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PubMed1983

A hybrid proteolytic fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I. Structure, inhibition pattern, dissociation properties, and generation of two homodimers.

A hybrid dimeric fragment of Escherichia coli aspartokinase I-homoserine dehydrogenase I (Fazel, A., Müller, K., Le Bras, G., Garel, J.-R., Véron, M., and Cohen, G. N. (1983) Biochemistry 22, 158-165) has been purified and shown to possess both aspartokinase and homoserine dehydrogenase activities and is rather stable in the presence of L-threonine. Its two activities are still inhibited by threonine, but noncooperatively in contrast to the native protein. The aspartokinase activity is found to be more sensitive to threonine than the dehydrogenase activity. In the absence of threonine, the different chains of the hybrid (Mr = 89,000 + 59,000) dissociate first into monomers, this being followed by the pairing of two homologous chains to form two homodimers. In the presence of L-threonine, the two homodimers do not dissociate to re-form the hybrid fragment. The NH2-terminal analysis of different chains of the hybrid shows that the homodimers correspond, respectively, to the dimer of the native protein (Mr = 2 X 89,000) and to a dimer already described (Véron, M., Falcoz-Kelly, F., and Cohen, G. N. (1972) Eur. J. Biochem. 28, 520-527).

Alcohol Oxidoreductases↗
PubMed2002

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

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.

Arabidopsis↗
PubMed1991

Effect of dimethyl sulfoxide on lysine production by a mutant of Bacillus subtilis with homoserine dehydrogenase activity.

Some Bacillus subtilis mutants with different levels of homoserine dehydrogenase were described. Strains that do not accumulate methionine have a high homoserine dehydrogenase activity. Low activity was detected in mutants where cell growth was completely inhibited by 0.7 mmol/L methionine. A low concentration of dimethyl sulfoxide had a stimulatory effect on lysine production by the methionine-sensitive mutant of Bacillus subtilis.

Bacillus subtilis↗
PubMed1991

Rapid purification and characterization of homoserine dehydrogenase from Saccharomyces cerevisiae.

Homoserine dehydrogenase of Saccharomyces cerevisiae has been rapidly purified to homogeneity by heat and acid treatments, ammonium sulfate fractionation, and chromatography on Matrex Gel Red A and Q-Sepharose columns. The final preparation migrated as a single entity upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr of 40,000. The Mr of the native enzyme was 81,000 as determined by gel filtration, suggesting that the enzyme is composed of two identical subunits. This feature was also confirmed by cross-linking analysis using the bifunctional reagent dimethyl suberimidate. Feedback inhibition by L-methionine and L-threonine was observed using the purified enzyme. The enzyme was markedly stabilized against heat treatment at high salt concentrations. Additions of feedback inhibitors or high concentrations of salts failed to cause any dissociation or aggregation of the enzyme subunits unlike enzymes from other sources such as Rhodospirillum rubrum. The enzyme denatured in 3 M guanidine-HCl was refolded by simple dilution with a concomitant restoration of the activity. Cross-linking analysis of the renaturation process suggested that the formation of the dimer is required for activity expression. Amino acid sequence analysis of peptides obtained by digestion of the enzyme protein with Achromobacter lyticus protease I revealed that several amino acid residues are strictly conserved among homoserine dehydrogenases from S. cerevisiae, Escherichia coli, and Bacillus subtilis.

Amino Acid Sequence↗
PubMed1984

E. coli aspartokinase II-homoserine dehydrogenase II polypeptide chain has a triglobular structure.

E.coli aspartokinase II-homoserine dehydrogenase II is, as aspartokinase I-homoserine dehydrogenase I, composed of three globular domains: the N-terminal domain is endowed with kinase activity; the C-terminal domain carries the dehydrogenase activity. These two parts of the polypeptide chain are separated by a central inactive domain. Thus, the polypeptide chains of the two multifunctional proteins are homologous not only in their sequence but also in their triglobular domain structure.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1985

Isolation of the aspartokinase domain of bifunctional aspartokinase I-homoserine dehydrogenase I from E.coli K12.

A proteolytic fragment (Mr approximately 25 000) carrying only the aspartokinase activity has been purified by chromatofocusing after limited proteolysis of aspartokinase I-homoserine dehydrogenase I from E.coli K12. The NH2-terminal sequence shows that it corresponds to the amino terminal peptide of the native enzyme. The results confirm a previous hypothesis about the organization of native aspartokinase I-homoserine dehydrogenase I.

Amino Acid Sequence↗
PubMed2001

Homoserine dehydrogenase from Saccharomyces cerevisiae: kinetic mechanism and stereochemistry of hydride transfer.

