Threonine-sensitive Homoserine Dehydrogenase Research Articles

Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant.

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.

Aspartate kinase regulation in maize: Evidence for co-purification of threonine-sensitive aspartate kinase and homoserine dehydrogenase

The threonine-sensitive and resistant forms of homoserine dehydrogenase activities were assayed in all fractions obtained during the purification of three aspartate kinase isoenzymes (threonine, lysine and lysine plus S-adenosylmethionine-sensitive) from maize cultures. The homoserine dehydrogenase isoenzyme resistant to inhibition by threonine, has a molecular weight of 70 000 and could be easily separated from the three aspartate kinase isoenzymes by ion-exchange chromatography and gel filtration; similarly the two lysine-sensitive forms of aspartate kinase could be separated from both forms of homoserine dehydrogenase. The homoserine dehydrogenase sensitive to threonine inhibition has a molecular weight of 180 000 and co-purified with the threonine-sensitive aspartate kinase isoenzyme. The hypothesis of the presence of a bifunctional protein containing activities of aspartate kinase-homoserine dehydrogenase is discussed.

Characterization of ligand-induced states of maize homoserine dehydrogenase

The threonine-sensitive homoserine dehydrogenase ( l-homoserine: NAD(P) + oxidoreductase), isolated from seedlings of Zea mays L., is characterized by variable kinetic and regulatory properties. Previous analysis of this enzyme suggested that it is capable of ligand-mediated interconversions among four kinetically distinct states ( S. Krishnaswamy and J. K. Bryan (1983) Arch. Biochem. Biophys. 222, 449–463). These forms of the enzyme have been identified and found to differ in oligomeric configuration and conformation. In the presence of KCl and threonine a rapid equilibrium among three species of the enzyme (B, T, and K) is established. Each of these species can undergo a unique slow transition to a steady-state form under assay conditions. Results obtained from gel-filtration chromatography and sucrose density centrifugation indicate that the B and steady-state forms are tetramers and the T and K states are dimers. Evidence is presented to indicate that the rapid conversion from one dimeric species to the other can only occur via formation of the tetrameric B state. Chromatography under reactingenzyme conditions provides direct support for the slow formation of a common steadystate species from any one of the other forms of the enzyme. The rate of transition is influenced by threonine, homoserine, NAD +, and, for transitions involving association reactions, by enzyme concentration. Small, reproducible differences in the apparent size of the T and K forms, and the B and steady-state species, are attributed to changes in conformation. This conclusion is supported by differential susceptibility of the enzymic states to proteolytic inactivation, by different rates of inactivation by dithio-bis-nitrobenzoate, and by alterations in their thermal stability. In addition, the B, T, and K states of the enzyme exhibit unique intrinsic fluorescence spectra. Spectral changes are shown to closely parallel changes in kinetic and hysteretic properties of the enzyme. The results of diverse methods of analysis are internally consistent, and provide considerable support for the conclusion that this pleiotropic regulatory enzyme can exist in any of several physically distinct states.

Ligand-induced interconversions of maize homoserine dehydrogenase among different states

The threonine-sensitive homoserine dehydrogenase has been isolated and extensively purified from shoots of Zea mays L. var. earliking. This enzyme is shown to be hysteretic under certain conditions. Progress curves of the NAD-dependent reaction catalyzed by the maize enzyme can be characterized by distinct lags prior to achievement of steady state velocities, reflecting transitions from less active species to a more active steady state form of the enzyme. Incubation of the enzyme for 1 min at 25 °C prior to initiation of the reaction profoundly influences the properties of the less active enzyme and the nature of the subsequent slow transitions during assay. When the feedback modifier, l-threonine, or KCl is included in the preincubation mixture, the transitions involve biomolecular association reactions. In the absence of either ligand, or in the presence of an appropriate mixture of both, a unimolecular transition occurs during assay. Three unique preincubation states of the enzyme have been identified on the basis of their response to substrates and effectors; whereas, the kinetic and regulatory properties of the steady state form of the enzyme are independent of preincubation conditions. Steady state can thus be achieved by three different transitions. Each transition is retarded by threonine and favored by substrates and potassium, although the effects of these compounds differ quantitatively. Under the conditions tested, monovalent cations have no effect on the steady state velocity of the enzyme. A model describing the relationships among the four unique states of the enzyme which is consistent with the present results and supported by previous observations is proposed.

