyme activity could be completely inhibited by isoleucine over a pH range of 7.4 to 9.0; the amount of isoleucine required for 50% inhibition increased with increasing pH. Isoleucine inhibition was pseudocompetitive with respect to substrate and increased the cooperative effects of threonine. l-Valine was found to reverse isoleucine inhibition; it also activated the enzyme in a pH range of 7.0 to 8.0 by eliminating the cooperative effects of threonine, thus normalizing the substrate saturation curves at these pH values. l-Leucine was shown to be a competitive inhibitor with respect to threonine, and to be able partially to reverse isoleucine inhibition. Treatment of TD with mercurials did not result in desensitization to isoleucine inhibition. However, at pH 10, virtually no sensitivity of the enzyme to isoleucine was observed while activity remained strong, which suggests the existence of separate sites on the TD molecule for binding threonine and isoleucine. A tentative model is presented which unifies the kinetic results reported here in terms of the interactions of TD with its effector molecules.     

CodY-Regulated Aminotransferases AraT and BcaT Play a Major Role in the Growth of Lactococcus lactis in Milk by Regulating the Intracellular Pool of Amino Acids

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.     

CodY-regulated aminotransferases AraT and BcaT play a major role in the growth of Lactococcus lactis in milk by regulating the intracellular pool of amino acids

Aminotransferases, which catalyze the last step of biosynthesis of most amino acids and the first step of their catabolism, may be involved in the growth of Lactococcus lactis in milk. Previously, we isolated two aminotransferases from L. lactis, AraT and BcaT, which are responsible for the transamination of aromatic amino acids, branched-chain amino acids, and methionine. In this study, we demonstrated that double inactivation of AraT and BcaT strongly reduced the growth of L. lactis in milk. Supplementation of milk with amino acids and keto acids that are substrates of both aminotransferases did not improve the growth of the double mutant. On the contrary, supplementation of milk with isoleucine or a dipeptide containing isoleucine almost totally inhibited the growth of the double mutant, while it did not affect or only slightly affected the growth of the wild-type strain. These results suggest that AraT and BcaT play a major role in the growth of L. lactis in milk by degrading the intracellular excess isoleucine, which is responsible for the growth inhibition. The growth inhibition by isoleucine is likely to be due to CodY repression of the proteolytic system, which is necessary for maximal growth of L. lactis in milk, since the growth of the CodY mutant was not affected by addition of isoleucine to milk. Moreover, we demonstrated that AraT and BcaT are part of the CodY regulon and therefore are regulated by nutritional factors, such as the carbohydrate and nitrogen sources.     

The mechanism of growth inhibition by threonine inEuglena gracilis: Regulation of isoleucine-valine biosynthesis by 2-oxobutyrate

InEuglena gracilis the growth inhibition by threonine was accompanied by a rapid accumulation of isoleucine in the cells. Among threonine-catabolizing enzymes only threonine dehydratase was detected in high activity inEuglena, and 2-oxobutyrate, the dehydratase products of threonine, also inhibited as did threonine. Threonine dehydratase was located in the cytosol, and its activity was not affected by isoleucine and related amino acids. 2-Oxobutyrate strongly inhibited the synthesis of valine from pyruvate while augmented the synthesis of isoleucine in mitochondria.     

Effects of mitochondrial protein synthesis inhibitors on the incorporation of isoleucine into Plasmodium falciparum in vitro

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [3H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [3H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.     

Effects of Mitochondrial Protein Synthesis Inhibitors on the Incorporation of Isoleucine into Plasmodium falciparum In Vitro1

Plasmodium falciparum was grown in human erythrocytes in vitro and the effect of chloramphenicol, erythromycin, and tetracycline on growth and maturation of the parasites and on their ability to incorporate [³H]isoleucine into protein was observed. Exposure of rings to high concentrations of chloramphenicol had little effect on subsequent maturation of the rings whereas brief (4 h) exposure of trophozoites caused a dose-dependent inhibition of subsequent ring formation. Incorporation of [³H]isoleucine into protein was not affected during at least 6 h of exposure to high concentration of the three drugs examined, but appreciable inhibition was observed after 21 h, with chloramphenicol being the least effective inhibitor. These results suggest that there is a stage-specific effect of inhibition of mitochondrial protein synthesis on subsequent development and that the mitochondria are essential for growth and development even though they lack a functional Krebs cycle.     

