Dissociation properties of aspartokinase I-homoserine dehydrogenase I extracted from a missense mutant of E. coli K12

The threonine sensitive aspartokinase-homoserine dehydrogenase devoid of aspartokinase activity has been extracted from a missense mutant of $\text{E. coli}$ K12 and some of its properties have been investigated. The genetic localization of the corresponding mutation indicated that the amino acid replacement lies in the kinase region of the molecule. The cooperativity of threonine inhibition of the homoserine dehydrogenase activity is lowered. The measurement of the molecular weight of the enzyme in presence or absence of threonine indicates that the molecule dissociates more easily than the wild type enzyme. These results are discussed in view of the recent structural model proposed for aspartokinase I-homoserine dehydrogenase I.     

Homoserine kinase from Escherichia coli K-12: properties, inhibition by L-threonine, and regulation of biosynthesis

We have partially purified homoserine kinase from a genetically derepressed strain of Escherichia coli K-12. The optimum pH of the enzyme-substrate reaction was 7.8 and the K(m) values for l-homoserine and adenosine 5'-triphosphate were both 3 x 10(-4) M. K(+) (or NH(4) (+)) as well as Mg(2+) were required for its activity. The sedimentation coefficient determined by ultracentrifugation in a sucrose density gradient was 5.0 +/- 0.25S. l-Homoserine was an excellent protector against heat inactivation of homoserine kinase. l-Threonine was a competitive inhibitor of homoserine kinase, suggesting that end-product inhibition of this enzyme plays a role in vivo in the overall regulation of threonine biosynthesis. The specific activity of aspartokinase I-homoserine dehydrogenase I and of homoserine kinase showed a strong positive correlation in extracts from strains under widely varying conditions of genetic or physiological derepression; it was concluded that these two enzymes are coordinately regulated in E. coli K-12.     

Mapping of the structural genes of the three aspartokinases and of the two homoserine dehydrogenases of Escherichia coli K-12

Mutants requiring threonine plus methionine (or homoserine), or threonine plus methionine plus diaminopimelate (or homoserine plus diaminopimelate) have been isolated from strains possessing only one of the three isofunctional aspartokinases. They have been classified in several groups according to their enzymatic defects. Their mapping is described. Several regions of the chromosome are concerned: thrA (aspartokinase I-homoserine dehydrogenase I) is mapped in the same region as thrB and thrC (0 min). lysC (aspartokinase III) is mapped at 80 min, far from the other genes coding for diaminopimelate synthesis. metLM (aspartokinase II-homoserine dehydrogenase II) lies at 78 min closely linked to metB, metJ, and metF.     

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.     

Bifunctional protein in carrot contains both aspartokinase and homoserine dehydrogenase activities

We have purified homoserine dehydrogenase to homogeneity and subjected polypeptide fragments derived from digests of the protein to amino acid sequencing. The amino acid sequence of homoserine dehydrogenase from carrot (Daucus carota) indicates that in carrot both aspartokinase and homoserine dehydrogenase activities reside on the same protein. Additional evidence that aspartokinase and homoserine dehydrogenase reside on a bifunctional protein is provided by coelution of activities during purification steps and by enzyme-specific gel staining techniques. Highly purified fractions containing aspartokinase activity were stained for aspartokinase activity, homoserine dehydrogenase activity, and protein. These gels confirmed that aspartokinase activity and homoserine dehydrogenase activity were present on the same protein. This arrangement of aspartokinase and homoserine dehydrogenase activities residing on the same protein is also found in Escherichia coli, which has two bifunctional enzymes, aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. The amino acid sequence of the major form of homoserine dehydrogenase from carrot cell suspension cultures most closely resembles that of the E. coli ThrA gene product aspartokinase I-homoserine dehydrogenase I.     

Immunochemical comparison of a bifunctional enzyme, aspartokinase II-homoserine dehydrogenase II, and its two proteolytic fragments.

We report here a comparison between immunochemical properties of the bifunctional enzyme aspartokinase II-homoserine dehydrogenase II of E.coli K12 and of its two isolated proteolytic fragments. Both fragments, one inactive and one endowed with homoserine dehydrogenase activity, react with antibodies raised against the native enzyme. Some of the antibodies elicited against the dehydrogenase fragment can recognize regions of this fragment which are not exposed in the entire enzyme.The immunochemical results are used to discuss a simple model in which this bifunctional enzyme is folded up in two domains. The organization of aspartokinase II-homoserine dehydrogenase II is compared to that of another bifunctional enzyme aspartokinase I-homoserine dehydrogenase I with which it shares some sequence homology.     

