Characterization of Aspartate Kinase from Corynebacterium pekinense and the Critical Site of Arg169.

Aspartate kinase (AK) is the key enzyme in the biosynthesis of aspartate-derived amino acids. Recombinant AK was efficiently purified and systematically characterized through analysis under optimal conditions combined with steady-state kinetics study. Homogeneous AK was predicted as a decamer with a molecular weight of ~48 kDa and a half-life of 4.5 h. The enzymatic activity was enhanced by ethanol and Ni(2+). Moreover, steady-state kinetic study confirmed that AK is an allosteric enzyme, and its activity was inhibited by allosteric inhibitors, such as Lys, Met, and Thr. Theoretical results indicated the binding mode of AK and showed that Arg169 is an important residue in substrate binding, catalytic domain, and inhibitor binding. The values of the kinetic parameter Vmax of R169 mutants, namely, R169Y, R169P, R169D, and R169H AK, with l-aspartate as the substrate, were 4.71-, 2.25-, 2.57-, and 2.13-fold higher, respectively, than that of the wild-type AK. Furthermore, experimental and theoretical data showed that Arg169 formed a hydrogen bond with Glu92, which functions as the entrance gate. This study provides a basis to develop new enzymes and elucidate the corresponding amino acid production.

A specialized aspartokinase enhances the biosynthesis of the osmoprotectants ectoine and hydroxyectoine in Pseudomonas stutzeri A1501.

The compatible solutes ectoine and hydroxyectoine are widely produced by bacteria as protectants against osmotic and temperature stress. l-Aspartate-beta-semialdehyde is used as the precursor molecule for ectoine/hydroxyectoine biosynthesis that is catalyzed by the EctABCD enzymes. l-Aspartate-beta-semialdehyde is a central intermediate in different biosynthetic pathways and is produced from l-aspartate by aspartokinase (Ask) and aspartate-semialdehyde-dehydrogenase (Asd). Ask activity is typically stringently regulated by allosteric control to avoid gratuitous synthesis of aspartylphosphate. Many organisms have evolved multiple forms of aspartokinase, and feedback regulation of these specialized Ask enzymes is often adapted to the cognate biochemical pathways. The ectoine/hydroxyectoine biosynthetic genes (ectABCD) are followed in a considerable number of microorganisms by an askgene (ask_ect), suggesting that Ask_Ect is a specialized enzyme for this osmoadaptive biosynthetic pathway. However, none of these Ask_Ect enzymes have been functionally characterized. Pseudomonas stutzeri A1501 synthesizes both ectoine and hydroxyectoine in response to increased salinity, and it possesses two Ask enzymes: Ask_Lys and Ask_Ect. We purified both Ask enzymes and found significant differences with regard to their allosteric control: Ask_LysC was inhibited by threonine and in a concerted fashion by threonine and lysine, whereas Ask_Ect showed inhibition only by threonine. The ectABCD_askgenes from P. stutzeri A1501 were cloned and functionally expressed in Escherichia coli, and this led to osmostress protection. An E. colistrain carrying the plasmid-based ectABCD_askgene cluster produced significantly more ectoine/hydroxyectoine than a strain expressing the ectABCDgene cluster alone. This finding suggests a specialized role for Ask_Ect in ectoine/hydroxyectoine biosynthesis.

Exploring the molecular basis for selective binding of Mycobacterium tuberculosis Asp kinase toward its natural substrates and feedback inhibitors: a docking and molecular dynamics study.

