Arginine Biosynthesis Pathway Research Articles

Gene expression of Pht cluster genes and a putative non-ribosomal peptide synthetase required for phaseolotoxin production is regulated by GacS/GacA in Pseudomonas syringae pv. phaseolicola

Pseudomonas syringae pv. phaseolicola is the causal agent of halo blight disease of beans ( Phaseolus vulgaris L.), which is characterized by watersoaked lesions surrounded by a chlorotic halo resulting from the action of a non-host specific toxin known as phaseolotoxin. This toxin inhibits the enzyme ornithine carbamoyltransferase involved in the arginine biosynthesis pathway. It was previously reported that genes within the Pht cluster were involved in the regulation and synthesis of phaseolotoxin. The GacS/GacA two-component signal transduction system controls important pathogenicity and virulence mechanisms in several Gram-negative bacteria. Tox − phenotype gacA − and gacS − mutants were obtained and gacA − transcriptome analysis revealed that this response activator controls expression of genes within the Pht cluster as well as another gene located in a different region in the bacterial chromosome and that has been unambiguously shown to be directly involved in phaseolotoxin biosynthesis. Results presented in this work suggest that phaseolotoxin biosynthesis involve elements within and outside the Pht Cluster, and that the GacS/GacA two-component system exerts control over them.

Evolution of the Effect of Arginine on Thermal Stability and Oligomerization of N-Acetylglutamate Synthase

N-acetylglutamate synthase (NAGS; E.C.2.3.1.1) catalyzes the formation of N-acetylglutamate (NAG) from acetyl coenzyme A and glutamate. In microorganisms and plants, NAG is the first intermediate of arginine biosynthesis pathway, while in animals, NAG acts as an allosteric activator of carbamylphosphate synthetase I and III. NAGS itself is allosterically regulated by arginine. In bacteria, fungi, and plants, arginine acts as an inhibitor, in fish, a partial inhibitor, but in mammals, arginine is an activator. We used Thermofluor methodology to determine if the effect of arginine on the thermal stability of NAGS parallels its effects on NAGS activity. Addition of arginine to bacterial NAGS, which is inhibited by arginine, resulted in a destabilized protein. Addition of arginine to the zebrafish and mouse NAGS stabilized both proteins, despite opposing effects of arginine on their enzymatic activity. We then used analytical gel chromatography to determine if changes in oligomerization state of NAGS could occur upon arginine binding. Our results indicate that bacterial and mammalian NAGS appear to be ensembles of molecules with different oligomerization states that are in rapid exchange with each other. Upon addition of arginine, the partition coefficient of both NAGS increased. The behavior of zebrafish NAGS was different. It eluted as two peaks suggesting two distinct oligomerization states. Upon addition of arginine to zebrafish NAGS the partition coefficients of both peaks decreased. These studies indicate that the effect of arginine on the biophysical properties of NAGS indeed changed during evolution and suggest that the inversion of the allosteric effect and stabilization effects of arginine on NAGS could be linked.

The Effect of ArgR–DNA Binding Affinity on Ornithine Production in Corynebacterium glutamicum

pEMBTL-SY1, which can over produce the ArgR protein in Corynebacterium glutamicum, was constructed. The DNA-binding affinity of ArgR was analyzed using a Chromatin Immunoprecipitation (ChIP) assay. The level of ArgR protein expression in the plasmid-carrying C. glutamicum (pEMBTL-SY1) was higher than that in the wild-type strain. On the other hand, there was no increase in the DNA-binding affinity of ArgR on the upstream of argB and the level of ornithine production. The DNA-binding affinity of ArgR on the arg operon and the level of ornithine production in the presence of three metabolites, ornithine, arginine, and proline, were examined as feedback controlling effectors in the arginine biosynthesis pathway in C. glutamicum. The ChIP assay showed that the supplemented metabolites altered the ArgR-binding affinity on the upstream of argB, which is consistent with the change in ornithine production. This suggests that the regulation of ornithine biosynthesis by the transcriptional regulator, ArgR, depends on the DNA-binding affinity of the arg operon, which is regulated by the feedback controlling effectors, rather than on the level of ArgR protein expression.

