Dihydrodipicolinic Acid Research Articles

Allosteric Signaling in Dihydrodipicolinate Synthase

Dihydrodipicolinate synthase (DHDPS) is an enzyme found in most bacterial species and regulates the production of cell wall precursors necessary for the life of the organism. Specifically, DHDPS catalyzes the condensation of pyruvate and aspartate-β-semialdehyde (ASA) to produce dihydrodipicolinic acid leading to the synthesis of cell wall precursors lysine and mesodiaminopimelate. DHDPS is regulated through feedback inhibition when lysine binds at a location distinct from the reactive site. The mechanism by which lysine remotely disrupts catalysis is not well understood. A clear understanding of how the natural inhibitor, lysine, binds to DHDPS and what effects this has on the machinery of the enzyme will be invaluable for development of novel antibiotic leads. Analysis of DHDPS crystals with and without inhibitors has revealed structural changes that appear to link the allosteric and active sites. Our ongoing research examines the validity of observed structural changes in the mechanism of allosteric inhibition.

EtfA catalyses the formation of dipicolinic acid inClostridium perfringens

Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5-15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis, and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens, Clostridium botulinum and Clostridium tetani, has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces < or = 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli, and this protein catalysed DPA formation in vitro. The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.

The Arabidopsis thaliana dhdps gene encoding dihydrodipicolinate synthase, key enzyme of lysine biosynthesis, is expressed in a cell-specific manner.

Lysine synthesis in prokaryotes, some phycomycetes and higher plants starts with the condensation of L-aspartate-beta-semialdehyde (L-ASA) and pyruvate into dihydrodipicolinic acid. The enzyme that catalyses this step, dihydrodipicolinate synthase (DHDPS), is inhibited by the end-product lysine and is therefore thought to have a regulatory control on lysine synthesis. We have cloned and sequenced an Arabidopsis thaliana DNA fragment containing 900 bases upstream of the dhdps coding sequence. A transcriptional fusion of this fragment with the beta-glucuronidase reporter gene (uidA. Gus) was used to study the transcription properties of this promoter fragment (DS). No lysine-induced repression on transcription could be detected. Expression of DS-Gus activity in transformed Arabidopsis thaliana and Nicotiana tabacum was found to be cell type-specific. In the vegetative parts of the plant, GUS activity was located in meristems and young vasculature of roots, in vasculature of stem and leaves and in the meristems of young shoots. In flowers, high expression was found in the carpels, style, stigma, developing embryos, tapetum of young anthers and pollen. We demonstrated that the Arabidopsis DS promoter can direct its cell type-specific expression in a heterologous plant, Nicotiana tuabacum. The importance of transcriptional regulation of the dhdps gene, and in more general genes involved in amino acid biosynthesis, is discussed.

Transgenic canola and soybean seeds with increased lysine.

We have increased the lysine content in the seeds of canola and soybean plants by circumventing the normal feedback regulation of two enzymes of the biosynthetic pathway, aspartokinase (AK) and dihydrodipicolinic acid synthase (DHDPS). Lysine-feedback-insensitive bacterial DHDPS and AK enzymes encoded by the Corynebacterium dapA gene and a mutant E. coli lysC gene, respectively, were linked to a chloroplast transit peptide and expressed from a seed-specific promoter in transgenic canola and soybean seeds. Expression of Corynebacterium DHDPS resulted in more than a 100-fold increase in the accumulation of free lysine in the seeds of canola; total seed lysine content approximately doubled. Expression of Corynebacterium DHDPS plus lysine-insensitive E. coli AK in soybean transformants similarly caused several hundred-fold increases in free lysine and increased total sed lysine content by as much as 5-fold. Accumulation of alpha-amino adipic acid (AA) in canola and saccharopine in soybean, which are intermediates in lysine catabolism, was also observed.

ChemInform Abstract: Pyridine and Piperidine Derivatives as Inhibitors of Dihydrodipicolinic Acid Synthase, a Key Enzyme in the Diaminopimelate Pathway to L‐Lysine.

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Biosynthesis of lysine in plants: the putative role of meso-diaminopimelate dehydrogenase.

