Cloning and characterization of argR, a gene that participates in regulation of arginine biosynthesis and catabolism in Pseudomonas aeruginosa PAO1

Gel retardation experiments indicated the presence in Pseudomonas aeruginosa cell extracts of an arginine-inducible DNA-binding protein that interacts with the control regions for the car and argF operons, encoding carbamoylphosphate synthetase and anabolic ornithine carbamoyltransferase, respectively. Both enzymes are required for arginine biosynthesis. The use of a combination of transposon mutagenesis and arginine hydroxamate selection led to the isolation of a regulatory mutant that was impaired in the formation of the DNA-binding protein and in which the expression of an argF::lacZ fusion was not controlled by arginine. Experiments with various subclones led to the conclusion that the insertion affected the expression of an arginine regulatory gene, argR, that encodes a polypeptide with significant homology to the AraC/XylS family of regulatory proteins. Determination of the nucleotide sequence of the flanking regions showed that argR is the sixth and terminal gene of an operon for transport of arginine. The argR gene was inactivated by gene replacement, using a gentamicin cassette. Inactivation of argR abolished arginine control of the biosynthetic enzymes encoded by the car and argF operons. Furthermore, argR inactivation abolished the induction of several enzymes of the arginine succinyltransferase pathway, which is considered the major route for arginine catabolism under aerobic conditions. Consistent with this finding and unlike the parent strain, the argR::Gm derivative was unable to utilize arginine or ornithine as the sole carbon source. The combined data indicate a major role for ArgR in the control of arginine biosynthesis and aerobic catabolism.     

Phosphatase-mediated enhancement of cardiac cAMP-activated Cl-conductance by a Cl- channel blocker, anthracene-9-carboxylate

The argR gene of Streptomyces clavuligerus has been located in the upstream region of argG. It encodes a protein of 160 amino acids with a deduced M(r) of 17117 for the monomer. Transformants containing the amplified argR gene showed lower activity (50%) of the biosynthetic ornithine carbamoyltransferase (OTC) activity and higher levels (380%) of the catabolic ornithine aminotransferase (OAT) activity than control strains. Amplification of an arginine (ARG) box-containing sequence results in a 2- to 2.5-fold derepression of ornithine acetyltransferase and OTC, suggesting that the repressor is titrated out. Footprinting experiments using the pure homologous arginine repressor (AhrC) of B. subtilis showed a protected 38 nt region (ARG box) in the coding strand upstream of argC. The protected region contained two tandemly repeated imperfect palindromic 18-nt ARG boxes. The repressor-operator interaction was confirmed by bandshift experiments of the DNA fragment containing the protected region. By computer analysis of the Streptomyces sequences available in the databases, a consensus ARG box has been deduced for the genus Streptomyces. This is the first example of a clear regulation of an amino acid biosynthetic pathway in Streptomyces species, challenging the belief that actinomycetes do not have a well-developed regulatory system of these pathways.     

Use of inducible feedback-resistant N-acetylglutamate synthetase (argA) genes for enhanced arginine biosynthesis by genetically engineered Escherichia coli K-12 strains

The goal of this work was to construct Escherichia coli strains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. coli strains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33-38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain "arg boxes," which titrate the ArgR repressor protein, with or without the E. coli carAB genes encoding carbamyl phosphate synthetase and the argI gene for ornithine transcarbamylase. The free arginine production rates of "arg-derepressed" E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt) argA. The expression system with fbr argA produced 7- to 35-fold more arginine than a system overexpressing carAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5 alpha strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carAB and argI genes. Plasmids containing wt or fbr argA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.     