The Complete Genome and Proteome of Laribacter hongkongensis Reveal Potential Mechanisms for Adaptations to Different Temperatures and Habitats

Laribacter hongkongensis is a newly discovered Gram-negative bacillus of the Neisseriaceae family associated with freshwater fish–borne gastroenteritis and traveler's diarrhea. The complete genome sequence of L. hongkongensis HLHK9, recovered from an immunocompetent patient with severe gastroenteritis, consists of a 3,169-kb chromosome with G+C content of 62.35%. Genome analysis reveals different mechanisms potentially important for its adaptation to diverse habitats of human and freshwater fish intestines and freshwater environments. The gene contents support its phenotypic properties and suggest that amino acids and fatty acids can be used as carbon sources. The extensive variety of transporters, including multidrug efflux and heavy metal transporters as well as genes involved in chemotaxis, may enable L. hongkongensis to survive in different environmental niches. Genes encoding urease, bile salts efflux pump, adhesin, catalase, superoxide dismutase, and other putative virulence factors—such as hemolysins, RTX toxins, patatin-like proteins, phospholipase A1, and collagenases—are present. Proteomes of L. hongkongensis HLHK9 cultured at 37°C (human body temperature) and 20°C (freshwater habitat temperature) showed differential gene expression, including two homologous copies of argB, argB-20, and argB-37, which encode two isoenzymes of N-acetyl-L-glutamate kinase (NAGK)—NAGK-20 and NAGK-37—in the arginine biosynthesis pathway. NAGK-20 showed higher expression at 20°C, whereas NAGK-37 showed higher expression at 37°C. NAGK-20 also had a lower optimal temperature for enzymatic activities and was inhibited by arginine probably as negative-feedback control. Similar duplicated copies of argB are also observed in bacteria from hot springs such as Thermus thermophilus, Deinococcus geothermalis, Deinococcus radiodurans, and Roseiflexus castenholzii, suggesting that similar mechanisms for temperature adaptation may be employed by other bacteria. Genome and proteome analysis of L. hongkongensis revealed novel mechanisms for adaptations to survival at different temperatures and habitats.

Genome‐scale model for Clostridium acetobutylicum: Part I. Metabolic network resolution and analysis

A genome-scale metabolic network reconstruction for Clostridium acetobutylicum (ATCC 824) was carried out using a new semi-automated reverse engineering algorithm. The network consists of 422 intracellular metabolites involved in 552 reactions and includes 80 membrane transport reactions. The metabolic network illustrates the reliance of clostridia on the urea cycle, intracellular L-glutamate solute pools, and the acetylornithine transaminase for amino acid biosynthesis from the 2-oxoglutarate precursor. The semi-automated reverse engineering algorithm identified discrepancies in reaction network databases that are major obstacles for fully automated network-building algorithms. The proposed semi-automated approach allowed for the conservation of unique clostridial metabolic pathways, such as an incomplete TCA cycle. A thermodynamic analysis was used to determine the physiological conditions under which proposed pathways (e.g., reverse partial TCA cycle and reverse arginine biosynthesis pathway) are feasible. The reconstructed metabolic network was used to create a genome-scale model that correctly characterized the butyrate kinase knock-out and the asolventogenic M5 pSOL1 megaplasmid degenerate strains. Systematic gene knock-out simulations were performed to identify a set of genes encoding clostridial enzymes essential for growth in silico.

A lysine accumulation phenotype of ScIpk2Δ mutant yeast is rescued by Solanum tuberosum inositol phosphate multikinase