Extracts from Chlamydomonas, corn, soybean and tobacco were tested for enzymes of the lysine biosynthetic pathway. Dihydrodipicolinic acid (DHD) synthase, DHD reductase, diaminopimelate (DAP) epimerase and DAP decarboxylase were present in all. However, in contrast to the report of Wenko et al., meso-DAP dehydrogenase could not be detected in extracts prepared from soybean. Moreover, it was not found in Chlamydomonas, corn and tobacco as well. In order to set an upper limit to the amount of meso-DAP dehydrogenase that might be present, reconstruction experiments were performed with soybean and corn extracts in which the conversion of dihydrodipicolinate to lysine was made dependent on the addition of limited amounts of the meso-DAP dehydrogenase purified from Bacillus sphaericus. The presence of DAP epimerase and the absence of meso-DAP dehydrogenase indicates that the meso-DAP dehydrogenase abbreviated pathway for lysine synthesis is not operative in plants.

Pyridine and piperidine derivatives as inhibitors of dihydrodipicolinic acid synthase, a key enzyme in the diaminopimelate pathway to l-lysine

A key step in the diaminopimelate (DAP) pathway to l-lysine ( 7) involves condensation of pyruvate with aspartic acid β-semialdehyde ( 1) to yield l-2,3-dihydrodipicolinic acid ( 2) (DHDPA) catalyzed by DHDPA synthase. The best inhibitors of DHDPA synthase of the thirty pyridine and piperidine derivatives prepared were the N-oxide ( 15) of dipicolinic acid and the di-imidate ( 13) of dimethyl pyridine-2,6-dicarboxylate each with an IC 50 value of 0.2 mM. The N-oxide ( 15) and dinitrile ( 12) are non-competitive inhibitors with K i values of 0.29 and 1.25 mM against aspartate semialdehyde and 0.06 and 0.34 mM against pyruvate, respectively.

The effect of dipicolinic acid on maize tissue culture growth is not solely due to inhibition of lysine biosynthesis

Dipicolinic acid, a known inhibitor of an enzyme (dihydrodipicolinic acid reductase) in the maize (Zea mays L.) lysine biosynthetic pathway, inhibits the growth of maize suspension and callus cultures. Inhibited cultures contain somewhat lower free lysine levels, but the inhibition of suspension culture growth was not reversible with simultaneous addition of L-lysine to the culture medium. It is concluded that dipicolinic acid does not act solely as an analog blocking lysine production. Dipicolinic acid thus appears to be unsuitable as a selection for maize tissue culture mutants with lysine overproduction.

Organelle DNA compositions and isoenzyme expression in an interspecific somatic hybrid of Daucus

Cell suspension cultures of Daucus carota, D. capillifolius and a somatic hybrid of these lines were analyzed to determine their chloroplast and mitochondrial DNA compositions. The plastid DNAs (pDNA) from the somatic hybrid and D. carota were identical and were different from that of D. capillifolius when analyzed on agarose electrophoretic gels after digestion by the restriction endonuclease HpaII. The endonuclease restriction patterns of the mitochondrial DNAs (mtDNA) from each cell line were different. Although the restriction pattern of the mtDNA from the somatic hybrid contained fragments in common with one or both parents, unique fragments not found in the restriction pattern of either parent were also present. The amounts and feedback regulation of aspartokinase, homoserine dehydrogenase and dihydrodipicolinic acid synthase were quantified to define the effects of somatic hybridization upon the pathway leading to the biosynthesis of lysine, threonine, methionine and isoleucine. Regulation of each enzyme by end product inhibitors was not altered in the somatic hybrid, but levels of each enzyme appeared to be increased. However, isoenzyme analysis indicated two major forms of homoserine dehydrogenase were present in the hybrid, including one unique form not present in either parent.

Partial Purification and Characterization of Dihydrodipicolinic Acid Reductase from Maize

Dihydrodipicolinic acid reductase, an enzyme which catalyzes the pyridine nucleotide-linked reduction of dihydrodipicolinic acid to tetrahydrodipicolinic acid in the biosynthetic pathway leading to l-lysine, has been partially purified from maize (Zea mays cv Pioneer 3145) kernels. The crude maize extract and the partially purified enzyme were assayed for dihydrodipicolinic acid reductase by their ability to restore the capability of crude extracts of a mutant Escherichia coli (CGSC 4549; defective in dihydrodipicolinic acid reductase) to synthesize diaminopimelic acid from aspartic acid and pyruvic acid.In a study of its properties, the Michaelis constant obtained for dihydrodipicolinic acid was 4.3 x 10(-4) and for NADPH the K(m) was 4.6 x 10(-5). The enzyme had a pH optimum close to 7 and was much more temperature labile than the bacterial enzyme. Its molecular weight was 8.0 x 10(4).Several compounds, viz., alpha-picolinic acid, l-pipecolic acid, isophthalic acid, and isocinchomeronic acid, with structures similar to dihydrodipicolinic acid inhibited the reductase reaction. Dipicolinic acid, the most potent of these inhibitors, acted as a competitive inhibitor with a K(i) of 9.0 x 10(-4). The competitive inhibition of the reductase reaction by dipicolinic acid (oxidized dihydrodipicolinic acid) suggests that the substrate for this enzyme was in the ring rather than the open chain form. Oxidized pyridine nucleotide inhibited the activity slightly.

