Aspartate kinase and homoserine dehydrogenase ofCandida utilis

Aspartate kinase and homoserine dehydrogenase activity were assayed in a dialyzed cell-free extract ofCandida utilis. Aspartate kinase was partly inhibited by ATP-Mg and by Mg²⁺ alone. There appear to be two isoenzymes of aspartate kinase in the yeast, one heatlabile, the other relatively heat-stable. The first is subject to feedback inhibition by threonine, the other is threonine-resistant. Neither aspartate kinase nor homoserine dehydrogenase is the rate-limiting enzyme in methionine biosynthesis. Homoserine dehydrogenase measured in the forward direction showed an activity five times higher than aspartate kinase. No regulatory interaction could be demonstrated for this enzyme. No repression of aspartate kinase and homoserine dehydrogenase synthesis by threonine, methionine or both amino acids was observed.     

New phenolic inhibitors of yeast homoserine dehydrogenase

A relatively unexploited potential target for antimicrobial agents is the biosynthesis of essential amino acids. Homoserine dehydrogenase, which reduces aspartate semi-aldehyde to homoserine in a NAD(P)H-dependent reaction, is one such target that is required for the biosynthesis of Met, Thr, and Ile from Asp. We report a small molecule screen of yeast homoserine dehydrogenase that has identified a new class of phenolic inhibitors of this class of enzyme. X-ray crystal structural analysis of one of the inhibitors in complex with homoserine dehydrogenase reveals that these molecules bind in the amino acid binding region of the active site and that the phenolic hydroxyl group interacts specifically with the backbone amide of Gly175. These results provide the first nonamino acid inhibitors of this class of enzyme and have the potential to be exploited as leads in antifungal compound design.     

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.     

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.     

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-sensitive aspartate kinase and homoserine dehydrogenase from Pisum sativum

Aspartate kinase and two homoserine dehydrogenases were partially purified from 4-day-old pea seedlings. A sensitive method for measuring aspartate kinase activity is described. Aspartate kinase activity was dependent upon ATP, Mg²⁺ or Mn²⁺, and aspartate. The aspartate kinase was inhibited in a sigmoidal manner by threonine and K_i for threonine was 0·57 mM. The enzyme could be desensitized to the inhibitor and threonine protected the enzyme against thermal inactivation. Aspartate kinase activity was enhanced by isoleucine, valine and alanine. Homoserine, methionine and lysine were without effect. The homoserine dehydrogenase activity which was associated with aspartate kinase during purification could be resolved into two peaks by gel filtration. The activity of both peaks was inhibited by aspartate and cysteine and one was inhibited by threonine.     

Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Haemophilus influenzae met2-encoded homoserine transacetylase

The first unique step in bacterial and plant methionine biosynthesis involves the acylation of the gamma-hydroxyl of homoserine. In Haemophilus influenzae, acylation is accomplished via an acetyl-CoA-dependent acetylation catalyzed by homoserine transacetylase. The activity of this enzyme regulates flux of homoserine into multiple biosynthetic pathways and, therefore, represents a critical control point for cell growth and viability. We have cloned homoserine transacetylase from H. influenzae and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the acetyl group of acetyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-acetylhomoserine. The maximal velocity and V/K(homoserine) were independent of pH over the range of values tested, while V/K(acetyl)(-)(CoA) was dependent upon the ionization state of a single group exhibiting a pK value of 8.6, which was required to be protonated. Solvent kinetic isotope effect studies yielded inverse effects of 0.75 on V and 0.74 on V/K(CoA) on the reverse reaction and effects of 1.2 on V and 1.7 on V/K(homoserine) on the forward reaction. Direct evidence for the formation of an acetyl-enzyme intermediate was obtained using rapid-quench labeling studies. On the basis of these observations, we propose a chemical mechanism for this important member of the acyltransferase family and contrast its mechanism with that of homoserine transsuccinylase.     

