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At least 19 recordsLinked to original sources
PubMed1977

Proteolysis of the bifunctional methionine-repressible aspartokinase II-homoserine dehydrogenase II of Escherichia coli K12. Production of an active homoserine dehydrogenase fragment.

The dimeric bifunctional enzyme aspartokinase II-homoserine dehydrogenase II (Mr = 2 X 88,000) of Escherichia coli K12 can be cleaved into two nonoverlapping fragments by limited proteolysis with subtilisin. These two fragments can be separated under nondenaturing conditions as dimeric species, which indicates that each fragment has retained some of the association areas involved in the conformation of the native protein. The smaller fragment (Mr = 2 X 24,000) is devoid of aspartokinase and homoserine dehydrogenase activity. The larger fragment (Mr = 2 X 37,000) is endowed with full homoserine dehydrogenase activity. These results show that the polypeptide chains of the native enzyme are organized in two different domains, that both domains participate in building up the native dimeric structure, and that one of these domains only is responsible for homoserine dehydrogenase activity. A model of aspartokinase II-homoserine dehydrogenase II is proposed, which accounts for the present results.

Alcohol Oxidoreductases↗
PubMed1990

[Development of the vector-host system in Corynebacterium. Cloning and expression of homoserine dehydrogenase and homoserine kinase genes in Corynebacterium cells].

Novel cloning vectors for glutamic acid producing bacteria have been constructed. The cryptic plasmid pBO1 (4.4 kb) from Brevibacterium sp. recombined with the plasmid pACYC184 (4.0 kb) from Escherichia coli was used to produce composite plasmid named pKA1. The plasmid could propagate and express the Cm-r phenotype in E. coli and coryneform glutamic acid producing bacteria Br. flavum, C. glutamicum, Br. lactofermentum. The pKA1 plasmid and its variants deleted within non-essential plasmid regions with unique restriction sites HindIII, SalGI, SphI were used in cloning experiments. The genes coding for threonine biosynthesis of C. glutamicum and Br. flavum were subcloned into shuttle vectors in C. glutamicum cells. Recombinant plasmids were introduced into protoplasts by polyethylenglycol-mediated transformation of plasmid DNAs. It was shown that the presence of plasmids containing the Br. flavum thrA2 gene in C. glutamicum (thrB) caused 10-fold increase in homoserine dehydrogenase activity, as compared to that of wild type strain, and in homoserine production.

Alcohol Oxidoreductases↗
PubMed1975

The threonine-sensitive homoserine dehydrogenase and aspartokinase activities of Escherichia coli K12. Specific inactivation of the homoserine dehydrogenase activity by the affinity label, 2-amino-4-oxo-5-chloropentanoic acid.

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.

Alcohol Oxidoreductases↗
PubMed1992

Expression of Escherichia coli homoserine kinase in mouse 3T3 cells.

The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

3T3 Cells↗
PubMed1975

Purification and properties of homoserine transacetylase from Bacillus polymyxa.

Homoserine transacetylase (EC 2.3.1.31), the first enzyme of methionine biosynthesis, has been purified to near homogeneity from extracts of a methionine auxotroph of Bacillus polymyxa. The enzyme is subject to rapid irreversible inactivation. Its half-life at 0 degrees is 15 min and much less at higher temperatures, but ethylene glycol affords some protection. In addition, Zn2+ reversibly inhibits the enzyme with a K-I of 3 muM. The enzyme has a molecular weight of about 40,000 and consists of a single polypeptide chain. Besides catalyzing the acetyl transfer from acetyl-CoA to L-homoserine, homoserine transacetylase promotes a homoserine-O-acetylhomoserine exchange reaction in the absence of CoA, suggesting the formation of an acetyl-enzyme intermediate. The results of kinetic studies are consistent with a ping-pong mechanism. Homoserine transacetylase is subject to multivalent feedback inhibition by L-methionine and S-adenosylmethionine. Analysis of the inhibition data and specificity studies suggest that the inhibitors bind to separate sites on the enzyme which are distinct from the active site. Inhibition is competitive with respect to both substrates, and the saturation curves for the inhibitors, as well as substrate saturation curves in the absence or presence of the inhibitors, are hyperbolic. The absence of cooperativity is, in fact, a property which would be expected in a monomeric allosteric enzyme such as homoserine transacetylase.

Acetyl Coenzyme A↗
PubMed1975

Aspartokinase I-homoserine dehydrogenase I of Escherichia coli K12 (lambda). Activation by monovalent cations and an analysis of the effect of the adenosine triphosphate-magnesium ion complex on this activation process.