Regulation of cephamycin C synthesis, aspartokinase, dihydrodipicolinic acid synthetase, and homoserine dehydrogenase by aspartic acid family amino acids in Streptomyces clavuligerus.

The effect of the cephalosporin precursors and amino acids of the aspartic acid family on antibiotic production by Streptomyces clavuligerus was investigated DL-meso-Diaminopimelate and L-lysine each stimulated specific antibiotic production by 75%. A fourfold increase in specific production was obtained by simultaneous addition of the two compounds. The stimulation could be further increased by adding valine to the two effectors. In the streptomycetes the alpha-aminoadipyl side chain of the cephalosporin antibiotics is derived from lysine. Streptomycetes, like other bacteria, are expected to produce lysine from aspartic acid; therefore, the feedback control mechanisms operating in the aspartic acid family pathway of S. clavuligerus, which may affect the flow of carbon to alpha-aminoadipic acid, were investigated. Threonine inhibited antibiotic production by 41% when added to minimal medium at a concentration of 10 mM. Simultaneous addition of 10 mM lysine completely reversed this inhibition. The aspartokinase of S. clavuligerus was found to be subject to concerted feedback inhibition by threonine and lysine. Threonine may act to limit the supply of lysine available for cephamycin C biosynthesis via this concerted mechanism. Single or simultaneous addition of any other amino acid of the aspartate family in the in vitro assay did not inhibit aspartokinase activity. Activity was stimulated by lysine. Aspartokinase biosynthesis was partially repressed by methionine or isoleucine at concentrations higher than 10 mM. Methionine, but not isoleucine, inhibited cephamycin C synthesis by 27% when added to minimal medium at a concentration of 10 mM. Dihydrodipicolinate synthetase, the first specific enzyme of the lysine branch, was not inhibited by lysine but was partially inhibited by high concentrations of 2,6-diaminopimelate and alpha-aminoadipate; it was slightly repressed by diaminopimelic acid. Homoserine dehydrogenase activity was inhibited by threonine and partially repressed by isoleucine. It appears that S. clavuligerus aspartokinase is a key step in the control of carbon flow toward alpha-aminoadipic acid.

Factors modulating transcription and translation in vitro of ribosomal protein S20 and isoleucyl-tRNA synthetase from Escherichia coli.

The DNA-dependent protein-synthesizing system developed by Zubay [Zubay, G. (1973) Annu. Rev. Genet. 7, 267--287] was optimized for the transcription and translation of genes from the 0.5-min region of the Escherichia coli chromosome carried by transducing lambda phages. The E. coli gene products synthesized were isoleucyl tRNA synthetase, ribosomal protein S20, dihydrodipicolinic acid reductase and (possibly) the two subunits carbamoyl-phosphate synthetase. Formation of ribosomal protein S20 is specifically stimulated by the addition of 16-S rRNA and not by 5-S or 23-S rRNA. 16-S rRNA increases the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added. Addition of 16-S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced. Furthermore, S20 synthesis is stimulated more than fourfold when concomitant synthesis of rRNA occurs from a plasmid carrying an rrn transcriptional unit. The results described are explained in terms of a model which suggests that ribosomal protein S20 feedback inhibits its synthesis at the translational level and that removal of S20 into ribosomal assembly (i.e. binding to 16-S rRNA) releases inhibition. The model postulates a direct link between synthesis of ribosomal RNA and ribosomal protein and between the rates of ribosomal assembly and ribosomal protein synthesis. The stimulatory effect of guanosine 3'-diphosphate 5'-diphosphate on isoleucyl-tRNA synthetase formation and its inhibition of the synthesis of ribosomal protein S20 in vitro occurs at the level of transcription. Its relevance in vivo, however, remains to be demonstrated. Formation of isoleucyl-tRNA synthetase in vitro is not influenced either by the addition of a surplus of purified enzyme nor by the limitation of protein synthesis by the addition of anti-(isoleucyl-tRNA synthetase) serum. There is no evidence, therefore, that isoleucyl-tRNA synthetase is autogenously regulated.