Homoserine dehydrogenase (HSD), which is required for the synthesis of threonine, isoleucine and methionine in fungi, is a potential target for novel antifungal drugs. In order to design effective inhibitors, the kinetic mechanism of Saccharomyces cerevisiae HSD and the stereochemistry of hydride transfer were examined. Product inhibition experiments revealed that yeast HSD follows an ordered Bi Bi kinetic mechanism, where NAD(P)H must bind the enzyme prior to aspartate semialdehyde (ASA) and homoserine is released first followed by NAD(P)+. H-(1,2,4-triazol-3-yl)-D,L-alanine was an uncompetitive inhibitor of HSD with respect to NADPH (K(ii)=3.04+/-0.18 mM) and a noncompetitive inhibitor with respect to ASA (K(is)=1.64+/-0.36 mM, K(ii)=3.84+/-0.46 mM), in agreement with the proposed substrate order. Both kinetic isotope and viscosity experiments provided evidence for a very rapid catalytic step and suggest nicotinamide release to be primarily rate limiting. Incubation of HSD with stereospecifically deuterated NADP[2H] and subsaturating amounts of aspartate semialdehyde revealed that the pro-S NADPH hydride is transferred to the aldehyde. The pH dependence of steady state kinetic parameters indicate that ionizable groups with basic pKs may be involved in substrate binding, consistent with the observation of Lys223 at the enzyme active site in the recently determined 3D structure [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. These findings provide the requisite foundation for future exploitation of fungal HSD in inhibitor design.

Homoserine Dehydrogenase↗
PubMed2001

Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity.

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.

Amino Acid Sequence↗
PubMed1997

Bacterial N-acyl-homoserine-lactone-dependent signalling and its potential biotechnological applications.

N-acyl homoserine lactones are bacterial signalling molecules involved in regulating diverse metabolic functions, particularly those relating to virulence, in concert with cell density. Each aspect of the signalling pathway, from production and recognition of the signal to expression of the target genes, offers a potential opportunity for exploitation. Attention is now focusing on the development of novel methods for bacterial enumeration, modulation of bacterial virulence and flexible, coordinated expression of heterologous genes through the use of N-acyl-homoserine-lactone-based systems.

Bacteria↗
PubMed1984

Stepwise inactivation of Escherichia coli aspartokinase-homoserine dehydrogenase I.

In the range of guanidine hydrochloride concentrations from 0.2 to 1.2 M, aspartokinase-homoserine dehydrogenase I loses its enzymatic properties, both kinase and dehydrogenase activities and their allosteric inhibition by L-threonine. Ligands which stabilize the tetrameric native structure protect the enzyme against inactivation. Under some conditions, all the functional properties do not disappear at the same rate: an intermediate species possessing only the kinase activity can be detected. Several arguments suggest that this partly active intermediate has a monomeric structure. These results show that deactivation of aspartokinase-homoserine dehydrogenase I is a stepwise process, compatible with the reverse of the previously described reactivation [Garel, J.-R., & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3379-3383]. The same measurements performed with a monofunctional fragment carrying the dehydrogenase activity show that the loss of dehydrogenase activity is the same whether or not the polypeptide chain is intact or lacks the kinase region; this finding suggests that the protein is composed of independent regions. The influence of protein aggregation in studying unfolding-refolding of oligomeric enzymes is also discussed.

Aspartokinase Homoserine Dehydrogenase↗
PubMed2003

Efficient synthesis of enantiopure beta-amino-gamma-keto acids from l-homoserine.

[reaction: see text] A variety of beta-amino-gamma-keto acids were prepared in four steps from commercially available l-homoserine. The reaction sequence is highlighted by a Ni-catalyzed Grignard addition to a N-protected derivative of l-homoserine. One of the beta-amino-gamma-keto acids was then used to create a beta-peptide trimer composed solely of beta-amino-gamma-keto acids.

Homoserine↗
PubMed2001

Haemodynamic effects of the bacterial quorum sensing signal molecule, N-(3-oxododecanoyl)-L-homoserine lactone, in conscious, normal and endotoxaemic rats.

N-acylhomoserine lactones (AHLs) are small, diffusible signalling molecules, employed by Gram-negative bacteria to coordinate gene expression with cell population density. Recent in vitro findings indicate that AHLs may function as virulence determinants per se, through modification of cytokine production by eukaryotic cells, and by stimulating the relaxation of blood vessels. In the present study, we assessed the influence of AHLs on cardiovascular function in conscious rats, and draw attention to the ability of the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), a signal molecule produced by P. aeruginosa, to cause marked bradycardia. This bradycardic effect was blocked by atropine and atenolol, and did not occur in vitro. Furthermore, modification of the acyl side chain length resulted in the loss of activity, whereas removal of the homoserine lactone ring, did not. The bradycardic effect of 3-oxo-C12-HSL was also observed in endotoxaemic animals, albeit attenuated. In normal rats, 3-oxo-C12-HSL caused initial mesenteric and hindquarters vasoconstriction, but only slight, and delayed signs of vasodilatation in the renal and mesenteric vascular beds. Furthermore, administration of 3-oxo-C12-HSL (pre-treatment or 2 h post-treatment) together with LPS, did not modify the established regional haemodynamic effects of the LPS, 6 h after the onset of its infusion. Our observations do not provide any clear evidence for an ability of 3-oxo-C12-HSL to modify the haemodynamic responses to LPS infusion. However, they are not inconsistent with the hypothesis that some of the cardiovascular sequelae of bacterial infection may be modulated by an influence of bacterial quorum sensing signalling molecules on the host.