Inositol phosphates and the enzymes that interconvert them are key regulators of diverse cellular processes including the transcriptional machinery of arginine synthesis [York (2006) Biochim. Biophys. Acta 1761, 552-559]. Despite considerable interest and debate surrounding the role of Saccharomyces cerevisiae inositol polyphosphate kinase (ScIPK2, ARG82, ARGRIII) and its inositol polyphosphate products in these processes, there is an absence of data describing how the transcripts of the arginine synthetic pathway, and the amino acid content of ScIpk2Delta, are altered under different nutrient regimes. We have cloned an IPMK (inositol phosphate multikinase) from Solanum tuberosum, StIPMK (GenBank(R) accession number EF362785), that despite considerable sequence divergence from ScIPK2, restores the arginine biosynthesis pathway transcripts ARG8, acetylornithine aminotransferase, and ARG3, ornithine carbamoyltransferase of ScIpk2Delta yeast to wild-type profiles. StIPMK also restores the amino acid profiles of mutant yeast to wild-type, and does so with ornithine or arginine as the sole nitrogen sources. Our data reveal a lysine accumulation phenotype in ScIpk2Delta yeast that is restored to a wild-type profile by expression of StIPMK, including restoration of the transcript profiles of lysine biosynthetic genes. The StIPMK protein shows only 18.6% identity with ScIPK2p which probably indicates that the rescue of transcript and diverse amino acid phenotypes is not mediated through a direct interaction of StIPMK with the ArgR-Mcm1 transcription factor complex that is a molecular partner of ScIPK2p.

The regulatory PII protein controls arginine biosynthesis in Arabidopsis

In higher plants, PII is a nuclear-encoded plastid protein which is homologous to bacterial PII signalling proteins known to be involved in the regulation of nitrogen metabolism. A reduced ornithine, citrulline and arginine accumulation was observed in two Arabidopsis PII knock-out mutants in response to NH 4 + resupply after N starvation. This difference could be explained by the regulation of a key enzyme of the arginine biosynthesis pathway, N-acetyl glutamate kinase (NAGK) by PII. In vitro assays using purified recombinant proteins showed the catalytic activation of Arabidopsis NAGK by PII giving the first evidence of a physiological role of the PII protein in higher plants. Using Arabidopsis transcriptome microarray (CATMA) and RT-PCR analyses, it was found that none of the genes involved in the arginine biosynthetic or catabolic pathways were differentially expressed in a PII knock-out mutant background. In conclusion, the observed changes in metabolite levels can be explained by the reduced activation of NAGK by PII.

Two arginine repressors regulate arginine biosynthesis in Lactobacillus plantarum.

The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine. This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway. Fourteen mutants were genetically characterized for constitutive arginine production. Mutations were located either in one of the arginine repressor genes (argR1 or argR2) present in L. plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involved in arginine biosynthesis. Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis. In L. plantarum, arginine repression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1. A123, equivalent to the conserved residue A124 in E. coli ArgR involved in arginine binding, was different in the wild-type ArgR2. Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which have only 35% identical residues. Other mutants harbored wild-type argR genes, and 20 mutants have lost their ability to grow in normal air without carbon dioxide enrichment; this revealed a link between arginine biosynthesis and a still-unknown CO2-dependent metabolic pathway. In many gram-positive bacteria, the expression and interaction of different ArgR-like proteins may imply a complex regulatory network in response to environmental stimuli.

Tsc1+ and tsc2+ Regulate Arginine Uptake and Metabolism in Schizosaccharomyces pombe

Mutations in either TSC1 or TSC2 cause tuberous sclerosis complex, an autosomal dominant disorder characterized by seizures, mental retardation, and benign tumors of the skin, brain, heart, and kidneys. Homologs for the TSC1 and TSC2 genes have been identified in mouse, rat, Fugu, Drosophila, and in the yeast Schizosaccharomyces pombe. Here we show that S. pombe lacking tsc1+ or tsc2+ have similar phenotypes including decreased arginine uptake, decreased expression of three amino acid permeases, and low intracellular levels of four members of the arginine biosynthesis pathway. Recently, the small GTPase Rheb was identified as a target of the GTPase-activating domain of tuberin in mammalian cells and in Drosophila. We show that the defect in arginine uptake in cells lacking tsc2+ is rescued by the expression of a dominant negative form of rhb1+, the Rheb homolog in S. pombe, but not by expressing wild-type rhb1+. Expression of the tsc2+ gene with a patient-derived mutation within the GAP domain did not rescue the arginine uptake defect in tsc2+ mutant yeast. Taken together, these findings support a model in which arginine uptake is regulated through tsc1+, tsc2+, and rhb1+ in S. pombe and also suggest a role for the Tsc1 and Tsc2 proteins in amino acid biosynthesis and sensing.

ArgR and AhrC are both required for regulation of arginine metabolism in Lactococcus lactis.