Tuberculosis (TB) is still a major public health problem, compounded by the human immunodeficiency virus (HIV)-TB co-infection and recent emergence of multidrug-resistant (MDR) and extensively drug resistant (XDR)-TB. In this context, aspartokinase of mycobacterium tuberculosis has drawn attention for designing novel anti-TB drugs. Asp kinase is an enzyme responsible for the synthesis of 4-phospho-L-aspartate from L-aspartate and involved in the branched biosynthetic pathway leading to the synthesis of amino acids lysine, threonine, methionine and isoleucine. An intermediate of lysine biosynthetic branch, mesodiaminopimelate is also a component of the peptidoglycan which is a component of bacterial cell wall. To interfere with the production of all these amino acids and cell wall, it is possible to inhibit Asp kinase activity. This can be achieved using Asp kinase inhibitors. In order to design novel Asp kinase inhibitors as effective anti-TB drugs, it is necessary to have an understanding of the binding sites of Asp kinase. As no crystal structure of the enzyme has yet been published, we built a homology model of Asp kinase using the crystallized Asp kinase from M. Jannaschii, as template structures (2HMF and 3C1M). After the molecular dynamics refinement, the optimized homology model was assessed as a reliable structure by PROCHECK, ERRAT, WHAT-IF, PROSA2003 and VERIFY-3D. The results of molecular docking studies with natural substrates, products and feedback inhibitors are in agreement with the published data and showed that ACT domain plays an important role in binding to ligands. Based on the docking conformations, pharmacophore model can be developed by probing the common features of ligands. By analyzing the results, ACT domain architecture, certain key residues that are responsible for binding to feedback inhibitors and natural substrates were identified. This would be very helpful in understanding the blockade mechanism of Asp kinase and providing insights into rational design of novel Asp kinase inhibitors for M.tuberculosis.

[Construction of recombinant plasmids containing threonine operon and their effects on L-threonine accumulation].

OBJECTIVE: We reconstructed two recombinant plasmids and studied their effects on L-threonine accumulation of Escherichia coli W3110. METHODS: We amplified the threonine operon containing ThrLp promoter, lead peptide thrL, thrA, thrB and thrC genes by PCR from E. coli W3110 chromosome and ligated it into the pMD19 T-vector. Site-directed mutation were carried out by gene splicing by overlap extension PCR to release the feedback inhibition of aspartokinase I (thrA). Two recombinant plasmids pWYE112 and pWYE134 were transformed into E. coli W3110 by electroporation. Fed-batch cultures of E. coli W3110 were carried out in 5-Liter fermentors and the L-threonine concentration was measured by HPLC. RESULTS: Fed-batch fermentation results showed that E. coli W3110 could accumulate little L-threonine (0.036 +/- 0.004 g/L) but recombinant E. coli W3110 harboring the plasmid pWYE112 containing a threonine operon exhibited a L-threonine production of 2.590 +/- 0.115 g/L. Furthermore, L-threonine production reached 9.223 +/- 1.279 g/L when the feedback inhibition of thrA was released. CONCLUSION: Overexpression of threonine operon can lead to the accumulation of L-threonine. Further release of feedback inhibition of aspartokinase I can enhance its accumulation.

Structural Insight into concerted inhibition of alpha 2 beta 2-type aspartate kinase from Corynebacterium glutamicum.

Aspartate kinase (AK) catalyzes the first step of the biosynthesis of the aspartic acid family amino acids, and is regulated via feedback inhibition by end-products including Thr and Lys. To elucidate the mechanism of this inhibition, we determined the crystal structure of the regulatory subunit of AK from Corynebacterium glutamicum at 1.58 A resolution in the Thr-binding form, the first crystal structure of the regulatory subunit of alpha(2)beta(2)-type AK. The regulatory subunit contains two ACT domain motifs per monomer and is arranged as a dimer. Two non-equivalent ACT domains from different chains form an effector-binding unit that binds a single Thr molecule, and the resulting two effector-binding units of the dimer associate perpendicularly in a face-to-face manner. The regulatory subunit is a monomer in the absence of Thr but becomes a dimer by adding Thr. The dimerization is eliminated in mutant AKs with changes in the Thr-binding region, suggesting that the dimerization induced by Thr binding is a key step in the inhibitory mechanism of AK from C. glutamicum. A putative Lys-binding site and the inhibitory mechanism of CgAK are discussed.

Purification, crystallization and preliminary X-ray analysis of the regulatory subunit of aspartate kinase from Thermus thermophilus.

Aspartate kinase (AK) from Thermus thermophilus, which catalyzes the first step of threonine and methionine biosynthesis, is regulated via feedback inhibition by the end product threonine. To elucidate the mechanism of regulation of AK, the regulatory subunit (the beta subunit of T. thermophilus AK) was crystallized in the presence of the inhibitor threonine. Diffraction data were collected to 2.15 A at a synchrotron source. The crystal belongs to the cubic space group P4(3)32 or P4(1)32, with unit-cell parameters a = b = c = 141.8 A.

A novel organization of ACT domains in allosteric enzymes revealed by the crystal structure of Arabidopsis aspartate kinase.

