A novel bifunctional aspartate kinase-homoserine dehydrogenase from the hyperthermophilic bacterium, Thermotoga maritima.

The orientation of the three domains in the bifunctional aspartate kinase-homoserine dehydrogenase (AK-HseDH) homologue found in Thermotoga maritima totally differs from those observed in previously known AK-HseDHs; the domains line up in the order HseDH, AK, and regulatory domain. In the present study, the enzyme produced in Escherichia coli was characterized. The enzyme exhibited substantial activities of both AK and HseDH. L-Threonine inhibits AK activity in a cooperative manner, similar to that of Arabidopsis thaliana AK-HseDH. However, the concentration required to inhibit the activity was much lower (K0.5 = 37 µM) than that needed to inhibit the A. thaliana enzyme (K0.5 = 500 µM). In contrast to A. thaliana AK-HseDH, Hse oxidation of the T. maritima enzyme was almost impervious to inhibition by L-threonine. Amino acid sequence comparison indicates that the distinctive sequence of the regulatory domain in T. maritima AK-HseDH is likely responsible for the unique sensitivity to L-threonine. Abbreviations: AK: aspartate kinase; HseDH: homoserine dehydrogenase; AK-HseDH: bifunctional aspartate kinase-homoserine dehydrogenase; AsaDH: aspartate-ß-semialdehyde dehydrogenase; ACT: aspartate kinases (A), chorismate mutases (C), and prephenate dehydrogenases (TyrA, T).

Analysis of Loss-of-Function Mutants in Aspartate Kinase and Homoserine Dehydrogenase Genes Points to Complexity in the Regulation of Aspartate-Derived Amino Acid Contents.

Biosynthesis of aspartate (Asp)-derived amino acids lysine (Lys), methionine (Met), threonine (Thr), and isoleucine involves monofunctional Asp kinases (AKs) and dual-functional Asp kinase-homoserine dehydrogenases (AK-HSDHs). Four-week-old loss-of-function Arabidopsis (Arabidopsis thaliana) mutants in the AK-HSDH2 gene had increased amounts of Asp and Asp-derived amino acids, especially Thr, in leaves. To explore mechanisms behind this phenotype, we obtained single mutants for other AK and AK-HSDH genes, generated double mutants from ak-hsdh2 and ak mutants, and performed free and protein-bound amino acid profiling, transcript abundance, and activity assays. The increases of Asp, Lys, and Met in ak-hsdh2 were also observed in ak1-1, ak2-1, ak3-1, and ak-hsdh1-1. However, the Thr increase in ak-hsdh2 was observed in ak-hsdh1-1 but not in ak1-1, ak2-1, or ak3-1. Activity assays showed that AK2 and AK-HSDH1 are the major contributors to overall AK and HSDH activities, respectively. Pairwise correlation analysis revealed positive correlations between the amount of AK transcripts and Lys-sensitive AK activity and between the amount of AK-HSDH transcripts and both Thr-sensitive AK activity and total HSDH activity. In addition, the ratio of total AK activity to total HSDH activity negatively correlates with the ratio of Lys to the total amount of Met, Thr, and isoleucine. These data led to the hypothesis that the balance between Lys-sensitive AKs and Thr-sensitive AK-HSDHs is important for maintaining the amounts and ratios of Asp-derived amino acids.

Application of a generalized MWC model for the mathematical simulation of metabolic pathways regulated by allosteric enzymes.

In our effort to elucidate the systems biology of the model organism, Escherichia coli, we have developed a mathematical model that simulates the allosteric regulation for threonine biosynthesis pathway starting from aspartate. To achieve this goal, we used kMech, a Cellerator language extension that describes enzyme mechanisms for the mathematical modeling of metabolic pathways. These mechanisms are converted by Cellerator into ordinary differential equations (ODEs) solvable by Mathematica. In this paper, we describe a more flexible model in Cellerator, which generalizes the Monod, Wyman, Changeux (MWC) model for enzyme allosteric regulation to allow for multiple substrate, activator and inhibitor binding sites. Furthermore, we have developed a model that describes the behavior of the bifunctional allosteric enzyme aspartate kinase I-homoserine dehydrogenase I (AKI-HDHI). This model predicts the partition of enzyme activities in the steady state which paves the way for a more generalized prediction of the behavior of bifunctional enzymes.

Transcriptional and biochemical regulation of a novel Arabidopsis thaliana bifunctional aspartate kinase-homoserine dehydrogenase gene isolated by functional complementation of a yeast hom6 mutant.