Enzyme-catalyzed acylation of homoserine: mechanistic characterization of the Escherichia coli metA-encoded homoserine transsuccinylase

The first unique step in bacterial and plant methionine biosynthesis involves the activation of the gamma-hydroxyl of homoserine. In Escherichia coli, this activation is accomplished via a succinylation reaction catalyzed by homoserine transsuccinylase. The activity of this enzyme is closely regulated in vivo and therefore represents a critical control point for cell growth and viability. We have cloned homoserine transsuccinylase from E. coli and present the first detailed enzymatic study of this enzyme. Steady-state kinetic experiments demonstrate that the enzyme utilizes a ping-pong kinetic mechanism in which the succinyl group of succinyl-CoA is initially transferred to an enzyme nucleophile before subsequent transfer to homoserine to form the final product, O-succinylhomoserine. The maximal velocity, V/K(succinyl)(-)(CoA), and V/K(homoserine) all exhibited a bell-shaped pH dependence with apparent pK's of 6.6 and approximately 7.9. The enzyme was inhibited by iodoacetamide in a pH-dependent manner, with an apparent pK of the group being inactivated of 6.4. This suggests the presence of an active site cysteine which forms a succinyl-cysteine intermediate during enzymatic turnover. Solvent kinetic isotope effect studies yielded inverse effects of 0.7 on V and 0.61 on V/K in the reverse reaction only. On the basis of these observations, we propose a detailed chemical mechanism for this important member of the acyltransferase family.     

The role of transamination in the synthesis of homoserine in peas

Incubation of intact pea plants (Pisum sativum), or detached shoots, in continuous light caused a substantial increase (up to 4-fold in 2 days) in levels of homoserine. Amino acids supplied to leaves in the transpiration stream enhanced the accumulation, with glutamate, aspartate, and asparagine causing similar enhancement. Aminooxyacetate (AOA), a transamination inhibitor, at 1 millimolar prevented the accumulation. ¹⁴C-labeling experiments showed that succinate was a good source of carbon for homoserine synthesis; carbon from aspartate or asparagine was also incorporated into homoserine. For each precursor, the transfer of label was prevented by AOA. The keto acid analog of homoserine was rapidly transaminated in leaves to give homoserine. The results suggest that accumulating homoserine is synthesised by transamination rather than being derived from aspartate via the aspartate kinase/homoserine dehydrogenase pathway. The latter pathway was shown to be operating in the chloroplasts, and was sensitive to threonine (but was not inhibited by AOA), suggesting that this path has a role in synthesis of aspartate-derived amino acids but is not involved in the accumulation of excess homoserine in the pea.     

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.     

Minor threonine dehydratase encoded within the threonine synthetic region of Bacillus subtilis

Challenging auxotrophs on metabolites that are precursors of a biosynthetic step involving a mutated enzyme has revealed a new class of suppressor mutations which act by derepressing a minor enzyme activity not normally detected in the wild-type strain. These indirect, partial suppressor mutations which allow isoleucine auxotrophs to grow on homoserine or threonine have been analyzed to determine their effect on enzymes involved in the biosynthesis of these amino acids. It has been found that one class of these suppressor mutations (sprA) leads to the derepression of homoserine kinase, homoserine dehydrogenase, and a minor threonine dehydratase that is not sufficiently active to be detected in the wild-type strain. The gene encoding this second threonine dehydratase activity has been found to be located between the structural genes for homoserine kinase and homoserine dehydrogenase. The results of these experiments indicate that plating of auxotrophs on precursors of a biosynthetic step involving mutated enzymes could prove to be a valuable method for the detection of regulatory mutants as well as a possible tool in studying the evolution of biochemical pathways.     