The DNA binding proteins ArgR and AhrC are essential for regulation of arginine metabolism in Escherichia coli and Bacillus subtilis, respectively. A unique property of these regulators is that they form hexameric protein complexes, mediating repression of arginine biosynthetic pathways as well as activation of arginine catabolic pathways. The gltS-argE operon of Lactococcus lactis encodes a putative glutamate or arginine transport protein and acetylornithine deacetylase, which catalyzes an important step in the arginine biosynthesis pathway. By random integration knockout screening we found that derepression mutants had ISS1 integrations in, among others, argR and ahrC. Single as well as double regulator deletion mutants were constructed from Lactococcus lactis subsp. cremoris MG1363. The three arginine biosynthetic operons argCJDBF, argGH, and gltS-argE were shown to be repressed by the products of argR and ahrC. Furthermore, the arginine catabolic arcABD1C1C2TD2 operon was activated by the product of ahrC but not by that of argR. Expression from the promoter of the argCJDBF operon reached similar levels in the single mutants and in the double mutant, suggesting that the regulators are interdependent and not able to complement each other. At the same time they also appear to have different functions, as only AhrC is involved in activation of arginine catabolism. This is the first study where two homologous arginine regulators are shown to be involved in arginine regulation in a prokaryote, representing an unusual mechanism of regulation.

The N-acetylglutamate synthase/N-acetylglutamate kinase metabolon of Saccharomyces cerevisiae allows co-ordinated feedback regulation of the first two steps in arginine biosynthesis.

In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869-42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N-acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.

Towards structural understanding of feedback control of arginine biosynthesis: cloning and expression of the gene for the arginine-inhibited N-acetyl-L-glutamate kinase from Pseudomonas aeruginosa, purification and crystallization of the recombinant enzyme and preliminary X-ray studies.

N-Acetyl-L-glutamate kinase (NAGK) catalyzes the second step in the pathway of arginine biosynthesis in microorganisms and plants. In many species, it is the pathway-controlling enzyme and is subject to feedback inhibition by arginine. The gene for the best characterized arginine-inhibitable NAGK, that from Pseudomonas aeruginosa, has been cloned in a pET22 plasmid and overexpressed in Escherichia coli. The enzyme was purified in three steps to 95% purity and was shown by cross-linking to form dimers. It was crystallized by the hanging-drop vapour-diffusion method at 277 K in the presence of ADP, Mg and N-acetyl-L-glutamate. The crystallization solution contained 0.1 M sodium cacodylate pH 6.5, 150-170 mM magnesium acetate and 13% polyethylene glycol 8000. Prismatic crystals of maximum dimension approximately 0.5 mm diffract to 2.75 A resolution and belong to space group P1 (unit-cell parameters a = 71.86, b = 98.78, c = 162.9 A, alpha = 91.49, beta = 92.03, gamma = 107.56 degrees ). Packing density considerations agree with 6-18 NAGK monomers in the asymmetric unit, with a corresponding solvent content of 79-36%. Self-rotation function calculations confirm the space group and suggest the presence of 3-7 dimers in the unit cell.

A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.

Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.

In vivo Mutational Analysis of Highly Conserved Amino Acid Residues of the Small Subunit Cpa1p of the Carbamylphosphate Synthetase ofSaccharomyces cerevisiae

The role of selected amino acid residues located in the putative catalytic domain and of two conserved histidine residues within the small subunit of the carbamylphosphate synthetase (CPS) specific to the arginine biosynthesis pathway of the yeast Saccharomyces cerevisiae was studied using site-directed mutagenesis to change all residues to aspartic acid. Carbamylphosphate synthesis catalysed by modified CPS was tested in vivo. The C264D, H307D and H349D mutants were unable to grow on minimal medium, indicating the importance of these three residues for efficient CPS activity, whereas, four other mutated residues located in the catalytic site (including a proline residue) do not affect the growth rate. These results in comparison to those obtained with the CPS of Escherichia coli, implicate residues Cys 264 and His 349 in the glutaminase catalytic activity, and His 307 in the binding of glutamine to the active site. Using these three defective mutants, we investigated the in vivo utilization of ammonia by CPS. C264D and H307D mutants are able to use ammonia as a substrate when provided in sufficiently high concentrations (up to 200 mM). The H349D mutant, however, did not grow even at ammonium sulfate concentrations above 400 mM, suggesting that this substitution is critical to NH3-dependent CPS activity although the ammonia binding site is presumably located within the large subunit of the enzyme.