Isolation and characterization of two homoserine dehydrogenases from maize suspension cultures.

Homoserine dehydrogenase in unpurified extracts of maize (Zea mays L.) cell suspensions is inhibited 73% by the feedback regulator threonine; the remaining 27% of the total activity is not affected even by high concentrations of threonine. The threonine-resistant and threonine-sensitive homoserine dehydrogenase activities were separated by affinity chromatography on Blue Sepharose columns, and the two distinct homoserine dehydrogenases were purified. The threonine-resistant enzyme is an Mr = 70,000 dimer of two Mr = 38,000 subunits and the threonine-sensitive enzyme is an Mr = 190,000 dimer containing two apparently different subunits with molecular weights of 89,000 and 93,000. The threonine-resistant enzyme exhibits normal Michaelis-Menten kinetics and its activity is not affected by any of the amino acid end products of the aspartate pathway. The threonine-sensitive enzyme exhibits positive cooperative kinetics with respect to NADPH and is inhibited by threonine and stimulated by isoleucine. All attempts to affect interconversion of the two purified enzymes have been unsuccessful. Because the purified enzymes correspond to activities present in crude extracts of various maize tissues, it is concluded that the two types of homoserine dehydrogenase are natural in vivo constituents of maize.

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.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Carboxymethylation of a unique cysteine induces a conformational change of the enzyme.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Carboxymethylation of a unique cysteine induces a conformational change of the enzyme.

Threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Kinetic and spectroscopic effects upon binding of serine and threonine.

The two threonine-sensitive activities aspartokinase and homoserine dehydrogenase are inhibited by L-serine. The inhibition of the aspartokinase by L-serine displays homotropic cooperative effects and is competitive versus aspartate. The inhibition by L-serine of the homoserine dehydrogenase displays Michaelis-Menten kinetics which are of a competitive nature versus homoserine. Characteristic effects of L-serine on the protein include a perturbation of its absorption and fluorescence spectra, with an increase in the fluorescence of the protein-NADPH complex. L-serine shifts the allosteric equilibrium of the protein to a "T-like" conformation to which L-threonine binds noncooperatively. L-Serine, a threonine analog, is not capable, as the physiological effector, of inducing a complete R to T transition of the enzyme; the aspartokinase globules show a cooperative conformation change upon serine binding, but this conformation change is not found in the homoserine dehydrogenase globules.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K-12. Incubation of the enzyme in alkaline conditions: dissociation and disulfide-bridge formation.

Aspartokinase I - homoserine dehydrogenase I from Escherichia coli K-12, a homotetrameric enzyme, dissociates into dimers upon alkaline treatment. Both aspartokinase and homoserine dehydrogenase inactivation, as well as desensitazion towards L-threonine, occur in a multi-step process. Dithiothreitol stabilizes a dimeric form retaining full activity and sensitivity; L-homoserine stabilizing another dimeric form devoid of aspartokinase activity and retaining a substantial dehydrogenase activity insensitive toward L-threonine. A model is proposed showing that dissociation into dimers occurs in a first step, the resulting dimer losing both aspartokinase and homoserine dehydrogenase sensitivity in two subsequent steps involving the formation of intrachain disulfide bonds.

The Threonine‐Sensitive Homoserine Dehydrogenase and Aspartokinase Activities of Escherichia coli K12

2-Amino-4-oxo-5-chloropentanoic acid inactivates specifically the homoserine dehydrogenase activity of the bifunctional enzyme, aspartokinase I--homoserine dehydrogenase I. The aspartokinase activity remains essentially untouched and retains its threonine sensitivity. The inactivation of the dehydrogenase requires the covalent binding of one equivalent of the analogue per subunit. Alkylation does not affect the tetrameric state of the protein. The alkylating agent, a substrate analogue, meets the qualitative and quantitative requirements of an affinity label.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Distribution and accessibility to antibodies of some epitopes of the bifunctional enzyme.

