Aspartokinase of Streptococcus mutans: purification, properties, and regulation.

Aspartokinase from Streptococcus mutans BHT was purified to homogeneity and characterized. The molecular weight of the native enzyme was estimated to be 242,000 by gel filtration. Cross-linking of aspartokinase with dimethyl suberimidate and polyacrylamide gel electrophoresis of the amidinated enzyme in the presence of sodium dodecyl sulfate showed the enzyme to be composed of six identical subunits with a molecular wieght of 40,000. The optimal pH range for enzyme activity was 6.5 to 8.5. The apparent Michaelis-Menten constants for aspartate and ATP were 5.5 and 2.2 mM, respectively. The enzyme was stable within the temperature range of 10 to 35 degrees C. Aspartokinase was not feedback inhibited by individual amino acids, but was concertedly inhibited by L-lysine and L-threonine (93.5% inhibition at 10 mM each). The inhibition was noncompetitive with respect to aspartate (Ki = 10 mM) and mixed with respect to ATP. L-Threonine methyl ester and L-threonine amide were able to substitute for L-threonine in feedback inhibition, but the requirement for L-lysine uas strict. The feedback inhibitor pair protected the enzyme against heat denaturation. Aspartokinase synthesis was repressed by L-threonine; this repression was enhanced by L-lysine, but was slightly attenuated by L-methionine.     

Kinetic and allosteric properties of highly-purified, biosynthetic L-threonine dehydrogenase from brewer's yeast Saccharomyces carlsbergensis

Kinetic and allosteric propeties of highly purified "biosynthetic" L-threonine dehydratase from brewer's yeast S. carlbergensis were studied at three pH values, using L-threonine and L-serine as substrates. It was shown that the plot of the initial reaction rate (v) versus initial substrate concentrations ([S]0 pH 6.5 is hyperbolic (Km=5.0.10-2M), while these at pH 7.8 and 9.5 have a faintly pronounced sigmoidal shape with fast occurring saturation plateaus ([S]0.5= 1.0.10-2 and 0.9.10-2M, respectively). the ratios between L-threonine and L-serine dehydratation rates depend on pH. The kinetic properties and the dependence of substrate specificity on pH suggest that the enzyme molecule undergoes pH-induced (at pH 7.0) conformational changes. The determination of pK values of the enzyme functional groups involved in L-threonine binding demonstrated that these groups have pK is approximately equal to 7.5 and 9.5. The latter group was hypothetically identified as a epsilon-NH2-group of the lysine residue. High concentrations of the allosteric inhibitor (L-isoleucine) decrease the rates of L-threonine and L-serine dehydratation and induce the appearance (at pH 6.5) or increase (at pH 7.9 and 9.5) of homotropic cooperative interactions between the active sites in the course of L-threonine dehydratation. The enzyme inhibition by L-isoleucine increases with a decrease of L-threonine concentrations. Low L-isoleucine concentrations, as well as the enzyme activator (L-valine) stimulate the enzyme at non-saturating substrate concentrations (when L-threonine or L-serine are used as substrates) without normalization of (v) versus [S]0 plots. The maximal activation of the enzyme is observed at pHG 8.5--9.0. It is assumed that the molecule of "biosynthetic" L-threonine dehydratase from brewer's yeast contains two types of sites responsible for the effector binding, i.e., "activatory" and "inhibitory" ones.     

Bacterial catabolism of threonine. Threonine degradation initiated by L-threonine-NAD+ oxidoreductase.