Isoleucine hydroxamate, an isoleucine antagonist

Isoleucine hydroxamate (Ile.Hdx) was found to inhibit the growth of Serratia marcescens and to antagonize isoleucine. At a low concentration of Ile.Hdx, at which the growth of the wild strain was completely inhibited, the growth of an isoleucine auxotroph was not inhibited in the medium containing a limiting amount of d-threonine as the isoleucine source. At a higher concentration, this antagonist exhibited a considerable inhibitory effect on the growth of the auxotroph. Ile.Hdx showed the same inhibitory effect as isoleucine on l-threonine dehydratase activity at the concentrations 10 times those of isoleucine. Ile.Hdx caused also derepression of isoleucine-valine biosynthetic enzymes and the derepression was overcome by isoleucine. These results indicate the Ile.Hdx causes growth inhibition by its effects on isoleucine metabolism.     

O-Methylthreonine Inhibition of Growth and of Threonine Deaminase in Escherichia coli

O-methylthreonine (OMT), an isosteric analogue of isoleucine, markedly inhibited growth of Escherichia coli 15. This inhibition was overcome most effectively by addition of isoleucine, valine, or leucine to the medium and less effectively by addition of threonine. The dipeptide, valylleucine, also relieved the OMT-induced inhibition but only after a lag period, suggesting that valine and leucine, liberated by dipeptidase action, compete with OMT for entry into the cell. OMT was activated and transferred to transfer ribonucleic acid (RNA) by isoleucyl-RNA synthetase in vitro. The rate of OMT incorporation into protein of intact cells was comparable to that of isoleucine. In contrast to isoleucine, very high concentrations of OMT were required to inhibit threonine deaminase, and the inhibition was strictly competitive with threonine. In addition, OMT inhibited a threonine deaminase preparation desensitized to isoleucine inhibition.     

Effects of amino acids on Thiobacillus acidophilus. I. Growth studies with special reference to valine.

The heterotrophic growth of Thiobacillus acidophilus was inhibited by branched-chain amino acids; valine, isoleucine, and leucine. The inhibition by valine and leucine were partially reversed by isoleucine, and the inhibition by isoleucine was partially reversed by valine. Inhibitions by methionine or threonine were partially reversed when both amino acids were present in the growth medium. Inhibition by tyrosine was increased by phenylalanine or tryptophan. Cystine completely inhibited growth. Other amino acids tested produced little or no inhibition. Acetohydroxy acid synthetase (AHAS) activity was demonstrated in crude extracts of T. acidophilus. In crude extracts the optimum pH was 8.5 with a shift to 9.0 in the presence of valine. Valine was the only branched-chain amino acid which inhibited the AHAS activity. The presence of only one peak of AHAS activity upon centrifugation in linear glycerol density gradients demonstrated that the AHAS activity sediments as one component.     

Differential effects of the sulfonylurea herbicides chlorsulfuron and sulfometuron methyl on microorganisms

The sulfonylurea herbicides exert their effects on cells via their inhibition of the acetohydroxy acid synthase (AHS) enzymes. Although chlorsulfuron and sulfometuron methyl often affected microbial growth differently their effects on the AHS activities of toluenised cells were similar. Sulfometuron methyl was always a more potent inhibitor than chlorsulfuron. We have postulated that sulfometuron methyl penetrated into microbial cells more readily then did chlorsulfuron. The effect of the herbicides on microbial growth was altered by the composition of the medium and in particular by valine or valine plus isoleucine. Different microorganisms had different complements of AHS isoenzymes which together with differences in permeability were the most likely explanations for the different responses observed. It was pointed out that application of these sulfonylurea herbicides would have significant effects on the microbial ecological balance of soil, and particularly so in alkaline soils. The consequences would be most evident in agricultural situations where the microbial population played an important role in maximising the productivity of crops.     