Asp kinase catalyzes the first step of the Asp-derived essential amino acid pathway in plants and microorganisms. Depending on the source organism, this enzyme contains up to four regulatory ACT domains and exhibits several isoforms under the control of a great variety of allosteric effectors. We report here the dimeric structure of a Lys and S-adenosylmethionine-sensitive Asp kinase isoform from Arabidopsis thaliana in complex with its two inhibitors. This work reveals the structure of an Asp kinase and an enzyme containing two ACT domains cocrystallized with its effectors. Only one ACT domain (ACT1) is implicated in effector binding. A loop involved in the binding of Lys and S-adenosylmethionine provides an explanation for the synergistic inhibition by these effectors. The presence of S-adenosylmethionine in the regulatory domain indicates that ACT domains are also able to bind nucleotides. The organization of ACT domains in the present structure is different from that observed in Thr deaminase and in the regulatory subunit of acetohydroxyacid synthase III.

Cloning, characterization and heterologous expression of the aspartokinase and aspartate semialdehyde dehydrogenase genes of cephamycin C-producer Streptomyces clavuligerus.

Carbon flow through the lysine branch of the aspartate biosynthetic pathway is a rate-limiting step in the formation of cephamycin C, a broad spectrum beta-lactam antibiotic produced by Streptomyces clavuligerus. In this study, genes which encode the enzymes catalyzing the first two steps of the aspartate pathway, ask (aspartokinase) and asd (aspartate semialdehyde dehydrogenase), in S. clavuligerus NRRL 3585 were cloned and sequenced. Nucleotide sequencing and codon preference analysis revealed three complete open reading frames (ORFs). ORF2 starts within ORF1 and terminates by utilizing the same stop codon as ORF1, an arrangement typical of many ask genes. ORF3 is located 2 nucleotides downstream of ORF1,2. Database comparisons with these proteins identified ORF1 as the large (alpha) subunit of aspartokinase, ORF2 as the small (beta) subunit of aspartokinase and ORF3 as the aspartate semialdehyde dehydrogenase. The cloned genes were functionally expressed in auxotrophic Escherichia coli strains, CGSC5074 (ask(-)) and E. coli CGSC5080 (asd(-)), the two enzymes were partially purified from E. coli cell extracts and their kinetic parameters were determined. The effects of end product amino acids and diaminopimelic acid on the activity of Ask and Asd enzymes were also described.

Small molecule functional discrimination of the kinases required for the microbial synthesis of threonine and isoleucine.

The biosynthesis of l-threonine and l-isoleucine in bacteria and in fungi requires the action of 2 amino acid kinases: aspartate kinase and homoserine kinase. Although these kinases bind similar substrates and catalyze analogous phosphotransfer chemistry, they do not show high amino acid sequence homology. We show that despite this difference, both kinases form a ternary complex consisting of enzyme- adenosine triphosphate- amino acid to accomplish phosphoryl transfer. With this similarity in mind, we set out to identify molecules that could lead to inhibitors with activity against both kinases in the pathway. We synthesized a series of aspartic acid-adenosine bisubstrate compounds separated by a variable length alkyl linker that we hypothesized could bind to these kinases. These bisubstrate compounds only inhibited the bacterial aspartate kinase. These results reveal unexpected differences in small molecule interactions among these functionally similar enzymes.

Mutation analysis of the feedback inhibition site of aspartokinase III of Escherichia coli K-12 and its use in L-threonine production.

Aspartokinase III (AKIII), one of three isozymes of Escherichia coli K-12, is inhibited allosterically by L-lysine. This enzyme is encoded by the lysC gene and has 449 amino acid residues. We analyzed the feedback inhibition site of AKIII by generating various lysC mutants in a plasmid vector. These mutants conferred resistance to L-lysine and/or an L-lysine analogue on their host. The inhibitory effects of L-lysine on and heat tolerance of 14 mutant enzymes were examined and DNA sequencing showed that the types of mutants were 12. Two hot spots, amino acid residue positions 318-325 and 345-352, were detected in the C-terminal region of AKIII and these enzyme regions may be important in L-lysine-mediated feedback inhibition of AKIII. Feedback resistant lysC relieved on L-threonine hyper-producing strain, B-3996, from reduced L-threonine productivity by addition of L-lysine, and furthermore increased L-threonine productivity even when no addition of L-lysine. It suggested that the bottleneck of L-threonine production of B-3996 was AK and feedback resistant lysC was effective because of the strict inhibition by cytoplasmic L-lysine.

Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32.

The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask-) and E. coli X6118 (asd-), respectively. The A. mediterranei U32 asparto-kinase and aspartate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for DL-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by L-threonine, L-lysine, and L-methionine, but not by L-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that asparto-kinase was subject to allosteric control by L-threonine. Repression of both aspartokinase and aspartate semi-aldehyde dehydrogenase gene transcription in A. mediterranei U32 by L-lysine, L-methionine, L-threonine, and L-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.

Analysis of photocontrol of aspartate kinase in barley (Hordeum vulgare L.) seedlings.

Aspartate kinase (AK) activity is regulated by light. The activity was more in light exposed barley seedlings than those grown in the dark. The light effect was manifested even with small exposures of 5 min duration and red light was more effective than white light in this respect. The effect of 5 min red light could be reversed by a 5 min pulse of far-red light indicating the involvement of phytochrome in this response. The phytochrome is also involved in long term light effects (24 hr exposures with white light). Ca++ takes part in the signal transduction pathway for this light response. Western blot analysis using antibodies raised against the purified lysine- and threonine-sensitive AK isoenzymes from spinach leaves showed no cross reaction with the antibodies to the threonine-sensitive AK in the dark and 5 min far-red light exposed seedlings. But the protein band was detected in the white and red lights. Northern blot analysis of seedlings grown under dark and exposed to white, red and far-red lights and probed with the gene encoding aspartokinase-homoserine dehydrogenase (AKHSD) protein indicated that the gene was differentially expressed. In dark grown seedlings, AKHSD transcript was in low concentration as compared to white light where the transcript concentration was high. A 5 min red light pulse increased the transcript concentration significantly in contrast to 5 min far-red light. The transcript concentration was reduced when 5 min red light was followed by a 5 min far-red light pulse. The AK activity in dark-raised seedlings is attributed to the presence of only one isoenzyme that is sensitive to lysine but insensitive to Ca++ and calmodulin (CAM). In both white and red light exposed seedlings, three isoenzymes of AK were detected. Two of these were sensitive to threonine while one was sensitive to lysine. Both of the threonine sensitive isoenzymes were activated by Ca++ and CAM. Also one of these isoenzymes seems to be located and synthesized in chloroplasts because its synthesis was completely inhibited by chloramphenicol but not by cycloheximide.

A mutant of Arabidopsis thaliana (L.) Heynh. with modified control of aspartate kinase by threonine.

Mutagenesis and subsequent selection of Arabidopsis thaliana plantlets on a growth inhibitory concentration of lysine has led to the isolation of lysine-resistant mutants. The ability to grown on 2 mM lysine has been used to isolate mutants that may contain an aspartate kinase with altered regulatory-feedback properties. One of these mutants (RL 4) was characterized by a relative enhancement of soluble lysine. The recessive monogenic nuclear transmission of the resistance trait was established. It was associated with an aspartate kinase less sensitive to feedback inhibition by threonine. Two mutants (RLT 40 and RL 4) in Arabidopsis, characterized by an altered regulation of aspartate kinase, were crossed to assess the effects of the simultaneous presence of these different aspartate kinase forms. A double mutant (RLT40 x RL4) was isolated and characterized by two feedback-desensitized isozymes of aspartate kinase to, respectively, lysine and threonine but no threonine and/or lysine overproduction was observed. Genetical analysis of this unique double aspartate kinase mutant indicated that both mutations were located on chromosome 2, but their loci (ak1 and ak2) were found to be unlinked.

Regulation of aspartokinase in Lactobacillus plantarum.

As a rational approach to the genetic development of a stable lysine overproducing strain of Lactobacillus plantarum for the fermentation of 'ogi', a Nigerian fermented cereal porridge, regulation of lysine biosynthesis in this species was investigated. Spontaneous lysine overproducing mutants of Lact. plantarum were obtained and their aspartokinase activities compared with those of wild-type strains under different conditions. Results showed that aspartokinase activity of Lact. plantarum cell extracts was not inhibited by either lysine, threonine, methionine or combinations of lysine and threonine. Instead, methionine enhanced aspartokinase activity in vitro. Results indicated that lysine biosynthesis in Lact. plantarum could be regulated by lysine via the control of aspartokinase production in a way different to that described for other bacteria.

