Regulation of enzymes of lysine biosynthesis in Corynebacterium glutamicum

The regulation of the six enzymes responsible for the conversion of aspartate to lysine, together with homoserine dehydrogenase, was studied in Corynebacterium glutamicum. In addition to aspartate kinase activity, the synthesis of diaminopimelate decarboxylase was also found to be regulated. The specific activity of this enzyme was reduced to one-third in extracts of cells grown in the presence of lysine. Aspartate-semialdehyde dehydrogenase, dihydrodipicolinate synthase, dihydrodipicolinate reductase, and diaminopimelate dehydrogenase were neither influenced in their specific activity, nor inhibited, by any of the aspartate family of amino acids. Homoserine dehydrogenase was repressed by methionine (to 15% of its original activity) and inhibited by threonine (4% remaining activity). Inclusion of leucine in the growth medium resulted in a twofold increase of homoserine dehydrogenase specific activity. The flow of aspartate semialdehyde to either lysine or homoserine was influenced by the activity of homoserine dehydrogenase or dihydrodipicolinate synthase. Thus, the twofold increase in homoserine dehydrogenase activity resulted in a decrease in lysine formation accompanied by the formation of isoleucine. In contrast, repression of homoserine dehydrogenase resulted in increased lysine formation. A similar increase of the flow of aspartate semialdehyde to lysine was found in strains with increased dihydrodipicolinate synthase activity, constructed by introducing the dapA gene of Escherichia coli (coding for the synthase) into C. glutamicum.     

Inhibition of homoserine dehydrogenase I by L-serine in Escherichia coli

We have reported that a major cause of growth inhibition of Escherichia coli by L-serine is its inhibition of homoserine dehydrogenase I (HDH I), which is involved in the biosyntheses of threonine and isoleucine [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. However, Patte et al. reported that L-serine does not inhibit HDH I [Patte, J.-C., Truffa-Bachi, P., & Cohen, G.N. (1966) Biochim. Biophys. Acta 128, 426-439]. In studies on the reason for these discrepant results, we found that the concentration of K+ and the pH in the assay mixture strongly influenced the inhibitory effect of L-serine. L-Serine strongly inhibited the HDH I activities in both the forward and reverse reactions between aspartate semialdehyde and homoserine at a physiological K+ concentration (100 to 200 mM) and physiological pH (7.5) for E. coli cells. On the other hand, two well-known inhibitors of HDH I, L-threonine and L-cysteine, strongly inhibited the activity regardless of the K+ concentration and pH.     

Isoleucine and Valine Metabolism of Escherichia coli XVI. Pattern of Multivalent Repression in Strain K-12

The levels of the five enzymes required for isoleucine and valine synthesis were examined under several growth conditions in strain K-12 of Escherichia coli and mutants derived from it. In strains with wild type repressibility, the same pattern of derepression was found on limiting isoleucine as is found to be constitutive in strain Tir-8, which has an altered isoleucine-activating enzyme. Homoserine dehydrogenase, which is essential for the biosynthesis of threonine and is normally derepressed on limiting isoleucine or threonine, is also derepressed in strain Tir-8. Threonine deaminase and homoserine dehydrogenase were partially repressed in strain Tir-8 by very high levels of isoleucine, but were not further derepressed over levels in minimal medium by limiting isoleucine.     

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.     

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.     

The kinetic mechanisms of the bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli

The kinetic mechanisms of the reactions catalyzed by the two catalytic domains of aspartokinase-homoserine dehydrogenase I from Escherichia coli have been determined. Initial velocity, product inhibition, and dead-end inhibition studies of homoserine dehydrogenase are consistent with an ordered addition of NADPH and aspartate beta-semialdehyde followed by an ordered release of homoserine and NADP+. Aspartokinase I catalyzes the phosphorylation of a number of L-aspartic acid analogues and, moreover, can utilize MgdATP as a phosphoryl donor. Because of this broad substrate specificity, alternative substrate diagnostics was used to probe the kinetic mechanism of this enzyme. The kinetic patterns showed two sets of intersecting lines that are indicative of a random mechanism. Incorporation of these results with the data obtained from initial velocity, product inhibition, and dead-end inhibition studies at pH 8.0 are consistent with a random addition of L-aspartic acid and MgATP and an ordered release of MgADP and beta-aspartyl phosphate.     

Metabolic Regulation by Homoserine in Escherichia coli B/r

A mathematical analysis of branched pathway regulation has led to the prediction of a novel homoserine control in Escherichia coli B. Experimental support for such control is presented in this paper. Homoserine, the precursor of both threonine and methionine, inhibits nicotinamide adenine dinucleotide phosphate (NADP(+))-specific glutamate dehydrogenase (EC 1.4.1.4), the enzyme catalyzing the first reaction in ammonia assimilation. Physiological and biochemical evidence for this effect are offered. Homoserine depresses the growth rate of the organism, and glutamate, the product of the inhibited reaction, reverses this effect. The NADP(+)-specific glutamate dehydrogenase activity in cell-free extracts is inhibited by homoserine, and this inhibition parallels the restriction of growth rate. These effects are found in other enteric bacteria which share a similar overall pattern of control for the amino acids derived from aspartate. On the other hand, a sampling of more distantly related species which have different pathways and/or regulatory patterns provides no evidence for homoserine inhibition of the glutamate dehydrogenase reaction.     

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.     