Isoleucine and valine metabolism of Escherichia coli. XIV. Effect of thiaisoleucine

Thiaisoleucine (2-amino-3-methylthiobutyrate) completely inhibited the growth of strain K-12 of Escherichia coli at a concentration of 5 x 10(-3)m. The inhibition was antagonized by growth-factor amounts of l-isoleucine. Thiaisoleucine inhibited the deamination of threonine and the transfer of (14)C-isoleucine to soluble ribonucleic acid and underwent transamination with alpha-ketoglutarate as the amino acceptor. In each case, the analogue appeared to be less effective than isoleucine as either an inhibitor or substrate.     

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.     

Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium.

The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured. The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter. The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition. This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism. The organization of the pathway thus potentiates the inhibitory effect of SMM. IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate. Valine completely protected strain TV105 from IpOHA at the MIC. A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested. IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration. The recovery is apparently due to induction of new KARI synthesis, as well as disappearance of IpOHA from the medium.     

Thrombin inhibition by cyclic peptides from thrombomodulin.

Peptides corresponding to the loop regions of the fourth, fifth, and sixth epidermal growth factor (EGF)-like domains of thrombomodulin (TM) have been synthesized and assayed for thrombin inhibition, as indicated by both inhibition of thrombin-mediated fibrinogen clotting and inhibition of the association of thrombin with TM that results in protein C activation. Peptides from the fifth EGF-like domain showed significant inhibition of fibrinogen clotting and protein C activation, whereas peptides from the fourth and sixth EGF-like domains were weak inhibitors in both assays. Two structural features were important for inhibitory potency of the peptides from the fifth EGF-like domain: cyclization by a disulfide bond and attachment of the "tail" amino acids C-terminal to the disulfide loop. Linear control peptides did not significantly inhibit clotting or protein C activation. The C-terminal loop alone, the "tail" peptide, or a mixture of the two were at least 10-fold less potent inhibitors of clotting or protein C activation. A more constrained peptide analog was designed by deletion of an isoleucine within the C5-C6 disulfide loop, TM52-1 + 5C. This analog was a better inhibitor in both assay systems, having a Ki for protein C activation of 26 microM.     

Target of serine inhibition in Escherichia coli

L-serine has long been known to inhibit growth of Escherichia coli cells cultured in minimal medium supplemented with glucose, lactate, or another carbohydrate as the sole source of carbon. However, the target of serine inhibition was not known. The growth inhibition was released by adding isoleucine, 2-ketobutyric acid, threonine or homoserine, but not by aspartate. Thus the inhibition site must be between aspartate and homoserine in the isoleucine biosynthetic pathway. We found that homoserine dehydrogenase I was strongly inhibited by serine. We isolated serine-resistant mutants, and found that in these mutants homoserine dehydrogenase I was resistant to serine. Thus, we conclude that the target of serine inhibition in Escherichia coli is homoserine dehydrogenase I.     

Regulation of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) growth in McCoy cells by amino acid antagonism

Chlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.     

Inhibition of phototrophically growing Chromatium vinosum D by glycine

Abstract The effect of glycine on phototrophically growing Chromatium vinosum D was investigated in the presence and absence of various amino acids. Inhibition of autotrophic cells was more pronounced than that of heterotrophic cells. Cells of growth-inhibited cultures were misshaped, showing vacuolation and partial protoplast formation. Cells exposed to glycine in the presence of equimolar amounts of either d - or l -alanine did not show any inhibition of growth. Neither serine, leucine, valine, isoleucine nor α-aminoisobutyric acid could counteract the inhibition of growth by glycine. The inhibitory effect of glycine on growth is thus likely to be caused by a competition between glycine and alanine during cell wall synthesis. The possible functions of glycine transport system(s) in the natural habitat of C. vinosum are discussed.     

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.