Mechanism of Resistance of a Selected Carrot Cell Suspension Culture to S(2-aminoethyl)-L-cysteine

Carrot ( Daucus carota L. var. Danvers ) cell suspension cultures sensitive and resistant to growth inhibition by the lysine analog S(2-aminoethyl)-L-cysteine (AEC) were characterized during cell culture growth. Throughout the cell culture period both lines contained similar levels of free lysine. The uptake of 14 C-lysine from the culture medium was greatly reduced in the AEC-resistant cell line. Key enzymes involved in lysine and threonine synthesis from aspartate were examined. Changes in the amount of enzyme activity present during the culture period were noted for the first enzyme in the pathway, aspartokinase (EC 2.7.2.4) and the branchpoint enzyme dihydrodipicolinic acid synthase (EC 4.2.1.52) leading to lysine synthesis. The branchpoint enzyme leading to threonine synthesis, homoserine dehydrogenase, (EC 1.1.1.3) was not altered in amount of activity when the sensitive and resistant cell lines were compared, but fluctuations in the amount of inhibition of the enzyme by threonine were observed.

Expression of Aspartokinase, Dihydrodipicolinic Acid Synthase and Homoserine Dehydrogenase During Growth of Carrot Cell Suspension Cultures on Lysine-and Threonine-Supplemented Media

Reduction in the amounts of activity of the first enzyme, aspartokinase (EC 2.7.2.4) and two branch-point enzymes, dihydrodipicolinic acid synthase (EC 4.2.1.52) and homoserine dehydrogenase (EC 1.1.1.3), located in the pathway for the synthesis of aspartate-family amino acids, occurred when cell suspension cultures of Daucus carota L. var. Danvers were grown in media containing 2 mM threonine or 2 mM lysine, endproducts of the pathway. Activity of the lysine-sensitive form of aspartokinase was decreased when cells were grown in medium containing lysine and the activity of the threonine-sensitive form was decreased when cells were grown in medium containing threonine. Activity of the branch-point enzyme leading to threonine synthesis, homoserine dehydrogenase, was decreased up to 70% in specific activity (units/mg protein) and relative activity (units/g fresh weight) when cells were grown in media containing lysine or threonine. Threonine had no effect on the relative activity of dihydrodipicolinic acid synthase, but decreased its specific activity. Lysine decreased the relative activity of the synthase by up to 40%, but had little effect on its specific activity. The decreased activities of the enzymes were apparently not due to binding of the inhibitory amino acids to the enzymes since homogenization of cells in buffer with 2 mM lysine and threonine did not decrease the measurable enzyme activities. These and other results presented suggest that both forms of the aspartokinase activity and homoserine dehydrogenase activity can be altered by supplementing the growth medium with lysine or threonine.

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic-β-semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.

Dihydrodipicolinic acid synthase of Bacillus licheniformis. Quaternary structure, kinetics, and stability in the presence of sodium chloride and substrates