A possible role of the effect of methionine on the activity of aspartokinase in sporulation of a Streptomyces fradiae mutant.

In addition to methionine, aspartate may also induce sporulation in the Streptomyces fradiae St3110 mutant that requires methionine for sporulation but not for growth. Partially purified aspartokinase (EC 2.7.2.4.) of the mutant was tested for feed-back control by methionine, threonine, and lysine. Methionine or threonine alone did not have any significant effect but they had a concerted inhibitory effect together that was further increased by lysine. The threonine-lysine combination also caused inhibition of the enzyme activity, while lysine alone activated the enzyme. Methionine was also found to partially repress the aspartokinase activity to about one third of the control. The sporulating aerial mycelia induced by aspartate were lacking the characteristic sporulation pigmentation present in the wild type or after induction of spores by methionine. No significant difference in heat resistance between the two types of the spores was detected. The number of spores produced by aspartate was only about one fourth of that after induction by methionine. The data may indicate a role of aspartokinase control by methionine in restoring the normal sporulation, although in addition to this methionine may act by a different mechanism, as well.

Regulation of two aspartokinase isozymes in Streptococcus bovis.

Streptococcus bovis has been found to contain two distinct aspartokinases that can be separated by gel filtration chromatography. One of these isozymes elutes on Sephadex G-200 gel filtration at a molecular weight greater than 250,000. The molecular weight of the other isozyme is approximately 125,000. The earlier peak of aspartokinase activity is slightly inhibited by meso-diaminopimelate, while the second peak is sensitive to inhibition by lysine. The latter aspartokinase is not formed when the organism is grown in a medium containing more than 1 mM lysine. The level of lysine-sensitive aspartokinase is decreased during the growth cycle, whereas diaminopimelate-sensitive activity is little affected by the growth conditions. The regulatory properties of the two aspartokinases suggest that they may play different physiological roles.

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 (DAPs) enzyme was not much affected by growth conditions, the activity of lysine-sensitive (Lyss) 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 the first and last stage of lysine biosynthesis in Streptococcus bovis and Enterococcus faecium]. / Reguliatsiia fermentov pervoi i poslednei stadii biosinteza lizina u Streptococcus bovis i Enterococcus faecium.

Regulation of aspartate kinase and diaminopimelate decarboxylase activities in Streptococcus bovis and Enterococcus faecium cell-free extracts was studied. The levels of synthesis of aspartate kinase and diaminopimelate decarboxylase in both microorganisms are growth-dependent. The synthesis of these enzymes is depressed by lysine, but the activity of aspartate kinase is induced by addition of this amino acid and threonine to the reaction system. Meso-diaminopimelate dehydrogenase activity was not found in the extracts of Streptococcus bovis and Enterococcus faecium. The data excludes the possibility of lysine formation via six enzyme reactions.

Reversal of enzyme regiospecificity with alternative substrates for aspartokinase I from Escherichia coli.

The substrate specificity of aspartokinase I has been examined by using both steady-state kinetic analyses and phosphorus-31 NMR spectroscopic studies. Analogues in which the alpha-amino group is either derivatized or replaced are not substrates or inhibitors for the enzyme, indicating the importance of the alpha-amino group as a binding determinant. The alpha-carboxyl group is not required for substrate recognition, and the alpha-amide or alpha-esters are competent alternative substrates. In addition, beta-derivatized structural analogues, such as the beta-hydroxamate, the beta-amide, or beta-esters, were found to be viable substrates. This was unexpected since the beta-carboxyl group is the usual site of phosphorylation. The nature of the acyl phosphate products obtained from these beta-derivatized alternative substrates has been characterized by coupled enzyme assays, oxygen-18-labeling studies, and phosphorus-31 NMR spectroscopy. These beta-derivatized analogues are capable of productive binding to aspartokinase through a reversal of regiospecificity to make the alpha-carboxyl group available as a phosphoryl acceptor. Many, but not all, of these alpha-acyl phosphates have also been shown to be viable substrates for the next two enzyme-catalyzed steps in this metabolic pathway. This raises the possibility of producing enzyme-generated alternative substrates that can serve as antimetabolites for the downstream reactions in this biosynthetic pathway.