Threonine Locus of Escherichia coli K-12: Genetic Structure and Evidence for an Operon

Three genes, thrA, thrB, and thrC, were previously defined and localized in the threonine locus of Escherichia coli K-12. thrA, thrB, and thrC specify the enzymes aspartokinase I-homoserine dehydrogenase I, homoserine kinase, and threonine synthetase, respectively. A complementation analysis of the threonine cluster using derivatives of a lambda phage carrying the threonine genes (lambdadthr(c)) demonstrates that: (i) thrB and thrC each consist of a single cistron; and (ii) thrA is composed of two cistrons, thrA(1) and thrA(2), although it specifies a single polypeptide chain. thrA(1) and thrA(2) correspond to aspartokinase I and homoserine dehydrogenase I, respectively. Their relative order is established. The demonstration of polar effects of mutations (nonsense or induced by phage Mu) in thrA and thrB is taken as evidence for the existence of a thrA thrB thrC operon, transcribed in this order.     

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.     

Regulation of aspartate-derived amino acid biosynthesis in the yeastSaccharomyces cerevisiae

The activity of three enzymes, aspartokinase, homoserine dehydrogenase, and homoserine kinase, has been studied in the industrial strainSaccharomyces cerevisiae IFI256 and in the mutants derived from it that are able to overproduce methionine and/or threonine. Most of the mutants showed alteration of the kinetic properties of the enzymes aspartokinase, which was less inhibited by threonine and increased its affinity for aspartate, and homoserine dehydrogenase and homoserine kinase, which both lost affinity for homoserine. Furthermore, they showed in vitro specific activities for aspartokinase and homoserine kinase that were higher than those of the wild type, resulting in accumulation of aspartate, homoserine, threonine, and/or methionine/S-adenosyl-methionine (Ado-Met). Together with an increase in the specific activity of both aspartokinase and homoserine kinase, there was a considerable and parallel increase in methionine and threonine concentration in the mutants. Those which produced the maximal concentration of these amino acids underwent minimal aspartokinase inhibition by threonine. This supports previous data that identify aspartokinase as the main agent in the regulation of the biosynthetic pathway of these amino acids. The homoserine kinase in the mutants showed inhibition by methionine together with a lack or a reduction of the inhibition by threonine that the wild type undergoes, which finding suggests an important role for this enzyme in methionine and threonine regulation. Finally, homoserine dehydrogenase displayed very similar specific activity in the mutants and the wild type in spite of the changes observed in amino acid concentrations; this points to a minor role for this enzyme in amino acid regulation.     

Identification and expression of a cDNA from Daucus carota encoding a bifunctional aspartokinase-homoserine dehydrogenase

Aspartokinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) catalyze steps in the pathway for the synthesis of lysine, threonine, and methionine from aspartate. Homoserine dehydrogenase was purified from carrot (Daucus carota L.) cell cultures and portions of it were subjected to amino acid sequencing. Oligonucleotides deduced from the amino acid sequences were used as primers in a polymerase chain reaction to amplify a DNA fragment using DNA derived from carrot cell culture mRNA as template. The amplification product was radiolabelled and used as a probe to identify cDNA clones from libraries derived from carrot cell culture and root RNA. Two overlapping clones were isolated. Together the cDNA clones delineate a 3089 bp long sequence encompassing an open reading frame encoding 921 amino acids, including the mature protein and a long chloroplast transit peptide. The deduced amino acid sequence has high homology with the Escherichia coli proteins aspartokinase I-homoserine dehydrogenase I and aspartokinase II-homoserine dehydrogenase II. Like the E. coli genes the isolated carrot cDNA appears to encode a bifunctional aspartokinase-homoserine dehydrogenase enzyme.Abbreviations AK aspartokinase - HSDH homoserine dehydrogenase - PCR polymerase chain reaction - SDS sodium dodecyl sulfate The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.     