An aspartate kinase-homoserine dehydrogenase (AK-HSDH) cDNA of Arabidopsis thaliana has been cloned by functional complementation of a Saccharomyces cerevisiae strain mutated in its homoserine dehydrogenase (HSDH) gene (hom6). Two of the three isolated clones were also able to complement a mutant yeast aspartate kinase (AK) gene (hom3). Sequence analysis showed that the identified gene (akthr2), located on chromosome 4, is different from the previously cloned A. thaliana AK-HSDH gene (akthr1), and corresponds to a novel bifunctional AK-HSDH gene. Expression of the isolated akthr2 cDNA in a HSDH-less hom6 yeast mutant conferred threonine and methionine prototrophy to the cells. Cell-free extracts contained a threonine-sensitive HSDH activity with feedback properties of higher plant type. Correspondingly, cDNA expression in an AK-deficient hom3 yeast mutant resulted in threonine and methionine prototrophy and a threonine-sensitive AK activity was observed in cell-free extracts. These results confirm that akthr2 encodes a threonine-sensitive bifunctional enzyme. Transgenic Arabidopsis thaliana plants (containing a construct with the promoter region of akthr2 in front of the gus reporter gene) were generated to compare the expression pattern of the akthr2 gene with the pattern of akthr1 earlier described in tobacco. The two genes are simultaneously expressed in meristematic cells, leaves and stamens. The main differences between the two genes concern the time-restricted or absent expression of the akthr2 gene in the stem, the gynoecium and during seed formation, while akthr1 is less expressed in roots.

Mechanism of control of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase by threonine.

The regulatory domain of the bifunctional threonine-sensitive aspartate kinase homoserine dehydrogenase contains two homologous subdomains defined by a common loop-alpha helix-loop-beta strand-loop-beta strand motif. This motif is homologous with that found in the two subdomains of the biosynthetic threonine-deaminase regulatory domain. Comparisons of the primary and secondary structures of the two enzymes allowed us to predict the location and identity of the amino acid residues potentially involved in two threonine-binding sites of Arabidopsis thaliana aspartate kinase-homoserine dehydrogenase. These amino acids were then mutated and activity measurements were carried out to test this hypothesis. Steady-state kinetic experiments on the wild-type and mutant enzymes demonstrated that each regulatory domain of the monomers of aspartate kinase-homoserine dehydrogenase possesses two nonequivalent threonine-binding sites constituted in part by Gln(443) and Gln(524). Our results also demonstrated that threonine interaction with Gln(443) leads to inhibition of aspartate kinase activity and facilitates the binding of a second threonine on Gln(524). Interaction of this second threonine with Gln(524) leads to inhibition of homoserine dehydrogenase activity.

Production and characterization of bifunctional enzymes. Substrate channeling in the aspartate pathway.

The direct channeling of an intermediate between enzymes that catalyze consecutive reactions in a pathway offers the possibility of an efficient, exclusive, and protected means of metabolite delivery. Aspartokinase-homoserine dehydrogenase I (AK-HDH I) from Escherichia coli is an unusual bifunctional enzyme in that it does not catalyze consecutive reactions. The potential channeling of the intermediate beta-aspartyl phosphate between the aspartokinase of this bifunctional enzyme and aspartate semialdehyde dehydrogenase (ASADH), the enzyme that catalyzes the intervening reaction, has been examined. The introduction of increasing levels of inactivated ASADH has been shown to compete against enzyme-enzyme interactions and direct intermediate channeling, leading to a decrease in the overall reaction flux through these consecutive enzymes. These same results are obtained whether these experiments are conducted with aspartokinase III, a naturally occurring monofunctional isozyme, with an artificially produced monofunctional aspartokinase I, or with a fusion construct of AK I-ASADH. These results provide definitive evidence for the channeling of beta-aspartyl phosphate between aspartokinase and aspartate semialdehyde dehydrogenase in E. coli and suggest that ASADH may provide a bridge to channel the intermediates between the non-consecutive reactions of AK-HDH I.

Homocysteine antagonism of nitric oxide-related cytostasis in Salmonella typhimurium.

Nitric oxide (NO) is associated with broad-spectrum antimicrobial activity of particular importance in infections caused by intracellular pathogens. An insertion mutation in the metL gene of Salmonella typhimurium conferred specific hypersusceptibility to S-nitrosothiol NO-donor compounds and attenuated virulence of the organism in mice. The metL gene product catalyzes two proximal metabolic steps required for homocysteine biosynthesis. S-Nitrosothiol resistance was restored by exogenous homocysteine or introduction of the metL gene on a plasmid. Measurement of expression of the homocysteine-sensitive metH gene indicated that S-nitrosothiols may directly deplete intracellular homocysteine. Homocysteine may act as an endogenous NO antagonist in diverse processes including infection, atherosclerosis, and neurologic disease.