1. Isolates representing seven bacterial genera capable of growth on L-threonine medium, and possessing high L-threonine 3-dehydrogenase activity, were examined to elucidate the catabolic route. 2. The results of growth, manometric and enzymic experiments indicated the catabolism of L-threonine by cleavage to acetyl-CoA plus glycine, the glycine being further metabolized via L-serine to pyruvate, in all cases. No evidence was obtained of a role for aminoacetone in threonine catabolism or for the metabolism of glycine by the glycerate pathway. 3. The properties of a number of key enzymes in L-threonine catabolism were investigated. The inducibly formed L-threonine 3-dehydrogenase, purified from Corynebacterium sp. B6 to a specific activity of about 30-35 mumol of product formed/min per mg of protein, exhibited a sigmoid kinetic response to substrate concentration. The half-saturating concentration of substrate, [S]0.5, was 20mM and the Hill constant (h) was 1.50. The Km for NAD+ was 0.8mM. The properties of the enzyme were studied in cell-free extracts of other bacteria. 4. New assays for 2-amino-3-oxobutyrate-CoA ligase were devised. The Km for CoA was determined for the first time and found to be 0.14mM at pH8, for the enzyme from Corynebacterium sp. B6. Evidence was obtained for the efficient linkage of the dehydrogenase and ligase enzymes. Cell-free extracts all possessed high activities of the inducibly formed ligase. 5. L-Serine hydroxymethyltransferase was formed constitutively by all isolates, whereas formation of the 'glycine-cleavage system' was generally induced by growth on L-threonine or glycine. The coenzyme requirements of both enzymes were established, and their linked activity in the production of L-serine from glycine was demonstrated by using extracts of Corynebacterium sp. B6. 6. L-Serine dehydratase, purified from Corynebacterium sp. B6 to a specific activity of about 4mumol of product formed/min per mg of protein, was found to exhibit sigmoid kinetics with an [S]0.5 of about 20mM and h identical to 1.4. Similar results were obtained with enzyme preparations from all isolates. The enzyme required Mg2+ for maximum activity, was different from the L-threonine dehydratase also detectable in extracts, and was induced by growth on L-threonine or glycine.     

Metabolism of 2-oxoaldehydes in yeasts. Possible role of glycolytic bypath as a detoxification system in L-threonine catabolism by Saccharomyces cerevisiae

L-Threonine catabolism by Saccharomyces cerevisiae was studied to determine the role of glycolytic bypath as a detoxyfication system of 2-oxoaldehyde (methylglyoxal) formed from L-threonine catabolism. During the growth on L-threonine as a sole source of nitrogen, a large amount of aminoacetone was accumulated in the culture. The enzymatic analyses indicated that L-threonine was converted into either acetaldehyde and glycine by threonine aldolase or 2-aminoacetoacetate by NAD-dependent threonine dehydrogenase. Glycine formed was condensed with acetyl-CoA by aminoacetone synthase to form 2-aminoacetoacetate, a labile compound spontaneously decarboxylated into aminoacetone. The enzyme activities of the glycolytic bypath of the cells grown on L-threonine were considerably higher than those of the cells grown on ammonium sulfate as a nitrogen source. The result indicated the possible role of glycolytic bypath as a detoxification system of methylglyoxal formed from L-threonine catabolism.     

Behavior of L-threonine-degrading enzymes during liver regeneration

We examined the effects of a two-thirds hepatectomy in the adult rat on the activities of the three L-threonine-degrading enzymes, L-threonine dehydratase, L-threonine aldolase and L-threonine dehydrogenase. Noticeable variations were observed which did not occur in either sham-operated or turpentine-treated rats and were not linked to food intake. They were considered specific to the regenerating liver. When the reactions were followed in vitro, L-threonine deaminase and L-threonine aldolase were significantly lower for the first 12-24 h: L-threonine dehydrogenase decreased only after 48 h. These results are linked to a decrease in the enzyme concentration in the tissue. L-Serine and L-threonine liver concentrations increased 2-3-fold during the same periods. When the activities were evaluated in vivo, the levels of the first two enzymes remained constant for 24 h, but increased after 48 h; L-threonine dehydrogenase increased between 12 and 48 h. The in vivo activity of the enzymes was reflected by total L-threonine degradation, which had a single sharp peak at 48 h. The asynchronous variations in enzyme activity are related to the differences in protein metabolism which occur in the regenerating liver, and are the consequence of a new transient differential control. The changes observed are significant in liver regeneration; they regulate the consumption and the serum and liver levels of L-serine and L-threonine, setting them aside for protein synthesis. They minutely control the flux of amino acids toward gluconeogenesis, since, during the first 48 h after partial hepatectomy, the production of glucose is ensured principally by lactate; the contribution of L-threonine seems to be more significant only at 48 h. These findings are useful in the study of the regulation of the enzymes involved in amino acid metabolism during liver regeneration.     