A triglobular model for the polypeptide chain of aspartokinase I-homoserine dehydrogenase I of Escherichia coli

Limited proteolysis of aspartokinase I-homoserine dehydrogenase I from Escherichia coli by type VI protease from Streptomyces griseus yields five proteolytic fragments, three of which are dimeric, the other two being monomeric. One of the monomeric fragments (27 kilodaltons) exhibits residual aspartokinase activity, while the second one (33 kilodaltons) possesses residual homoserine dehydrogenase activity. The smallest of the dimeric species (2 X 25 kilodaltons) is inactive; the two other dimers exhibit either only homoserine dehydrogenase activity (2 X 59 kilodaltons) or both activities (hybrid fragment, 89 + 59 kilodaltons). This characterization of the proteolytic species in terms of molecular weight, subunit structure, and activity leads to the proposal of a triglobular model for the native enzyme. In addition, the time course of the formation of the various fragments was followed by measuring enzymatic activity and performing gel electrophoretic analysis of the protein mixture at defined time intervals during proteolysis. On the basis of the results of these studies, a reaction scheme describing the succession of events during proteolysis is given.     

Transient regulation of enzyme synthesis in Escherichia coli

Summary After lysine addition to an exponentially growing culture of Escherichia coli K12, the kinetics of repression of aspartokinase III synthesis show a transient regulatory phenomenon: during one generation, enzyme synthesis is practically equal to zero (Fig. 1). A similar phenomenon appears to be involved during repression of aspartokinase I-homoserine dehydrogenase I synthesis by threonine and isoleucine (Fig. 2). This sort of phenomenon has been previously reported in another system and interpreted as an indication of regulation at the translational level.     

Interaction of substrates and inhibitors with the homoserine dehydrogenase of kinase-inactivated aspartokinase I.

The aspartokinase activity of the aspartokinase-homoserine dehydrogenase complex of Escherichia coli was affinity labeled with substrates ATP, aspartate, and feedback inhibitor threonine. Exchange-inert ternary adducts of Co(III)-aspartokinase and either ATP, aspartate or threonine were formed by oxidation of corresponding Co(II) ternary complexes with H2O2. The ternary enzyme-Co(III)-threonine adduct (I) had 3.8 threonine binding sites per tetramer, one-half that of the native enzyme. The binding of threonine to I was still cooperative as determined by equilibrium dialysis (nH = 2.2) or by studying inhibition of residual dehydrogenase activity (nH = 2.7). Threonine still protected the SH groups of I against 5,5'-dithiobis(2-nitrobenzoate) (DTNB) reaction but the number of SH groups reacting with thiol reagents (DTNB) was reduced by 1-2 per subunit in the absence of threonine. This suggests either that Co(III) is bound to the enzyme via sulfhydryl groups or that 1-2SH groups are buried or rendered inaccessible in I. The binding of threonine to sites not blocked by the affinity labeling produced changes in the circular dichroism of the complex comparable to changes produced by threonine binding to native enzyme and also protected against proteolytic digestion. The major conformational changes produced by threonine are thus ascribable to binding at this one class of regulatory sites. The interactions of kinase substrates with various aspartokinase-Co(III) complexes containing ATP, aspartate, or threonine and a threonine-insensitive homoserine dehydrogenase produced by mild proteolysis were studied. The inhibition of homoserine dehydrogenase by kinase substrates is not due to binding of these inhibitors at the kinase active site but was shown to be due to binding to sites within the dehydrogenase domain of the enzyme. L-alpha-Aminobutyrate, a presumed threonine analogue, also inhibits the dehydrogenase by binding at the same or similar sites in the dehydrogenase domain and not at threonine regulatory site.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Transductional construction of a threonine-producing strain of Serratia marcescens.

A threonine-producing strain of Serratia marcescens Sr41 was constructed according to the following process. Thr- strain E-60 was derived from strain HNr59 having constitutive levels of threonine-sensitive aspartokinase and homoserine dehydrogenase. Thr+ transductant T-570 was constructed from strain E-60 and phage grown on strain HNr21 having feedback-resistant threonine-sensitive aspartokinase and homoserine dehydrogenase. This transductant lacked both feedback inhibition and repression for the two enzymes. Thr- strain N-11 was derived from strain AECr174 lacking feedback inhibition and repression of lysine-sensitive aspartokinase. Subsequently, the threonine region of strain T-570 was transduced into strain N-11. One of the THR+ transductants, strain T-693, produced markedly high levels of the two aspartokinases and homoserine dehydrogenase, which were insensitive to feedback inhibition. This strain produced about 25 mg of threonine per ml in the medium containing sucrose and urea.     

