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.

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.

Overproduction, purification, and characterization of recombinant bifunctional threonine-sensitive aspartate kinase-homoserine dehydrogenase from Arabidopsis thaliana.

In plant, the first and the third steps of the synthesis of methionine and threonine are catalyzed by a bifunctional enzyme, aspartate kinase-homoserine dehydrogenase (AK-HSDH). In this study, we report the first purification and characterization of a highly active threonine-sensitive AK-HSDH from plants (Arabidopsis thaliana). The specific activities corresponding to the forward reaction of AK and reverse reaction of HSDH of AK-HSDH were 5.4 micromol of aspartyl phosphate produced min(-1) mg(-1) of protein and 18.8 micromol of NADPH formed min(-1) mg(-1) of protein, respectively. These values are 200-fold higher than those reported previously for partially purified plant enzymes. AK-HSDH exhibited hyperbolic kinetics for aspartate, ATP, homoserine, and NADP with K(M) values of 11.6 mM, 5.5 mM, 5.2 mM, and 166 microM, respectively. Threonine was found to inhibit both AK and HSDH activities by decreasing the affinity of the enzyme for its substrates and cofactors. In the absence of threonine, AK-HSDH behaved as an oligomer of 470 kDa. Addition of the effector converted the enzyme into a tetrameric form of 320 kDa.

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.

Molecular analysis of the aspartate kinase-homoserine dehydrogenase gene from Arabidopsis thaliana.

The gene encoding Arabidopsis thaliana aspartate kinase (ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) was isolated from genomic DNA libraries using the carrot ak-hsdh gene as the hybridizing probe. Two genomic libraries from different A. thaliana races were screened independently with the ak probe and the hsdh probe. Nucleotide sequences of the A. thaliana overlapping clones were determined and encompassed 2 kb upstream of the coding region and 300 bp downstream. The corresponding cDNA was isolated from a cDNA library made from poly(A)(+)-mRNA extracted from cell suspension cultures. Sequence comparison between the Arabidopsis gene product and an AK-HSDH bifunctional enzyme from carrot and from the Escherichia coli thrA and metL genes shows 80%, 37.5% and 31.4% amino acid sequence identity, respectively. The A. thaliana ak-hsdh gene is proposed to be the plant thrA homologue coding for the AK isozyme feedback inhibited by threonine. The gene is present in A. thaliana in single copy and functional as evidenced by hybridization analyses. The apoprotein-coding region is interrupted by 15 introns ranging from 78 to 134 bp. An upstream chloroplast-targeting sequence with low sequence similarity with the carrot transit peptide was identified. A signal sequence is proposed starting from a functional ATG initiation codon to the first exon of the apoprotein. Two additional introns were identified: one in the 5' non-coding leader sequence and the other in the putative chloroplast targeting sequence. 5' sequence analysis revealed the presence of several possible promoter elements as well as conserved regulatory motifs. Among these, an Opaque2 and a yeast GCN4-like recognition element might be relevant for such a gene coding for an enzyme limiting the carbon-flux entry to the biosynthesis of several essential amino acids. 3' sequence analysis showed the occurrence of two polyadenylation signals upstream of the polyadenylation site. This work is the first report of the molecular cloning of a plant ak-hsdh genomic sequence. It describes a promoter element that may bring new insights to the regulation of the biosynthesis of the aspartate family of amino acids.

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.

Cloning and nucleotide sequence of the Bacillus subtilis hom gene coding for homoserine dehydrogenase. Structural and evolutionary relationships with Escherichia coli aspartokinases-homoserine dehydrogenases I and II.

The Bacillus subtilis hom gene, encoding homoserine dehydrogenase (L-homoserine:NADP+ oxidoreductase, EC 1.1.1.3) has been cloned and its nucleotide sequence determined. The B. subtilis enzyme expressed in Escherichia coli is sensitive by inhibition by threonine and allows complementation of a strain lacking homoserine dehydrogenases I and II. Nucleotide sequence analysis indicates that the hom stop codon overlaps the start codon of thrC (threonine synthase) suggesting that these genes, as well as thrB (homoserine kinase) located downstream from thrC, belong to the same transcription unit. The deduced amino acid sequence of the B. subtilis homoserine dehydrogenase shows extensive similarity with the C-terminal part of E. coli aspartokinases-homoserine dehydrogenases I and II; this similarity starts at the exact point where the similarity between E. coli or B. subtilis aspartokinases and E. coli aspartokinases-homoserine dehydrogenases stops. These data suggest that the E. coli bifunctional polypeptide could have resulted from the direct fusion of ancestral aspartokinase and homoserine dehydrogenase. The B. subtilis homoserine dehydrogenase has a C-terminal extension of about 100 residues (relative to the E. coli enzymes) that could be involved in the regulation of the enzyme activity.

Internal homologies in the two aspartokinase-homoserine dehydrogenases of Escherichia coli K-12.

In Escherichia coli, AK I- HDH I and AK II- HDH II are two bifunctional proteins, derived from a common ancestor, that catalyze the first and third reactions of the common pathway leading to threonine and methionine. An extensive amino acid sequence comparison of both molecules reveals two main features on each of them: (i) two segments, each of about 130 amino acids, covering the first one-third of the polypeptide chain, are similar to each other and (ii) two segments, each of about 250 amino acids and covering the COOH-terminal 500 amino acids also present a significant homology. These findings suggest that these two regions may have evolved independently of each other by a process of gene duplication and fusion previous to the appearance of an ancestral aspartokinase-homoserine dehydrogenase molecule.

Nucleotide sequence of the thrA gene of Escherichia coli.

The thrA gene of Escherichia coli codes for a single polypeptide chain having two enzymatic activities required for the biosynthesis of threonine, aspartokinase I and homoserine dehydrogenase I. This gene was cloned in a bacterial plasmid and its complete nucleotide sequence was established. It contains 2460 base pairs that encode for a polypeptide chain of 820 amino acids. The previously determined partial amino acid sequence of this protein is in good agreement with that predicted from the nucleotide sequence. The gene contains an internal sequence that resembles the structure of bacterial ribosome-binding sites, with an AUG preceded by four triplets, each of which can be converted to a nonsense codon by a single mutation. This suggests that the single polypeptide chain was formed by the fusion of two genes and that initiation of translation may occur inside the gene to give a protein fragment having only the homoserine dehydrogenase activity.

Fine structure analysis of the threonine operon in Escherichia coli K-12.

A fine structure analysis of the threonine operon in Escherichia coli K-12 was performed by deletion mapping. Lambda transducing bacteriophages carrying various parts of the threonine operon were isolated from strains in which the lacZ gene was fused to a thr gene. We tested for recombination between deletions of the threonine promotor extending into the threonine operon, carried by the phage, and bacterial thr auxotrophs. The relative order of thrO (operator) mutations was established. We propose that an operator region is located between a promoter region and the structural genes. Mutations leading to the desensitization of the aspartokinase I-homoserine dehydrogenase I towards threonine were localized in two different regions of the thrA gene.