Threonine deaminase from Escherichia coli. II. Maturation and physical properties of the enzyme from a mutant altered in its regulation of gene expression.

The biosynthetic L-threonine deaminase (L-threonine hydrolase deaminating, EC 4.2.1.16) has been purified from Escherichia coli K12 regulatory mutant CU18. This mutant has properties that follow the predictions of the autogregulatory model previously proposed for the control of synthesis of the isoleucine-valine biosynthetic enzymes. The autoregulatory model specifies that L-threonine deaminase participates in the control of the expression of the ilv ADE gene cluster as well as the ilv B gene and ilv C gene, which constitute three separate units of regulation. The single mutation in strain CU18 results in altered regulation of ilv gene expression and in the production of an altered L-threonine deaminase. The immature form of the enzyme purified from mutant CU18 exhibits an altered response to L-valine, a maturation-inducing ligand. The native form of the mutant is altered in its apparent Km for L-threonine and in its response to the effects of L-valine and L-isoleucine upon catalytic activity. The mutant and wild type L-threonine deaminases differ in the apoenzyme formed as a consequence of alkaline dialysis. Dialysis of the mutant enzyme yields an apoenzyme mixture, apparently of dimers and monomers, while the wild type enzyme yields only dimers. The CU18 L-threonine deaminase, is however, indistinguishable from the wild type enzyme in molecular weight and subunit composition.     

Novel psychrophilic and thermolabile L-threonine dehydrogenase from psychrophilic Cytophaga sp. strain KUC-1

A psychrophilic bacterium, Cytophaga sp. strain KUC-1, that abundantly produces a NAD(+)-dependent L-threonine dehydrogenase was isolated from Antarctic seawater, and the enzyme was purified. The molecular weight of the enzyme was estimated to be 139,000, and that of the subunit was determined to be 35,000. The enzyme is a homotetramer. Atomic absorption analysis showed that the enzyme contains no metals. In these respects, the Cytophaga enzyme is distinct from other L-threonine dehydrogenases that have thus far been studied. L-Threonine and DL-threo-3-hydroxynorvaline were the substrates, and NAD(+) and some of its analogs served as coenzymes. The enzyme showed maximum activity at pH 9.5 and at 45 degrees C. The kinetic parameters of the enzyme are highly influenced by temperatures. The K(m) for L-threonine was lowest at 20 degrees C. Dead-end inhibition studies with pyruvate and adenosine-5'-diphosphoribose showed that the enzyme reaction proceeds via the ordered Bi Bi mechanism in which NAD(+) binds to an enzyme prior to L-threonine and 2-amino-3-oxobutyrate is released from the enzyme prior to NADH. The enzyme gene was cloned into Escherichia coli, and its nucleotides were sequenced. The enzyme gene contains an open reading frame of 939 bp encoding a protein of 312 amino acid residues. The amino acid sequence of the enzyme showed a significant similarity to that of UDP-glucose 4-epimerase from Staphylococcus aureus and belongs to the short-chain dehydrogenase-reductase superfamily. In contrast, L-threonine dehydrogenase from E. coli belongs to the medium-chain alcohol dehydrogenase family, and its amino acid sequence is not at all similar to that of the Cytophaga enzyme. L-Threonine dehydrogenase is significantly similar to an epimerase, which was shown for the first time. The amino acid residues playing an important role in the catalysis of the E. coli and human UDP-glucose 4-epimerases are highly conserved in the Cytophaga enzyme, except for the residues participating in the substrate binding.     