Activities and regulation of the enzymes involved in the first and the third steps of the aspartate biosynthetic pathway in Enterococcus faecium

The enzymes aspartokinase and homoserine dehydrogenase catalyze the reaction at key branching points in the aspartate pathway of amino acid biosynthesis. Enterococcus faecium has been found to contain two distinct aspartokinases and a single homoserine dehydrogenase. Aspartokinase isozymes eluted on gel filtration chromatography at molecular weights greater than 250,000 and about 125,000. The molecular weight of homoserine dehydrogenase was determined to be 220,000. One aspartokinase isozyme was slightly inhibited by meso-diaminopimelic acid. Another aspartokinase was repressed and inhibited by lysine. Although the level of diaminopimelate-sensitive (DAP^s) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lys^s) aspartokinase disappeared rapidly during the stationary phase and was depressed in rich media. The synthesis of homoserine dehydrogenase was controlled by threonine and methionine. Threonine also inhibited the specific activity of this enzyme. The regulatory properties of aspartokinase isozymes and homoserine dehydrogenase from E. faecium are discussed and compared with those from Bacillus subtilis.     

The primary structure of Escherichia coli K12 aspartokinase I-homoserine dehydrogenase I. Site of limited proteolytic cleavage by subtilisin.

The sequence of the first 25 residues of the homoserine dehydrogenase fragment, produced by limited proteolysis of aspartokinase I-homoserine dehydrogenase I with substilisin, has been determined. The sequence of a cyanogen bromide peptide (CB5, 59 residues), isolated from the entire protein, is also presented. Residues 1 to 18 of the subtilisin homoserine dehydrogenase fragment match the sequence 42 to 59 of peptide CB5.     

Regulation of the Biosynthesis of Amino Acids of the Aspartate Family in Coliform Bacteria and Pseudomonads

The control of aspartokinase and homoserine dehydrogenase activities was compared in aerobic and fermentative pseudomonads (genera Pseudomonas and Aeromonas), and in coliform bacteria representative of the principal genera of the Enterobacteriaceae. Isofunctional aspartokinases subject to independent end-product control occur in the Enterobacteriaceae and in Aeromonas. In Pseudomonas, there appears to be a single aspartokinase, subject to concerted feedback inhibition by lysine and threonine. Within this genus, the sensitivity of aspartokinase to the single allosteric inhibitors varies considerably: the aspartokinase of the acidovorans group is little affected by the single inhibitors, whereas that of the fluorescent group is severely inhibited by either amino acid at high concentration. In all bacteria examined, homoserine dehydrogenase activity is inhibited by threonine; inhibition is more severe in aerobic pseudomonads than in the other groups. In most of the bacteria examined, either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate can serve as a cofactor for this enzyme, though the relative activity with the two pyridine nucleotides varies widely. Aerobic pseudomonads of the acidovorans group contain a homoserine dehydrogenase that is absolutely specific for NAD. The taxonomic implications of these findings are discussed.     

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.     

Immunological cross reactivity of four enzymes involved in the biosynthetic pathway of lysine, methionine and threonine in Escherichia coli K12.

In Escherichia coli K12 the biosynthetic pathway of lysine, methionine and threonine is characterized by three isofunctional aspartokinases and two homoserine dehydrogenases. A single polypeptide chain carries the threonine-sensitive aspartokinase and homoserine dehydrogenase (AK I-HDH I), and a different polypeptide chain carries the methionine-repressible aspartokinase and homoserine dehydrogenase (AK II-HDH II). Immuno-adsorbants prepared with rabbit antibodies against AK I-HDH I bind the lysine-sensitive aspartokinase (AK III), the AK II-HDH II, and the homoserine kinase (HSK), an enzyme of the threonine biosynthetic pathway. Saturation of the immunoadsorbant with AK I-HDH I results in a decreased binding capacity for the other enzymes. Displacement of bound AK III or HSK can be obtained with pure AK I-HDH I, showing that the affinity of the antibodies to homologous antigens is higher than to heterologous ones. Immunoadsorbants prepared with anti-HSK antibodies show the same type of recognition: binding of the three aspartkinases and a capacity to displace the heterologous antigens bound. Accordingly, the same antibodies, implicated in the binding of the homologous antigen, bind the other enzymes. None of the other enzymes of the pathway, or the other kinases tested are recognized by the two immunoadsorbants. It can be postulated that in E. coli K12, duplication of a common ancestor gene gave rise to the three aspartokinases and to the homoserine kinase; two of the genes coding for the aspartokinases fused with those coding for the homoserine dehydrogenases. Indicating that only few epitopes are shared by these enzymes, by conventional immuno-diffusion techniques no precipitation lines appeared with antibodies against AK I-HDH I and the other proteins.