Publications

• Characterization of aspartate kinase III of Bacillus subtilis.

  Kobashi N., Nishiyama M., Yamane H.

  A search in the Bacillus subtilis genome sequence found that the gene designated yclM encode(s) a protein showing significant identity in amino acid sequence to aspartate kinases. When yclM was introduced into Escherichia coli cells deficient in all three aspartate kinase genes, production of a pr ... >> More

  A search in the Bacillus subtilis genome sequence found that the gene designated yclM encode(s) a protein showing significant identity in amino acid sequence to aspartate kinases. When yclM was introduced into Escherichia coli cells deficient in all three aspartate kinase genes, production of a protein with molecular size 50 kDa, which was similar to the value deduced from the nucleotide sequence of the gene, was observed. Expectedly, the protein purified to homogeneity had aspartate kinase activity. The enzyme was significantly inhibited by simultaneous addition of both threonine and lysine, which is a typical feature of aspartate kinase III of B. subtilis. The enzyme was very unstable in 10 mM tris-HCl (pH 7.5) buffer, but was stabilized by addition of 500 mM ammonium sulfate. Although all the aspartate kinases so far investigated are oligomeric enzymes, this aspartate kinase was suggested to be a monomer. << Less

  Biosci. Biotechnol. Biochem. 65:1391-1394(2001) [PubMed] [EuropePMC]

• An integrated study of threonine-pathway enzyme kinetics in Escherichia coli.

  Chassagnole C., Rais B., Quentin E., Fell D.A., Mazat J.P.

  We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equ ... >> More

  We have determined the kinetic parameters of the individual steps of the threonine pathway from aspartate in Escherichia coli under a single set of experimental conditions chosen to be physiologically relevant. Our aim was to summarize the kinetic behaviour of each enzyme in a single tractable equation that takes into account the effect of the products as competitive inhibitors of the substrates in the forward reaction and also, when appropriate (e.g. near-equilibrium reactions), as substrates of the reverse reactions. Co-operative feedback inhibition by threonine and lysine was also included as necessary. We derived the simplest rate equations that describe the salient features of the enzymes in the physiological range of metabolite concentrations in order to incorporate them ultimately into a complete model of the threonine pathway, able to predict quantitatively the behaviour of the pathway under natural or engineered conditions. << Less

  Biochem. J. 356:415-423(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase from Arabidopsis thaliana.

  Paris S., Wessel P.M., Dumas R.

  In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from ... >> More

  In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants (Arabidopsis thaliana). The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 micromol of aspartyl phosphate produced min(-1) mg(-1) of protein and 18.8 micromol of NADPH formed min(-1) mg(-1) of protein, respectively. These values are 200-fold higher than those reported previously for partially purified plant enzymes. AK-HSDH exhibited hyperbolic kinetics for aspartate, ATP, homoserine, and NADP with K(M) values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 microM, respectively. Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors. In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa. Addition of the effector converted the enzyme into a tetrameric form of 320 kDa. << Less

  Protein Expr. Purif. 24:105-110(2002) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• The aspartate kinase of Pseudomonas putida. Regulation of synthesis and activity.

  Robert-Gero M., Poiret M., Cohen G.N.

  Biochim. Biophys. Acta 206:17-30(1970) [PubMed] [EuropePMC]

• The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. The two catalytic activities are carried by two independent regions of the polypeptide chain.

  Veron M., Falcoz-Kelly F., Cohen G.N.

  Eur J Biochem 28:520-527(1972) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• A specialized aspartokinase enhances the biosynthesis of the osmoprotectants ectoine and hydroxyectoine in Pseudomonas stutzeri A1501.

  Stoveken N., Pittelkow M., Sinner T., Jensen R.A., Heider J., Bremer E.

  The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. l-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. l-Aspartate-bet ... >> More

  The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. l-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. l-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from l-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an askgene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_askgenes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. colistrain carrying the plasmid-based ectABCD_askgene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCDgene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis. << Less

  J. Bacteriol. 193:4456-4468(2011) [PubMed] [EuropePMC]

• Identification of six novel allosteric effectors of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase isoforms. Physiological context sets the specificity.

  Curien G., Ravanel S., Robert M., Dumas R.

  The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. W ... >> More

  The Arabidopsis genome contains two genes predicted to code for bifunctional aspartate kinase-homoserine dehydrogenase enzymes (isoforms I and II). These two activities catalyze the first and the third steps toward the synthesis of the essential amino acids threonine, isoleucine, and methionine. We first characterized the kinetic and regulatory properties of the recombinant enzymes, showing that they mainly differ with respect to the inhibition of the homoserine dehydrogenase activity by threonine. A systematic search for other allosteric effectors allowed us to identify an additional inhibitor (leucine) and 5 activators (alanine, cysteine, isoleucine, serine, and valine) equally efficient on aspartate kinase I activity (4-fold activation). The six effectors of aspartate kinase I were all activators of aspartate kinase II activity (13-fold activation) and displayed a similar specificity for the enzyme. No synergy between different effectors could be observed. The activation, which resulted from a decrease in the Km values for the substrates, was detected using low substrates concentrations. Amino acid quantification revealed that alanine and threonine were much more abundant than the other effectors in Arabidopsis leaf chloroplasts. In vitro kinetics in the presence of physiological concentrations of the seven allosteric effectors confirmed that aspartate kinase I and II activities were highly sensitive to changes in alanine and threonine concentrations. Thus, physiological context rather than enzyme structure sets the specificity of the allosteric control. Stimulation by alanine may play the role of a feed forward activation of the aspartate-derived amino acid pathway in plant. << Less

  J. Biol. Chem. 280:41178-41183(2005) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• Kinetic and mutation analyses of aspartate kinase from Thermus flavus.

  Kobashi N., Nishiyama M., Tanokura M.

  To reveal the catalytic mechanism of Thermus aspartate kinase, each of 29 amino acid residues that were highly conserved in the sequenced aspartate kinases, was replaced with alanine or leucine by PCR site-directed mutagenesis. Comparison of the kinetic parameters of these mutants with those of th ... >> More

  To reveal the catalytic mechanism of Thermus aspartate kinase, each of 29 amino acid residues that were highly conserved in the sequenced aspartate kinases, was replaced with alanine or leucine by PCR site-directed mutagenesis. Comparison of the kinetic parameters of these mutants with those of the wild-type aspartate kinase suggested that Thr47 was involved in binding aspartate and that Lys7 and Glu74 were involved in catalysis. Analysis of the effective concentrations of magnesium ion on the activity showed that the mutants with replacements at Ser41, Thr47, Asp154 and Asp182 required higher concentrations of magnesium ion. This suggests that these four residues play important roles in the binding of magnesium ions which are required for enzymatic activity. << Less

  J. Biosci. Bioeng. 87:739-745(1999) [PubMed] [EuropePMC]