4-Butyrolactone↗
PubMed2002

L-Homoserine: a novel excreted metabolic marker of hepatitis B abnormally produced in liver from methionine.

The hepatitis B infection leads to various profound pathological processes in liver metabolism. Some biochemical alterations detectable by blood analysis are currently used for a preliminary evaluation of the infection. Based on existing data we present here evidence that non-protein amino acid L-homoserine is a pathological, hepatitis B-induced metabolite that is formed and excreted into urine from methionine via splitting S-adenosylmethionine. The urine L-homoserine is proposed as a new marker in the pre-diagnosis examinations that is easier for the clinical analysis than currently used blood test, and is applicable to large-scale epidemiological surveys of the probability of hepatitis B.

Alkyl and Aryl Transferases↗
PubMed2006

A structurally unrelated mimic of a Pseudomonas aeruginosa acyl-homoserine lactone quorum-sensing signal.

The pathogenic bacterium Pseudomonas aeruginosa uses acyl-homoserine lactone quorum-sensing signals to coordinate the expression of a battery of virulence genes in a cascade of regulatory events. The quorum-sensing signal that triggers the cascade is N-3-oxo-dodecanoyl homoserine lactone (3OC12-HSL), which interacts with two signal receptor-transcription factors, LasR and QscR. This signal is base labile, and it is degraded by mammalian PON lactonases. We have identified a structurally unrelated triphenyl mimic of 3OC12-HSL that is base-insensitive and PON-resistant. The triphenyl mimic seems to interact specifically with LasR but not with QscR. In silico analysis suggests that the mimic fits into the 3OC12-HSL-binding site of LasR and makes key contacts with LasR. The triphenyl mimic is an excellent scaffold for developing quorum-sensing inhibitors, and its stability and potency make it ideal for biotechnology uses such as heterologous gene expression.

4-Butyrolactone↗
PubMed1977

Homoserine dehydrogenase: spontaneous reactivation by dissociation of p-mercuribenzoate from an inactive enzyme--p-mercuribenzoate complex.

Incubation of Rhodospirillum rubrum homoserine dehydrogenase (L-homoserine:NAD+ oxidoreductase, EC 1.1.1.3) with p-mercuribenzoate (PMB) in the presence of 0.2 M KCl and 2 mM L-threonine resulted in complete loss of enzyme activity. Upon removal of excess PMB, KCl, and L-threonine, a time-dependent recovery of enzyme activity was observed in 25 mM phosphate/I mM EDTA buffer, pH 7.5. Circular dichroism studies indicated that the transition from inactive to reactivated form of the enzyme was accompanied by a conformational change in the protein. Experiments with [14C]PMB revealed loss of enzyme-bound radioactivity during reactivation. Increase in ionic strength of the phosphate buffer and/or addition of L-threonine, leading to enzyme aggregation, decreased the rate of enzyme reactivation, aggregated enzyme that remained inactive retained [14C]PMB on the enzyme. Sulfhydryl titration of various forms of the enzyme suggested a preferential release of PMB from a sulfhydryl group essential to enzymic activity. We conclude that reactivation of the inactive enzyme is due to dissociation of PMB from an "active-site" sulfhydryl group and that changes in the protein structure influence the rate of dissociation of the enzyme-PMB complex.

Alcohol Oxidoreductases↗
PubMed2005

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

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.

Adenosine Triphosphate↗
PubMed2003

Production of acylated homoserine lactones by different serotypes of Vibrio anguillarum both in culture and during infection of rainbow trout.

Onehundred and forty-eight out of onehundred and fifty strains of Vibrio anguillarum isolated from vibriosis in Danish marine aquaculture produced bacterial communication signals, acylated homoserine lactones, eliciting a response in the Agrobacterium tumefaciens (pZLR4) monitoring system. One strain, a serotype O4, induced a strong response in the Chromobacterium violaceum (CV026) monitoring system. Profiles of AHLs determined by TLC separation revealed the presence of at least four AHLs and a compound similar to N-3-oxo-decanoyl homoserine lactone (3-oxo-C10-HSL) was present in all strains. The production rate of the presumed 3-oxo-C10-HSL followed the growth rate of V. anguillarum whereas the production rate of a small AHL (Rf value of 0.74) increased faster than the growth rate of V. anguillarum indicating autoinduction. AHLs were produced by all serotypes (O1 to O10) and by non-typable strains. During infection with V. anguillarum, AHLs could be extracted from liver, kidney and muscle of rainbow trout and AHLs were detected both in vitro and in vivo when cell numbers reached 10(7) per ml or gram. Preliminary investigations of interactions between AHLs and the fish immune system were carried out determining oxidative burst of fish macrophages exposed to 3-oxo-C10-HSL. No activation or suppression of the superoxide anion production in the head kidney macrophages was seen when treated with the AHL compound in concentrations of 1 nM-10 microM. Our data show that AHLs are produced by almost all V. anguillarum strains and that no clear pattern relating AHL production to disease or virulence appear.

4-Butyrolactone↗