The dehydrogenase activity of the aspartokinase I-homoserine dehydrogenase I complex isolated from Escherichia coli K12 is subject to a cooperative activation by K+ or Rb+, which is characterized by a Hill coefficient of approximately 2. Ionic strength has little effect on the Hill coefficient for this activation process; however, high ionic strength appears to increase the enzyme's affinity for K+ and decrease its affinity for Rb+. The Vmax of the K+-activated dehydrogenase is greater than that of the Rb+-activated dehydrogenase. The results of a study of the competition between K+ and Rb+ in the activation process suggest the presence of an activated species containing both K+ and Rb+. The cooperative activation by K+ is antagonized by Na+ via a process that is noncooperative with respect to Na+. The MgATP-2- complex, a substrate for the kinase activity of aspartokinase I-homoserine dehydrogenase I, has a marked effect on the K+ activation of the dehydrogenase activity. Kinetic studies of this effect of MgATP-2- on the K+ requirement of the dehydrogenase at pH 8.9 indicate that: (a) activation by a monovalent cation is essential in the presence as well as in the absence of MgATP-2-; (b) the concentration of K+ required to activate fully the dehydrogenase is reduced in the presence of MgATP-2-; (c) activation of the dehydrogenase by K+ is noncooperative in the presence of MgATP-2-; and (d) the maximum velocity for the dehydrogenase catalyzed oxidation of homoserine is greater in the presence of MgATP-2- than in its absence. Based on these results, a simple model consistent with these data is proposed. Destruction of the kinase activity and the threonine sensitivity of the aspartokinase-homoserine dehydrogenase complex by treatment with 5,5'-dithiobis(2-nitrobenzoic acid) or by incubation at pH 9 also converts the K+ activation of the dehydrogenase from a cooperative to a noncooperative process. Marked protection of the enzyme against loss of threonine sensitivity at pH 9 is afforded by MgATP-2- plus K+ and homoserine. The apparent molecular radius of the enzyme complex as determined by gel filtration at pH 8.85 in the presence of threonine or MgATP-2- plus K+ and homoserine is dependent on the enzyme concentration. The observed apparent molecular radii of 70 A at high enzyme concentrations and 61 A at low enzyme concentrations are consistent with the enzyme's undergoing a concentration-dependent dissociation from a tetrameric to a dimeri

Adenosine Triphosphate↗
PubMed1975

Cobalt(III) labeled aspartokinase-homoserine dehydrogenase of Escherichia coli.

The kinase activity of the threonine-sensitive aspartokinase-homoserine dehydrogenase enzyme complex of Escherichia coli was selectively inactivated by Co(III) incorporation. Incubation of the enzyme with Co(II) in the presence of oxygen or H2O2 resulted in incorporation of one Co(III) per subunit. The cobalt(III) bound to the enzyme was not removable by dialysis and presumably results from formation of "inert" coordination complexes with ligands contributed by the enzyme. Cobalt was released from the enzyme by incubation with dithiothreitol but not by metal chelating agents. The Co(III)-labeled enzyme was aspartokinase inactive but still retained 60% of its original homoserine dehydrogenase activity. Studies of the time course of inactivation showed aspartokinase inactivation paralleled Co(III) incorporation. The residual dehydrogenase activity of aspartokinase inactive enzyme was still inhibited by threonine Thus, Co(III) incorporation seems to result in a specific inactivation of kinase activity which permits enumeration of the number of aspartokinase sites. Limited alpha-chymotrypsin digestion of Co(III)-enzyme produced homoserine dehydrogenase-active fragments devoid of Co(III), further confirming the specificity of the labeling procedure. Aspartokinase inactivation obtained without concomitant desensitization of homoserine dehydrogenase to threonine inhibition suggests that kinase active site integrity is not required for threonine binding and inhibition of homoserine dehydrogenase.

Aspartokinase Homoserine Dehydrogenase↗
PubMed1975

Regulation of homoserine transacetylase in whole cells of Bacillus polymyxa.