A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by L-threonine.

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by L-threonine. To investigate the regulation of the C. glutamicum HD enzyme by L-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, homdr. L-Threonine inhibits the wild-type (wt) enzyme with a Ki of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM L-threonine. The homdr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned homdr gene is genetically unstable. The cloned homdr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM L-threonine. Sequence analysis revealed that the homdr mutation is a single nucleotide (G1964) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the L-threonine allosteric response. The homdr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.     

Characterization of L-threonine deaminase activity of guinea pig liver induced by a high protein diet

In a foregoing paper, we demonstrated that under equilibrated diet conditions, guinea pig liver L-threonine deaminase activity should be allocated to two distinct enzymes: a specific L-threonine deaminase without activity toward L-serine and a L-serine deaminase having a secondary activity toward L-threonine. In the present work, we observed that a high protidic diet caused an elevation of total threonine deaminase activity. Thus purification of guinea pig liver L-threonine deaminase was attempted, using ultracentrifugation, salt precipitation, heat treatment, ion exchange chromatography on DEAE Sephacel, Sephadex G 200 molecular sieve, 2 amino-2 methyl-1 propanol linked CH 4B Sepharose chromatography. The weak variations of the ratios of specific activities respectively toward L-threonine and L-serine observed at each stage of the purification procedure indicated that both activities are very likely supported by a single enzyme preexisting in the liver of guinea pigs fed an equilibrated diet. No isoenzyme was evidenced by polyacrylamide gel electrophoresis or DEAE Sephacel chromatography. Moreover, our purification procedure demonstrated that not only inducible L-threonine deaminase guinea pig liver activity was due to L-serine deaminase, but also that an initially existing specific L-threonine deaminase activity paradoxically disappeared with a protein rich diet.     

L-threonine aldolase is not a genuine enzyme in rat liver.

Activity of L-threonine aldolase in rat liver cytosolic extract was not affected by the omission of alcohol dehydrogenase in a previously established NADPH-linked alcohol dehydrogenase-coupled assay. The liver extract was able to catalyse the dehydrogenation of NADPH with either acetaldehyde (a product of L-threonine aldolase action) or 2-oxobutyrate (a product of L-threonine dehydratase action). When the liver extract was chromatographed on a Sephacryl S-200 column, no threonine aldolase activity was detected in the eluate. However, activity of threonine aldolase re-appeared when the fractions with highest activity of lactate dehydrogenase and threonine dehydratase were mixed. Activity of threonine aldolase could also be abolished by removing threonine dehydratase from the liver extract with a specific antibody. Hence L-threonine aldolase should not be a genuine enzyme in the rat liver, and the apparent enzyme activity may result from a combined effect of threonine dehydratase and lactate dehydrogenase (or an oxo acid-linked NADPH dehydrogenase) in the liver cytosolic extract.     

Mobilization of the Proteus morganii chromosome by R plasmids

Mutants of Pseudomonas aeruginosa PAC1 which could grow on L-threonine were isolated. These mutants, like the parent strain, synthesized a biosynthetic threonine deaminase, but its apparent Km value for threonine was higher than that of the enzyme from strain PAC1. These mutants also synthesized an inducible NAD-dependent threonine dehydrogenase, which was not present in the parent strain. No threonine aldolase activity could be detected. The results suggest that the threonine deaminase with lowered affinity for L-threonine, together with L-threonine dehydrogenase, enabled these mutants to utilize L-threonine as the sole source of carbon for growth.     

Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production.

Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.     