Pathways of arginine biosynthesis in extreme thermophilic archaeo- and eubacteria

The pathway of arginine biosynthesis was investigated in two thermophilic eubacteria, Thermus aquaticus and Thermotoga maritima, and in two thermophilic archaeobacteria, Sulfolobus solfataricus and Pyrococcus furiosus. In the first three organisms, arginine biosynthesis proceeds via N-acetylated intermediates as in mesophilic microorganisms. Only the enzymes catalysing the three last steps of the pathway could be detected in P. furiosus. The two eubacterial strains possess an ornithine acetyltransferase and are thus able to recycle the acetyl group from acetylornithine to glutamate. The archaeobacterium, S. solfataricus, uses the linear pathway in which the formation of ornithine is mediated by the hydrolytic enzyme acetylornithinase. Repression of enzyme synthesis by arginine was observed for most of the enzymes tested in T. aquaticus and S. solfataricus. Feedback inhibition by arginine was shown only on the ornithine acetyltransferase from T. aquaticus. This inhibition pattern is of interest since it would be the first example of control of arginine biosynthesis at this particular step. Data concerning the thermal stability of the arginine biosynthetic enzymes are presented.

Arginine in Growing Cells of Sugarcane. Nutritional Effects, Uptake and Incorporation into Proteins

AbstractThe growth of sugarcane cell cultures derived from parenchyma tissues of three different varieties of sugarcane showed a marked dependence on the presence of arginine when the cells were cultured in a synthetic liquid medium containing a mixture of amino acids. The cells appeared lo have access to n normal biosynthetic pathway of arginine biosynthesis but took up exogenously supplied arginine rapidly and efficiently. Arginine was not limiting for protein formation, nor were proteins with AN unusually high number of arginine residues evident. The low level of arginine effective in bringing about the growth response is suggestive of a regulatory function for this amino acid in sugarcane cells.

Effect of the α-Hydrazino Analogue of Histidine on Histidine Transport and Arginine Biosynthesis

Abstract The α-hydrazino analogue of histidine was found to be an effective inhibitor of the growth of Salmonella typhimurium wild type as well as Escherichia coli W and E. coli K-12. A mutant, hisP1650, which is resistant to the inhibitory action of the compound was isolated and found to be defective in the histidine-specific transport system. The hydrazino analogue reacts rapidly with pyridoxal phosphate in a nonenzymatic reaction to give a product which does not have the spectral properties expected of a hydrazone. The analogue inactivates several pyridoxal phosphate enzymes. Evidence is presented suggesting that the primary point of inhibition in the cell is the pyridoxal phosphate moiety of acetylornithine transaminase in the pathway of arginine biosynthesis.

A biosynthetic ornithine decarboxylase in [formula omitted

Putrescine (1,4-diaminobutane) is found in E., coli , under a variety of growth conditions, at a level of approximately 15 micromoles per gram of wet weight. In spite of this relatively high intracellular concentration, very little is known about its biosynthesis. Tabor, Rosenthal and Tabor (1958) demonstrated the incorporation of 14C-ornithine into putrescine in intact E., coli , indicating that one of the late intermediates of the pathway of arginine biosynthesis is a precursor of putrescine. Gale (1940) demonstrated an induced ornithine decarboxylase in E., coli which catalyzed the formation of putrescine. However, since highly specialized culture conditions are required for the formation of this enzyme, it cannot be responsible for the large amounts of putrescine which are synthesized during growth in minimal medium. This induced ornithine decarboxylase probably serves a catabolic function. In this paper we report the presence of an ornithine decarboxylase in E., coli which appears to function in a biosynthetic rather than a catabolic capacity. This biosynthetic enzyme is distinct from the induced decarboxylase described by Gale, differing in its pH optimum, heat stability and conditions for formation.

Pathway of arginine biosynthesis in yeast

Pathway of arginine biosynthesis in yeast