The levels of homoserine transacetylase (EC 2.3.1.31) in Bacillus polymyxa grown in minimal medium can vary over a 40-fold range, depending on whether methionine limits growth or is present in excess. This suggests that the synthesis of the enzyme is under control by methionine or one of its metabolites. The stability of homoserine transacetylase in growing cells was measured after repression of further synthesis by the addition of methionine. At 30 degrees, the enzyme was stable for 2 hours, whereas at 37 degrees it decayed with a half-life of 40 min. This contrasts with the striking instability in cell-free extracts described in the preceding paper (Wyman, A., and Paulus, H. (1975) J. Biol. Chem. 250, 3897-3903). The properties of homoserine transacetylase were also studied in cells of B. polymyxa that had been made permeable to small molecules by treatment with toluene. They differed in two important respects from those of the enzyme in cell-free extracts described in the preceding paper: the enzyme was relatively stable, with a half-life of 15 min at 37 degrees, and responded in a sigmoid manner to increasing concentrations of the inhibitors L-methionine and S-adenosylmethionine. These observations suggest that homoserine transacetylase is an oligomeric protein within the bacterial cell but dissociates into monomers in cell-free extracts. When B. polymyxa was transferred at 39 degrees from a rich medium to one without amino acids, growth resumed only very slowly. The growth lag after shift-down was not observed at 37 degrees or in the presence of methionine or cystathionine. This phenomenon appears to be due to a need for derepression of homoserine transacetylase upon shift-down which is thwarted at 39 degrees by the rapid thermal inactivation of the enzyme. A possible physiological function of the striking thermolability of the first enzyme in methionine biosynthesis is discussed.

Acetyltransferases↗
PubMed1990

Yeast homoserine kinase. Characteristics of the corresponding gene, THR1, and the purified enzyme, and evolutionary relationships with other enzymes of threonine metabolism.

THR1, the gene from Saccharomyces cerevisiae, encoding homoserine kinase, one of the threonine biosynthetic enzymes, has been cloned by complementation. The nucleotide sequence of a 3.1-kb region carrying this gene reveals an open reading frame of 356 codons, corresponding to about 40 kDa for the encoded protein. The presence of three canonical GCN4 regulatory sequences in the upstream flanking region suggests that the expression of THR1 is under the general amino acid control. In parallel, the enzyme was purified by four consecutive column chromatographies, monitoring homoserine kinase activity. In SDS gel electrophoresis, homoserine kinase migrates like a 40-kDa protein; the native enzyme appears to be a homodimer. The sequence of the first 15 NH2-terminal amino acids, as determined by automated Edman degradation, is in accordance with the amino acid sequence deduced from the nucleotide sequence. Computer-assisted comparison of the yeast enzyme with the corresponding activities from bacterial sources showed that several segments among these proteins are highly conserved. Furthermore, the observed homology patterns suggest that the ancestral sequences might have been composed from separate (functional) domains. A block of very similar amino acids is found in the homoserine kinases towards the carboxy terminus that is also present in many other proteins involved in threonine (or serine) metabolism; this motif, therefore, may represent the binding site for the hydroxyamino acids. Limited similarity was detected between a motif conserved among the homoserine kinases and consensus sequences found in other mono- or dinucleotide-binding proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Biologically active ether lipids: incorporation of long-chain precursors into 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N-trimethyl)homoserines and other lipids of Chlorella fusca.

The lipids of Chlorella fusca are composed of the ester lipids typical of photosynthetically active cells. In addition, there occurs a class of less common ether lipids, the biologically active 1(3),2-diacylglycero-3(1)-O-4'-(N,N,N- trimethyl)homoserines, at a level of about 1.3% of total lipids. The acyl moieties of the total lipids include saturated as well as mono-, di- and tri-unsaturated species with chain lengths of 16 and 18 carbon atoms, the major constituents being palmitic and oleic acids. In both the diacylglycerophosphocholines, i.e., the major class of ester phospholipids, and the diacylglycero-4'-O-(N,N,N-trimethyl)homoserines palmitic acid is located predominantly at position 1 of the glycerol backbone, whereas oleic acid is almost equally distributed between positions 1 and 2; palmitoleic and polyunsaturated fatty acids are esterified preferentially at position 2. Incubation of C. fusca cultures with 14C-labeled fatty acids leads to their rapid incorporation into various lipid classes. Oleic and palmitic acids are incorporated at a faster rate than stearic acid (18:1 greater than 16:0 much greater than 18:0). 1,2- and 1,3-Diacylglycerols are the most prominent intermediates of early metabolism of the exogenous fatty acids. In the course of time, a steady decrease of radioactive 1,2-diacylglycerols is observed that is accompanied by an increase in labeled triacylglycerols, diacylglycerophosphocholines, and diacylglycero-O-(N,N,N-trimethyl)homoserines. The stereospecific distribution of acyl moieties in the diacylglycero-O-(N,N,N-trimethyl)homoserines in C. fusca indicates that these ether lipids are derived from 1,2-diacylglycerol intermediates. This notion is supported by the finding that during incubation with radioactively labeled fatty acids the formation of diacylglycero-O-(N,N,N-trimethyl)homoserines parallels the biosynthesis of both diacylglycerophosphocholines and diacylglycerophosphoethanolamines, two classes of phospholipids which are known to be derived from 1,2-diacylglycerols. The mechanism of the formation of the ether bond, however, is as yet unknown. Incubation of C. fusca cultures with 14C-labeled fatty acids or alcohols leads to the formation of fair proportions of wax esters that are labeled in both the acyl and the alkyl moieties, indicating that in these algae fatty acids and alcohols are interconverted. 14C-Labeled long-chain alcohols are not incorporated into the alkyl moieties of ether lipids, whereas labeled 1-O-alkylglycerols are used, though to a very small extent, as precursors of ether phospholipids.