Kinetic and allosteric properties of L-threonine-L-serine dehydratase from human liver]

It was shown that low concentrations of ATP (1..10(-4)M) and 10-fold concentrations of AMP (1.10(-3)M) at three constant L-threonine concentrations activated the L-threonine dehydratase activity of L-threonine-L-serine dehydratase from human liver, but had no effect on the L-serine dehydratase activity of this enzyme. Higher concentrations of both nucleotides inhibited the enzyme. The effects of ATP and AMP were specific. The activating and inhibiting effects of various concentrations of ATP and AMP were revealed as changes in the shapes of the curves for the initial reaction rate of the L-threonine dehydratase reaction versus initial substrate concentration. For this reaction the curves were not hyperbolic and were characterized by intermediary plateaux. ATP and AMP also influenced the maximal rate of the enzymatic reaction. Using the desensitization method it was shown that the activating effects of ATP and AMP are of allosteric nature. Thus, human liver L-threonine-L-serine dehydratase is an allosteric enzyme, for which positive allosteric effectors are low concentrations of ATP and AMP and negative allosteric effectors are high concentrations of these nucleotides. A possible mechanism of allosteric regulation of the enzyme under catalysis of the L-threonine dehydratase reaction and the lack of regulation under catalysis of the L-serine dehydratase reaction as well as specificity of the allosteric sites of this enzyme to the two nucleotides and the physiological significance of this process are discussed.     

Inhibition and regulation of rat liver L-threonine dehydrogenase by different fatty acids and their derivatives.

Rat liver L-threonine dehydrogenase is a mitochondrial enzyme which transforms L-threonine either into aminoacetone or into acetyl-CoA. We show that it is inhibited by several fatty acids and their derivatives: short chain fatty acids, L-2-hydroxybutyrate and D-3-hydroxybutyrate, long chain fatty acids, such as lauric acid, myristic acid, palmitic and stearic acids, bicarboxylic acids such as malonic acid and its derivatives methyl- and hydroxymalonic acids. The inhibition occurs at low and physiological concentrations of such compounds, which are normally present and metabolized in mitochondria. It presumably plays a role in the physiology of acetyl-CoA-dependent formation of fatty acids and ketobodies, in L-threonine-dependent gluconeogenesis, and in the regulation of L-threonine metabolism by L-threonine dehydrogenase and L-threonine deaminase.     

The human L-threonine 3-dehydrogenase gene is an expressed pseudogene

Background  

L-threonine is an indispensable amino acid. One of the major L-threonine degradation pathways is the conversion of L-threonine via 2-amino-3-ketobutyrate to glycine. L-threonine dehydrogenase (EC 1.1.1.103) is the first enzyme in the pathway and catalyses the reaction: L-threonine + NAD⁺ = 2-amino-3-ketobutyrate + NADH. The murine and porcine L-threonine dehydrogenase genes (TDH) have been identified previously, but the human gene has not been identified.     

The PduX enzyme of Salmonella enterica is an L-threonine kinase used for coenzyme B12 synthesis

Here, the PduX enzyme of Salmonella enterica is shown to be an L-threonine kinase used for the de novo synthesis of coenzyme B(12) and the assimilation of cobyric acid (Cby). PduX with a C-terminal His tag (PduX-His(6)) was produced at high levels in Escherichia coli, purified by nickel affinity chromatography, and partially characterized. (31)P NMR spectroscopy established that purified PduX-His(6) catalyzed the conversion of l-threonine and ATP to L-threonine-O-3-phosphate and ADP. Enzyme assays showed that ATP was the preferred substrate compared with GTP, CTP, or UTP. PduX displayed Michaelis-Menten kinetics with respect to both ATP and l-threonine and nonlinear regression was used to determine the following kinetic constants: V(max) = 62.1 +/- 3.6 nmol min(-1) mg of protein(-1); K(m)(, ATP) = 54.7 +/- 5.7 microm and K(m)(,Thr) = 146.1 +/- 8.4 microm. Growth studies showed that pduX mutants were impaired for the synthesis of coenzyme B(12) de novo and from Cby, but not from cobinamide, which was the expected phenotype for an L-threonine kinase mutant. The defect in Cby assimilation was corrected by ectopic expression of pduX or by supplementation of growth medium with L-threonine-O-3-phosphate, providing further support that PduX is an L-threonine kinase. In addition, a bioassay showed that a pduX mutant was impaired for the de novo synthesis of coenzyme B(12) as expected. Collectively, the genetic and biochemical studies presented here show that PduX is an L-threonine kinase used for AdoCbl synthesis. To our knowledge, PduX is the first enzyme shown to phosphorylate free L-threonine and the first L-threonine kinase shown to function in coenzyme B(12) synthesis.     