The biosynthesis of threonine by mammalian cells: expression of a complete bacterial biosynthetic pathway in an animal cell.

The coding regions for the Escherichia coli gene for aspartokinase I/homoserine dehydrogenase I (thrA) and the Corynebacterium glutamicum gene for aspartic semialdehyde dehydrogenase (asd) have been subcloned into a Simian Virus 40 (SV40)-based mammalian expression vector. Both enzyme activities are expressed in mouse 3T3 cells after transfer of the corresponding chimaeric gene. The kinetic parameters are similar to those of the native bacterial enzymes, and aspartokinase I/homoserine dehydrogenase I retains its allosteric regulation by threonine. An extract of the cells expressing aspartokinase I/homoserine dehydrogenase I, mixed with one from cells expressing aspartic semialdehyde dehydrogenase, produced homoserine when the mixture was incubated with aspartic acid, ATP and NADPH. The thrA and asd expression cassettes were combined into a single plasmid which, when transfected into 3T3 cells, enabled them to produce homoserine from aspartic acid. Homoserine-producing 3T3 cells were transfected with the plasmid pSVthrB/C (homoserine kinase and threonine synthase) and selected for growth on homoserine. Cell lines isolated from these cells expressed the complete bacterial threonine pathway, were independent of threonine for growth and could be maintained in medium which contained no free threonine. The threonine in the proteins of these cells became enriched in 15N when the culture medium contained [15N]aspartic acid. The production of homoserine and the growth of cells was at a maximum when there was more than 2.5 mM aspartate in the medium. Below this concentration the high Km of aspartokinase limited the flux through the pathway. In the presence of additional aspartic acid the new pathway could sustain a cell cycle time close to that of the same cells cultured in threonine-containing medium.

Homoserine dehydrogenase-I (Escherichia coli): action of monovalent ions on catalysis and substrate association-dissociation.

Changes in the kinetic properties of homoserine dehydrogenase-I (HD-I) from Escherichia coli, caused by substitution of Na+ for the normal activating monovalent ion, K+, has been investigated by equilibrium isotope exchange kinetics (EIEK). HD-I, part of the aspartokinase/homoserine dehydrogenase-I complex, is one of the few dehydrogenases to exhibit allosteric feedback regulation and cation activation. EIEK methods are especially useful for definitively identifying which rate constants are altered by bound modifiers. Saturation curves for the [14C]Hse<-->ASA and [3H]NADP+<-->NADPH exchanges were compared in the presence of K+ vs Na+, varying different combinations of substrate pairs in constant ratio at equilibrium. Kinetic differences between the K+ and Na+ enzymes were analyzed systematically by simulations with the ISOBI program. This analysis clearly demonstrates that substituting Na+ for K+ shifts the kinetic mechanism from preferred order random to a nearly random order scheme, along with causing significant rate limitation at catalysis between the central complexes. Initial velocity kinetics demonstrate that HD-I has a 10-fold higher affinity for Na+ than K+, but that the Na(+)-enzyme is 10-fold less active and exhibits higher substrate Km values, especially for L-Hse.

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.

Mechanism of renaturation of a large protein, aspartokinase-homoserine dehydrogenase.

The renaturation of aspartokinase-homoserine dehydrogenase and of some of its smaller fragments has been investigated after complete unfolding by 6 M guanidine hydrochloride. Fluorescence measurements show that a major folding reaction occurs rapidly (in less than a few seconds) after the protein has been transferred to native conditions and results in the shielding of the tryptophan residues from the aqueous solvent; this step also takes place in the fragments and probably corresponds to the independent folding of different segments along the polypeptide chain. The reappearance of the kinase activity, which is an index of the formation of "native" structure within a single chain, is much slower (a few minutes) and has the following properties: it is involved in a kinetic competition with the formation of aggregates; it has an activation energy of 22 +/- 5 kcal/mol; it is not related to a slow reaction in unfolding and thus probably not controlled by the cis-trans isomerization of X-Pro peptide bonds; its rate is inversely proportional to the solvent viscosity. It seems as if this reaction is limited by the mutual arrangement of the regions that have folded rapidly and independently. It is proposed that the mechanism where a fast folding of domains is followed by a slow pairing of folded domains could be generalized to other long chains composed of several domains; such a slow pairing of folded domains would correspond to a rate-limiting process specific to the renaturation of large proteins. The reappearance of the dehydrogenase activity measures the formation of a dimeric species. The dimerization can occur only after each chain has reached its "native" conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversible dissociation of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12. The active species is the tetramer.