Feedback-insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine

The regulatory properties of aspartate kinase (EC 2.7.2.4) and homoserine dehydrogenase (EC 1.1.1.3) in two barley (Hordeum vulgare L.) mutants resistant to growth inhibition by lysine plus threonine, Rothamsted (R) 3004 and R3202, were compared with those in the normal, sensitive parent line cv. Bomi. Three forms of aspartate kinase (AKI, AKII, AKIII) were chromatographically separated and were considered to represent at least three independently regulated isoenzymes. Aspartate kinase I was inhibited by threonine; AKII and AKIII by lysine or lysine plus S-adenosylmethionine. The characteristics of AKI were unchanged in the mutants. Aspartate kinase II and AKIII from Bomi were both inhibited by lysine and by lysine plus S-adenosylmethionine. Aspartate kinase II from mutant R3202 was altered in its properties such that it was insensitive to lysine or lysine plus S-adenosylmethionine; AKII from mutant R3004 did not differ in its properties from AKII of Bomi. The concentration of lysine required to give half maximal inhibition of AKIII from R3004 was ten times that required for AKIII of Bomi; AKIII from R3202 did not differ from that of Bomi in this regard. There was no change in the properties of homoserine dehydrogenase of the mutants as compared with that of Bomi. We conclude that the lt1 and lt2 loci code for structural genes for lysine- and lysine plus S-adenosylmethionine-sensitive aspartate kinase isoenzymes. The mutant genes Lt1b and Lt2 in R3202 and R3004 respectively code for feedback-desensitized isoenzymes. The presence of one of these is sufficient to allow the synthesis of methionine to overcome the growth inhibition by lysine plus threonine.     

Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate

The control of isocitrate dehydrogenase through phosphorylation is necessary for growth of Escherichia coli on acetate as the sole carbon source. To understand the mechanism by which phosphorylation inactivates isocitrate dehydrogenase, the sequence of icd, the isocitrate dehydrogenase structural gene, was determined and this information was used to construct mutants at the site of phosphorylation. Introduction of a negatively charged aspartate for the serine that is phosphorylated completely inactivates isocitrate dehydrogenase. Substitution of the serine with other amino acids results in a partially active enzyme in which both maximal velocity and interaction with substrates has been altered. Neither threonine nor tyrosine, when substituted for the serine at the phosphorylation site, is detectably phosphorylated by isocitrate dehydrogenase kinase.     

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.     

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.     

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.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Threonine production by regulatory mutants of Serratia marcescens.

beta-Hydroxynorvaline (alpha-amino-beta-hydroxyvaleric acid)-resistant mutants of Serratia marcescens deficient in both threonine dehydrogenase and threonine deaminase were isolated and characterized. One of the mutants, strain HNr21, lacked feedback inhibition of threonine-sensitive aspartokinase and homoserine dehydrogenase, was repressed for the two enzymes, and produced 11 mg of threonine per ml of medium containing a limiting amount of isoleucine. The other mutant, strain HNr59, was constitutively derepressed for aspartokinase and homoserine dehydrogenase. Its kinase was sensitive to feedback inhibition, but its dehydrogenase was insensitive to feedback inhibition. This strain produced 5 mg of threonine per ml of medium containing either a limiting or an excess amount of isoleucine. Diaminopimelate auxotrophs derived from strain HNr59 produced more threonine (13 mg/ml) than the parent strain. However, similar auxotrophs derived from strain HNr21 produced the same amount of threonine as that produced by the parent strain.     

Metabolism of aspartate in Mycobacterium smegmatis

Mycobacterium smegmatis grows best on L-asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol.mg dry cells-1.h-1 from 1 mM L-asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen. Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso-diaminopimilate, had no discernable effect on either enzyme activity.     

FKBP12 controls aspartate pathway flux in Saccharomyces cerevisiae to prevent toxic intermediate accumulation

FKBP12 is a conserved member of the prolyl-isomerase enzyme family and serves as the intracellular receptor for FK506 that mediates immunosuppression in mammals and antimicrobial actions in fungi. To investigate the cellular functions of FKBP12 in Saccharomyces cerevisiae, we employed a high-throughput assay to identify mutations that are synthetically lethal with a mutation in the FPR1 gene, which encodes FKBP12. This screen identified a mutation in the HOM6 gene, which encodes homoserine dehydrogenase, the enzyme catalyzing the last step in conversion of aspartic acid into homoserine, the common precursor in threonine and methionine synthesis. Lethality of fpr1 hom6 double mutants was suppressed by null mutations in HOM3 or HOM2, encoding aspartokinase and aspartate beta-semialdehyde dehydrogenase, respectively, supporting the hypothesis that fpr1 hom6 double mutants are inviable because of toxic accumulation of aspartate beta-semialdehyde, the substrate of homoserine dehydrogenase. Our findings also indicate that mutation or inhibition of FKBP12 dysregulates the homoserine synthetic pathway by perturbing aspartokinase feedback inhibition by threonine. Because this pathway is conserved in fungi but not in mammals, our findings suggest a facile route to synergistic antifungal drug development via concomitant inhibition of FKBP12 and Hom6.     

Biosynthesis of amino acids in Clostridium pasteurianum

1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) (14)C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus (14)C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of (14)C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate --> threonine --> glycine right harpoon over left harpoon serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine.     

Enhancement of isoleucine hydroxamate-mediated growth inhibition and improvement of isoleucine-producing strains of Serratia marcescens.

Growth inhibition by isoleucine hydroxamate in Serratia marcescens was significantly enhanced by adding valine plus leucine and by using glycerol as the carbon source. Isoleucine hydroxamate-resistant mutants were isolated under conditions in which growth inhibition was enhanced. One of the mutants, strain GIHVLr2179, lacked both feedback inhibition and repression of threonine deaminase. An alpha-aminobutyric acid-resistant mutant derived from strain GIHVLr2179, strain GIHVLAr2795, produced 12 mg of isoleucine per ml in the medium containing glucose and urea as carbon and nitrogen sources (a twofold increase over prior reports). This strain had increased activities of threonine deaminase, acetohydroxy acid synthase, aspartokinase, and homoserine dehydrogenase.