Chlorella↗
PubMed1990

The mechanism of antifungal action of (S)-2-amino-4-oxo-5-hydroxypentanoic acid, RI-331: the inhibition of homoserine dehydrogenase in Saccharomyces cerevisiae.

We have explored the mechanism by which an antifungal antibiotic, (S)-2-amino-4-oxo-5-hydroxypentanoic acid, RI-331, preferentially inhibits protein biosynthesis in Saccharomyces cerevisiae, by inhibiting the biosynthesis of the aspartate family of amino acids, methionine, isoleucine and threonine. This inhibition was effected by inhibiting the biosynthesis of their common intermediate precursor homoserine. The target enzyme of RI-331 was homoserine dehydrogenase (EC.1.1.1.3) which is involved in converting aspartate semialdehyde to homoserine in the pathway from aspartate to homoserine. The enzyme is lacking in animals. So the antibiotic is selectively toxic to prototrophic fungi.

Alcohol Oxidoreductases↗
PubMed1976

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.

Alcohol Oxidoreductases↗
PubMed1978

Detection of the homology among proteins by immunochemical cross-reactivity between denatured antigens. Application to the threonine and methionine regulated aspartokinases-homoserine dehydrogenases from Escherichia coli K 12.

The two isofunctional enzymes aspartokinases-homoserine dehydrogenases I and II from Escherichia coli K 12 are compared using immunochemical techniques. The antibodies raised against one of these two proteins when in its native state can only recognize the homologous antigen, whether it is native or denatured. Contrarily, the antibodies raised against one of these two proteins when in its denatured state can recognize both the homologous and heterologous denatured antigens. The existence of this cross-reaction only between the two denatured aspartokinases-homoserine dehydrogenases suggests that these two enzymes have some similarity since such a reaction is not detected with several other denatured proteins. The regions involved in this similarity are buried inside the native proteins, and become exposed only upon denaturation. The same results, the existence of a cross-reaction between denatured species and none between the native ones, is obtained with proteolytic fragments derived from these two proteins and endowed with homoserine dehydrogenase activity. This resemblance between the two aspartokinases-homoserine dehydrogenases suggests that these proteins derive from a common ancestor. It is also proposed that such a cross-reaction between two denatured proteins is evidence for an homology between their amino acid sequences, and that the use of denatured proteins as both immunogens and antigens could be useful in detecting sequence homologies.

Alkylation↗
PubMed1976

Homoserine kinase from Escherichia coli K12.

Homoserine kinase was purified to apparent homogeneity from a derepressed strain of Escherichia coli K12, using standard fractionation techniques. It is a dimer (Mr = 60000) composed of apparently identical polypeptide chains (Mr = 29000). Its amino acid composition and N-terminal sequence have been determined. L-Threonine is a competitive inhibitor of the substrate L-homoserine; this inhibition is straighforward and shows no sign of co-operativity. Evidence is presented that homoserine and threonine bind to the same site of this non-allosteric enzyme. The binding of homoserine and threonine can also be studied by difference spectroscopy; the latter studies reveal an unexpected effect of magnesium ions, which might be the basis for the unusual high Mg2+ requirement for optimal enzyme reaction.

Amino Acid Sequence↗
PubMed1992

Mechanism of action of an antifungal antibiotic, RI-331, (S) 2-amino-4-oxo-5-hydroxypentanoic acid; kinetics of inactivation of homoserine dehydrogenase from Saccharomyces cerevisiae.

An antifungal antibiotic (S) 2-amino-4-oxo-5-hydroxypentanoic acid, inhibited the biosynthesis of the aspartate family of amino acids (methionine, isoleucine and threonine) followed by the inhibition of protein biosynthesis in Saccharomyces cerevisiae. This inhibition was effected by impeding the biosynthesis of their common intermediate precursor, homoserine. The inhibition of biosynthesis of homoserine by the antibiotic was attributable to inactivation of homoserine dehydrogenase [EC 1.1.1.3], which is involved in the conversion of aspartate semialdehyde to homoserine in the metabolic pathway leading to threonine, methionine and isoleucine. Since such enzymic activity is not present in animal cells, the selective antifungal activity of the antibiotic is thus explained.

Aminolevulinic Acid↗
PubMed1977

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.

Aspartokinase Homoserine Dehydrogenase↗