Dimers of aspartokinase I/homoserine dehydrogenase I from Escherichia coli K 12 have been isolated under very mild conditions. The dimers which cannot be distinguished from the tetramers by their kinetic properties, reassociate in the presence of potassium ions or L-aspartate. The selective sensitivity of aspartokinase I/homoserine dehydrogenase I to mild proteolytic digestion of dimers has been used to probe the reassociation reaction under the conditions of aspartokinase assay. We demonstrate that rapid reassociation occurs and that the protein species present in the assay when dimers are used to test the activity is tetrameric. These results confirm the previously proposed model for the subunit association of aspartokinase I/homoserine dehydrogenase I.

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

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

Role of subunit interactions in the self-assembly of oligomeric proteins.

In oligomeric proteins, the native conformation and its functional properties depend on the interactions which exist between the different chains. The role of these subunit interactions can be studied using either the unfolded state or the native state as a starting point. During the folding process, the properties which appear following a bimolecular reaction are related to the formation of an association area. Similarly, the properties which are lost upon partial dissociation of the native state are related to the association area which is disrupted. Four examples are presented in this article: phosphofructokinase and aspartokinase-homoserine dehydrogenase from E. coli are studied through their folding process, and fatty acid synthetase from B. ammoniagenes and reptilian ovomacroglobulin are studied through their dissociated forms. In all cases, the function of the protein is a sensitive index of the formation of the subunit interactions, and can be more conveniently measured than other size/shape parameters. The extrapolation from the folding of small proteins to the assembly of large and complex structures can be reasonably achieved by admitting that subunit interactions are coupled to the subtle adjustments required by the protein to exert its biological function.

Fluorescence studies of threonine-promoted conformational transitions in aspartokinase I using the substrate analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate.

The trinitrophenyl derivative of ATP, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, has been used as a spectroscopic probe to investigate threonine-promoted conformational changes in the aspartokinase region of aspartokinase-homoserine dehydrogenase I in an attempt to relate the structural effects of threonine binding to inhibition of enzymatic activity. Binding of this analogue substrate to the enzyme is characterized by a 9-fold enhancement in probe fluorescence. Saturating levels of the feedback inhibitor, threonine, produce a 77% increase in fluorescence enhancement, indicating an increase in the rigidity or hydrophobicity of the nucleotide-binding site in the inhibited form of the enzyme. Threonine titration studies indicate that the two inhibitor-binding sites found on each subunit do not contribute equally to the fluorescence-detected conformational change. Comparison of the spectral change with the inhibition of dehydrogenase activity has revealed the exclusive involvement of the non-kinase threonine sites. No transition can be detected as a consequence of inhibitor binding at the kinase subsites. The results of the 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate study have provided further evidence for a concerted kinase-dehydrogenase conformational change which is induced by threonine interaction with the high affinity binding sites and which provides maximal inhibition of homoserine dehydrogenase and the majority of aspartokinase inhibition. The failure to observe a distinct enzyme form produced by threonine occupation of the low affinity kinase sites suggests that no large structural reorganization of the kinase active site is produced as a result of this binding event. The conformational change, suggested by the cooperativity of threonine binding, must instead involve only a subtle or highly localized alteration which does not perturb the environment of the ATP-binding cleft.

Independent folding regions in aspartokinase-homoserine dehydrogenase.

The folding of two monofunctional fragments of aspartokinase-homoserine dehydrogenase I has been studied. One of these fragments corresponds to the kinase activity and the N-terminal part of the polypeptide chain; the other one corresponds to the dehydrogenase activity and to the C-terminal part of the chain. Both fragments are able to refold into an enzymatically active conformation after complete disruption of their native structure. The kinase fragment folds up into an active monomeric species. The dehydrogenase fragment first folds up into an inactive monomeric species and then associates into an active dimeric species. These two fragments thus correspond to regions capable of autonomous folding. The folding of each of these fragments is compared to that of the corresponding region in the intact aspartokinase--homoserine dehydrogenase I reported previously [Garel, J.R., & Dautry-Varsat, A. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3379-3383]. It is concluded that the N-and C-terminal regions of the intact polypeptide chain behave as independent folding units. A model of the sequence of steps involved in the folding process of aspartokinase-homoserine dehydrogenase I is presented; its relevance to the evolution of this protein is also discussed.

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.

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.

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.