Production and characterization of bifunctional enzymes. Domain swapping to produce new bifunctional enzymes in the aspartate pathway

The bifunctional enzyme aspartokinase-homoserine dehydrogenase I from Escherichia coli catalyzes non-consecutive reactions in the aspartate pathway of amino acid biosynthesis. Both catalytic activities are subject to allosteric regulation by the end product amino acid L-threonine. To examine the kinetics and regulation of the enzymes in this pathway, each of these catalytic domains were separately expressed and purified. The separated catalytic domains remain active, with each of their catalytic activities enhanced in comparison to the native enzyme. The allosteric regulation of the kinase activity is lost, and regulation of the dehydrogenase activity is dramatically decreased in these separate domains. To create a new bifunctional enzyme that can catalyze consecutive metabolic reactions, the aspartokinase I domain was fused to the enzyme that catalyzes the intervening reaction in the pathway, aspartate semialdehyde dehydrogenase. A hybrid bifunctional enzyme was also created between the native monofunctional aspartokinase III, an allosteric enzyme regulated by lysine, and the catalytic domain of homoserine dehydrogenase I with its regulatory interface domain still attached. In this hybrid the kinase activity remains sensitive to lysine, while the dehydrogenase activity is now regulated by both threonine and lysine. The dehydrogenase domain is less thermally stable than the kinase domain and becomes further destabilized upon removal of the regulatory domain. The more stable aspartokinase III is further stabilized against thermal denaturation in the hybrid bifunctional enzyme and was found to retain some catalytic activity even at temperatures approaching 100 degrees C.     

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.     

Simultaneous separation by high-performance liquid chromatography of carbamoyl aspartate, carbamoyl phosphate and dihydroorotic acid

Leflunomide is an immunomodulatory drug which acts by inhibiting dihydroorotic acid dehydrogenase, the fourth enzyme of pyrimidine biosynthesis. We modified our high-performance liquid chromatography method to demonstrate that the principal metabolite in mitogen-stimulated human T-lymphocytes incubated with leflunomide was not dihydroorotic acid, but carbamoyl aspartate. Identification involved preparation of [¹⁴C]carbamoyl aspartate from [¹⁴C]aspartic acid and mammalian aspartate transcarbamoylase. Accumulation of carbamoyl aspartate indicates that under these conditions the equilibrium constant for dihydroorotase favours the reverse reaction. This HPLC method, enabling simultaneous separation of the first four intermediates in the de novo pyrimidine pathway may be of use in a variety of experimental situations.     

Intracellular localization of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves

The intracellular localization of several aspartate pathway enzymes has been studied in pea (Pisum sativum cv Feltham First) and barley (Hordeum vulgare cv Julia) leaves. Protoplast lysates were fractionated by differential or sucrose density gradient centrifugation, in media optimized for each enzyme. The results show that aspartate kinase, homoserine kinase, threonine synthase, and cystathionine γ-synthase are confined to the chloroplast. Cystathionine β-lyase appears to be present in several fractions, though more than 50% of the total activity is associated with the chloroplasts. In contrast, neither methionine synthase nor methionine adenosyl-transferase were significantly associated with chloroplasts, and only a small proportion of the methionine synthase was associated with the mitochondrial fraction. Methionine adenosyltransferase, and hence S-adenosylmethionine synthesis, is not found in any organelle fraction. The conclusion is that whereas threonine, like lysine, is synthesized only in the chloroplast, the last step in methionine biosynthesis occurs largely in the cytoplasm.     

Metabolic engineering of Escherichia coli W3110 for efficient production of homoserine from glucose

Efficient microbial cell factory for the production of homoserine from glucose has been developed by iterative and rational engineering of Escherichia coli W3110. The whole pathway from glucose to homoserine was divided into three groups, namely, glucose transport and glycolysis (‘up-stream’), TCA and glyoxylate cycles (‘mid-stream’), and homoserine module (conversion of aspartate to homoserine and its secretion; ‘down-stream’), and the carbon flux in each group as well as between the groups were accelerated and balanced. Altogether, ∼18 genes were modified for active and consistent production of homoserine during both the actively-growing and non-growing stages of cultivation. Finally, fed-batch, two-stage bioreactor experiments, separating the growth from the production stage, were conducted for 61 h, which gave the high titer of 110.8 g/L, yield of 0.64 g/g glucose and volumetric productivity of 1.82 g/L/h, with an insignificant amount of acetate (<0.5 g/L) as the only noticeable byproduct. The metabolic engineering strategy employed in this study should be applicable for the biosynthesis of other amino acids or chemicals derived from aspartic acid.     

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.     

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.     

Effect of gene amplification on threonine production by yeast

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.