Requirements for induction of the biodegradative threonine dehydratase in Escherichia coli.

Synthesis of the biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, was prevented by dissolved oxygen concentrations of 6 micrometer or greater. This effect was shown to be exerted solely on synthesis, rather than being the result of enzyme inactivation in vivo. In addition to an anaerobic environment, maximum enzyme synthesis was dependent upon the presence of a complete complement of amino acids, with omission of L-threonine, L-valine, or L-leucine producing the largest decreases in enzyme formation. L-Threonine, the most essential of the amino acid requirements, could be partially replaced by DL-allothreonine or alpha-ketobutyrate. Half-maximal stimulation of enzyme synthesis occurred with 0.4 mM threonine in the medium. The roles of anaerobiosis and amino acids are interpreted as being in accord with the concept that threonine dehydratase functions in anaerobic energy production under conditions of amino acid sufficiency.     

The sulfhydryl content of L-threonine dehydrogenase from Escherichia coli K-12: relation to catalytic activity and Mn2+ activation

When oxidized to cysteic acid by performic acid or converted to carboxymethylcysteine by alkylation of the reduced enzyme with iodoacetate, a total of six half-cystine residues/subunit are found in L-threonine dehydrogenase (L-threonine: NAD+ oxidoreductase, EC 1.1.1.103; L-threonine + NAD(+)----2-amino-3-oxobutyrate + NADH) from Escherichia coli K-12. Of this total, two exist in disulfide linkage, whereas four are titratable under denaturing conditions by dithiodipyridine, 5,5'-dithiobis(2-nitrobenzoic acid), or p-mercuribenzoate. The kinetics of enzyme inactivation and of modification by the latter two reagents indicate that threonine dehydrogenase has no free thiols that selectively react with bulky compounds. While incubation of the enzyme with a large excess of iodoacetamide causes less than 10% loss of activity, the native dehydrogenase is uniquely reactive with and completely inactivated by iodoacetate. The rate of carboxymethylation by iodoacetate of one -SH group/subunit is identical with the rate of inactivation and the carboxymethylated enzyme is no longer able to bind Mn2+. NADH (0.5 mM) provides 40% protection against this inactivation; 60 to 70% protection is seen in the presence of saturating levels of NADH plus L-threonine. Such results coupled with an analysis of the kinetics of inactivation caused by iodoacetate are interpreted as indicating the inhibitor first forms a reversible complex with a positively charged moiety in or near the microenvironment of a reactive -SH group in the enzyme before irreversible alkylation occurs. Specific alkylation of one -SH group/enzyme subunit apparently causes protein conformational changes that entail a loss of catalytic activity and the ability to bind Mn2+.     

L-Threonine dehydrogenase from goat liver. Feedback inhibition by methylglyoxal

L-Threonine dehydrogenase, which forms aminoacetone from L-threonine and NAD, has been extensively purified from goat liver. A feedback inhibition of this enzyme has been observed with methylglyoxal. Kinetic data and other experiments indicate that methylglyoxal acts at a site other than the active site of the enzyme. The enzyme contains a single subunit of Mr 89,000. The apparent Km values of the enzyme for L-threonine and NAD were found to be 5.5 and 1 mM, respectively.