In the presence of l-threonine, the allosteric effector, most of the antigenic determinants situated in the aspartokinase region of the wild-type enzyme become unavailable to the antibodies raised against a fragment of the enzyme containing this region and devoid of homoserine dehydrogenase activity. The cross-reactivities of the antibodies raised against this fragment (extracted from a nonsense mutant) and a fragment endowed with homoserine dehydrogenase activity but devoid of aspartokinase activity (obtained by limited proteolysis) with the corresponding antigens were studied. The conclusion is drawn that the two fragments, which share an overlapping sequence of molecular weight about 17,000, share at least two antigenic determinants.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K 12. Intra and intersubunit interactions between the catalytic regions of the bifunctional enzyme.

The interactions between the catalytic regions of the subunit of the bifunctional enzyme aspartokinase I‐homoserine dehydrogenase I, although not necessary for the catalytic functions, are shown to be of utmost importance for the regulation of the activities of the enzyme.The study of the effect of a number of –SH reagents on the protein shows that the inhibition of the homoserine dehydrogenase activity by the allosteric effector l‐threonine is closely correlated with the existence of an active aspartokinase region.The kinetic study of the proteolytic reaction leading to the previously described homoserine dehydrogenase fragment indicates that a dissociation is a necessary requirement for the digestion of the aspartokinase region.Cross‐linking with dimethylsuberimidate and proteolytic degradation of the fully cross‐linked tetramer have been used to show that the oligomeric interactions, necessary to build the tetrameric structure, involve the kinase region only.A tentative model is proposed showing aspartokinase I‐homoserine dehydrogenase I as an isologous tetramer in which the aspartokinase regions, organized as a “core”, are surrounded by the homoserine dehydrogenase regions.

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.

Aspartokinase I‐homoserine dehydrogenase I from Escherichia coli K 12 was subjected to mild proteolysis. A fragment carrying only the desensitized homoserine dehydrogenase activity was purified. It is a dimer having a subunit molecular weight of 55000 as opposed to 86000 for the subunit of the native tetrameric enzyme.On the other hand, a threonine sensitive aspartokinase devoid of homoserine dehydrogenase activity was extracted from the mutant Gif 108. The purified protein is shown to have subunits shorter than those of the wild‐type enzyme (molecular weight 47000).It was shown that the two catalytic activities of aspartokinase I‐homoserine dehydrogenase I are sequentially distributed on the single polypeptide chain: the aspartokinase activity is located in the amino‐terminal section and the homoserine dehydrogenase in the carboxyl‐terminal section.The results are discussed in relation to the configuration of the native enzyme and with the possible origin of the bifunctional protein.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K 12. Binding of threonine and of pyridine nucleotides: stoichiometry and optical effects.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K 12. Binding of threonine and of pyridine nucleotides: stoichiometry and optical effects.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K 12. Subunit structure of the protein catalyzing the two activities.

The complex protein carrying the two threonine sensitive activities aspartokinase I and homoserine dehydrogenase I is composed of six subunits of a molecular weight 60,000. Furthermore, quantitative determination of the N‐terminal amino acids indicates the presence of six methionine residues per mole of native enzyme (mol. wt. 360,000).Equilibrium sedimentation studies of N‐ethylmaleimide treated protein shows that its six disulfide bridges, revealed by chemical analysis, are intra‐chain.Fingerprints of tryptic digests of the protein fail to reveal any difference between the subunits.

Regulation of the methionine-specific aspartokinase and homoserine dehydrogenase of Salmonella typhimurium.

SUMMARY: Salmonella typhimurium, like Escherichia coli, has a methionine-regulated β-aspartokinase and homoserine dehydrogenase in addition to threonine-sensitive and lysine-sensitive aspartokinase isoenzymes and a threonine-sensitive homoserine dehydrogenase. These methionine-regulated enzymes are not subject to feedback inhibition by methionine (or by methionine and S-adenosylmethionine), but the homoserine dehydrogenase is inhibited by the methionine precursor cysteine and to a lesser extent by homocysteine. Studies of enzyme concentrations in methionine analogue-resistant strains show that mutations which lead to de-repression of the later methionine-forming enzymes also result in de-repressed synthesis of the methionine-regulated β-aspartokinase and homoserine dehydrogenase, although the methionine-forming enzymes as a whole are not subject to co-ordinate control.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K 12. 4. Isolation, molecular weight, amino acid analysis and behaviour of the sulfhydryl groups of the protein catalyzing the two activities.