Dihydrodipicolinic acid synthase ( l-aspartate-β-semialdehyde hydro-lyase (adding pyruvate and cyclising), EC 4.2.1.52) obtained from Bacillus licheniformis was purified to homogeneity. Its molecular weight was 108 000 to 117 500, depending on the concentration of NaCl and substrates present, and it contained four subunits of identical molecular weight (28 000). The K m values for pyruvate and l-aspartic semialdehyde were approximately 5.3 and 2.6 mM, respectively. It was previously shown that pyruvate and a high sodium chloride concentration contributed to the stability of the enzyme. The effect of these substances and the other substrate, l-aspartic semialdehyde, on molecular weight was determined. None of these three substances significantly affected the apparent molecular weight. The effect of sodium chloride, pyruvate, and l-aspartic semialdehyde on enzyme structure was studied by determining the effect of their presence on inactivation of the enzyme by several chemical denaturants and heat. Pyruvate dramatically protected against inactivation by all of the denaturants. Sodium chloride protected against inactivation by sodium dodecyl sulfate, guanidine · HCl, urea, and heat, but somewhat facilitated inactivation by ethanol. l-Aspartic semialdehyde had no significant effect on inactivation by sodium dodecyl sulfate and ethanol; it rendered the enzyme slightly more sensitive to inactivation by guanidine · HCl and urea. The thermal melting curve obtained for the enzyme in the presence of l-aspartic semialdehyde was biphasic. The activity was reduced approximately 50% by heating for 30 min at temperatures between 50 and 80°C. Only by heating at temperatures above 80°C did the inactivation become complete. The partially inactivated enzyme could be reactivated by heating after removal of the l-aspartic semialdehyde. Pyruvate prevented the partial inactivation and facilitated reactivation. The only difference detected between the native enzyme and the partially inactivated form of the enzyme was that the latter had a reduced V. It is known that in other spore-formers, dihydrodipicolinate synthase increases in activity late in sporulation. This increase may be important for normal sporulation to occur. The possibility is discussed that the intracellular pool sizes of pyruvate and l-aspartic semialdehyde might have an influence on the level of dihydrodipicolinate synthase activity, by controlling the amount of partial inactivation of the enzyme that occurs in vivo.

Regulation of dihydrodipicolinate synthase during growth and sporulation of Bacillus cereus.

A four- to sixfold increase in specific activity of dihydrodipicolinic acid synthase was observed during sporulation of Bacillus cereus. The enzyme from cells harvested before and after the increase in specific activity appeared to be very similar as judged by pH optima, heat denaturation kinetics, apparent Michaelis constants, chromatography on diethylaminoethyl-cellulose and Sephadex G-200, and polyacrylamide gel electrophoresis. Studies with various combinations of amino acids and one of the enzyme substrates, pyruvate, failed to give evidence for control of the enzyme by activation, inhibition, repression, induction, or stabilization. Omission of calcium from the sporulation medium had no significant effect on the specific activity pattern of the enzyme as a function of age of culture.

Pyridine-2, 6-dicarboxylic Acid (Dipicolinic Acid) Formation in Bacillus subtilis

Non-enzymatic formation of dipicolinic acid (DPA) from diketopimelic acid and ammonia was clearly demonstrated using a new method for DPA analysis. The reaction rates of DPA formation were almost the same under aerobic and anaerobic conditions. Nearly equimolecular quantities of DPA and tetrahydrodipicolinic acid were detected in spontaneous reaction mixture. The spontaneous reaction seemed to be due to dismutation of dihydrodipicolinic acid, resulting in DPA and tetrahydrodipicolinic acid. The apparent optimum pH of the spontaneous reaction was 8.2 and the maximal rate of DPA formation was observed with a 1 : 4 molar ratio of diketopimelic acid to ammonia. The rate of the spontaneous reaction was stimulated by ferrous sulfate, FMN, and riboflavin. Dihydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinate, prepared from pyruvate and aspartic beta-semialdehyde, with NADPH as reductant. The reductase was isolated from Bacillus subtilis, and found to stimulate DPA formation from diketopimelic acid and ammonia. The enzymatic DPA formation was absolutely dependent on oxygen, and optimum pH was 6.4. The catalytic action of the enzyme was similar to that of the oxidase. Possible mechanisms of DPA formation from diketopimelic acid and ammonia are proposed.

The control of lysine biosynthesis in maize

Aspartate kinase has been partially purified and characterised from germinating maize seedlings. The K m for aspartate was 9 mM. Out of several amino acids which are potential feedback regulators of the enzymes, only lysine is markedly inhibitory, having a K i of 13 μM and causing 100% inhibition at 0.5 mM. Lysine also protects the enzyme against heat inactivation. Dihydrodipicolinic acid synthase isolated from the same tissue is also inhibited by lysine, 1 mM causing 95% inhibition.

Overproduction of lysine by mutant strains of Escherichia coli with defective lysine transport systems.

Mutants selected on the basis of their resistance to S-(beta-aminoethyl) cysteine and overproduction of lysine were found to be defective in the lysine transport system. The overproduction of lysine was not due to mutation affecting either of the two regulatory enzymes aspartokinase and dihydrodipicolinic acid synthetase. Uptake of labeled lysine by the lysine-specific transport system was reduced to a negligible level, while uptake by the lysine, ornithine, arginine system was also affected. A hypothesis regarding the nature of these mutations and their effects on the regulation of lysine biosynthesis is discussed.