The interactions between the catalytic regions of the subunit of the bifunctional enzyme aspartokinase I-homoserine dehydrogenase I, although not necessary for the catalytic functions, are shown to be of utmost importance for the regulation of the activities of the enzyme. The study of the effect of a number of –SH reagents on the protein shows that the inhibition of the homoserine dehydrogenase activity by the allosteric effector l-threonine is closely correlated with the existence of an active aspartokinase region. The kinetic study of the proteolytic reaction leading to the previously described homoserine dehydrogenase fragment indicates that a dissociation is a necessary requirement for the digestion of the aspartokinase region. Cross-linking with dimethylsuberimidate and proteolytic degradation of the fully cross-linked tetramer have been used to show that the oligomeric interactions, necessary to build the tetrameric structure, involve the kinase region only. A tentative model is proposed showing aspartokinase I-homoserine dehydrogenase I as an isologous tetramer in which the aspartokinase regions, organized as a “core”, are surrounded by the homoserine dehydrogenase regions.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli

In Escherichia coli K12, 1. 1. A single mutation can lead to the concomitant modification or to the concomitant loss of the two activities, threonine-sensitive β-aspartokinase (ATP: L-aspartate 4-phosphotransferase, EC 2.7.2.4) and threonine-sensitive homoserine dehydrogenase (L-homoserine: NADP+ oxidoreductase, EC 1.1.1.3). 2. 2. The two activities cannot be separated and the ratio of the specific activities remains constant throughout a 600-fold purification. 3. 3. The substrates of one of the activities are inhibitors of the otther activity. The observed inhibitions are specific. 4. 4. The threonine-sensitive aspartokinase is protected against thermal inactivation by NADPH, a substrate of the homoserine dehydrogenase. 5. 5. The conclusion is drawn that the two activities under study are carried by a single protein molecule. The apparent molecular mass of this complex protein is changed in some mutants. There is no apparent correlation between the apparent molecular mass and the cooperativity of inhibitor molecules. 6. 6. The significance of these findings is discussed in terms of metabolic regulation and intracellular topology.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli

1.1. Homoserine dehydrogenase I (L-homoserine: NADP+ oxidoreductase, EC 1.1.1.3) of Escherichia coli is inactivated in two steps by p-mercuribenzoic acid (PMB). The first inactivation step is accompanied by desensitization of the enzyme activity towards its allosteric effector, L-threonine. The desensitized dehydrogenase activity is protected against further action of PMB by its own substrates and by the substrates of the associated activity, aspartokinase I (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4).2.2. ATP and aspartate, the substrates of the kinase reaction induce conformational changes in the part of the complex enzyme molecule responsible for the dehydrogenase atalytic activity. 1. Homoserine dehydrogenase I (L-homoserine: NADP+ oxidoreductase, EC 1.1.1.3) of Escherichia coli is inactivated in two steps by p-mercuribenzoic acid (PMB). The first inactivation step is accompanied by desensitization of the enzyme activity towards its allosteric effector, L-threonine. The desensitized dehydrogenase activity is protected against further action of PMB by its own substrates and by the substrates of the associated activity, aspartokinase I (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4). 2. ATP and aspartate, the substrates of the kinase reaction induce conformational changes in the part of the complex enzyme molecule responsible for the dehydrogenase atalytic activity.

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli

1. 1. Homoserine dehydrogenase I (l-homoserine:NADP+ oxidoreductase, EC 1.1.1.3) of Escherichia coli is inactivated with apparent first-order kinetics by exposure at pH 9 in Tris buffer. This inactivation is accompanied by desensitization of the enzyme towards threonine. 2. 2. L-Aspartate and ATP, the substrates of the associated activity, β-aspartokinase I (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) protect against the desensitization of homoserine dehydrogenase.