Arginine catabolism and the arginine succinyltransferase pathway in Escherichia coli

Arginine catabolism produces ammonia without transferring nitrogen to another compound, yet the only known pathway of arginine catabolism in Escherichia coli (through arginine decarboxylase) does not produce ammonia. Our aims were to find the ammonia-producing pathway of arginine catabolism in E. coli and to examine its function. We showed that the only previously described pathway of arginine catabolism, which does not produce ammonia, accounted for only 3% of the arginine consumed. A search for another arginine catabolic pathway led to discovery of the ammonia-producing arginine succinyltransferase (AST) pathway in E. coli. Nitrogen limitation induced this pathway in both E. coli and Klebsiella aerogenes, but the mechanisms of activation clearly differed in these two organisms. We identified the E. coli gene for succinylornithine aminotransferase, the third enzyme of the AST pathway, which appears to be the first of an astCADBE operon. Its disruption prevented arginine catabolism, impaired ornithine utilization, and affected the synthesis of all the enzymes of the AST pathway. Disruption of astB eliminated succinylarginine dihydrolase activity and prevented arginine utilization but did not impair ornithine catabolism. Overproduction of AST enzymes resulted in faster growth with arginine and aspartate. We conclude that the AST pathway is necessary for aerobic arginine catabolism in E. coli and that at least one enzyme of this pathway contributes to ornithine catabolism.     

Enzymes related to catecholamine biosynthesis in Tetrahymena pyriformis. Presence of GTP cyclohydrolase I

Arginine biosynthesis in Escherichia coli is negatively regulated by the hexameric repressor protein ArgR and the corepressor L-arginine. L-Arginine binds to ArgR in the C-terminal domain of the repressor. Binding to operator DNA occurs in the N-terminal domain. The molecular structures of both domains have recently been elucidated. The known stereochemistry of the arginine binding pocket was used for the rational design of a mutant ArgR with altered ligand specificity. Our prediction was that a replacement of Asp128 by asparagine would preferentially lead to the binding of L-citrulline, rather than L-arginine. The D128N mutant was constructed and was shown to fulfill our expectation by several experimental approaches. By isothermal titration calorimetry it was found to bind L-citrulline much more strongly than L-arginine, in contrast to wild-type ArgR. Exchange between the mutant trimers of the hexamer was inhibited by L-citrulline, as it is by L-arginine in the wild-type. The mutant protein was precipitated by L-citrulline but not by L-arginine, whereas the reverse is true for the wild-type protein. Demonstration of a corepressor action was, however, precluded by the superrepressor effect of the D128N mutation by itself. The mutant protein, in the absence of L-citrulline or L-arginine is as strong a repressor as the wild-type protein in the presence of L-arginine. We discuss two possible mechanisms, in terms of the known domain structures that could explain our observations.     

Endogenous synthesis of arginine plays an important role in maintaining arginine homeostasis in postweaning growing pigs

This study was conducted to determine whether endogenous synthesis of arginine plays a role in regulating arginine homeostasis in postweaning pigs. Pigs were fed a sorghum-based diet containing 0. 98% arginine and were used for studies at 75 d of age (28.4 kg body weight). Mitochondria were prepared from the jejunum and other major tissues for measuring the activities of Delta1-pyrroline-5-carboxylate (P5C) synthase and proline oxidase (enzymes catalyzing P5C synthesis from glutamate and proline, respectively) and of ornithine aminotransferase (OAT) (the enzyme catalyzing the interconversion of P5C into ornithine). For metabolic studies, jejunal enterocytes were incubated at 37 degrees C for 30 min in Krebs-Henseleit bicarbonate buffer containing 2 mmol/L L-glutamine, 2 mmol/L L-[U-14C]proline, and 0-200 micromol/L gabaculine (an inhibitor of OAT). The activities of P5C synthase, proline oxidase and OAT were greatest in enterocytes among all of the tissues studied. Incubation of enterocytes with gabaculine resulted in decreases (P < 0.05) in the synthesis of ornithine and citrulline from glutamine and proline. When gabaculine was orally administered to pigs (0.83 mg/kg body weight) to inhibit intestinal synthesis of citrulline from glutamine and proline, plasma concentrations of citrulline (-26%) and arginine (-22%) decreased (P < 0.05), whereas those of alanine (+21%), ornithine (+17%), proline (+107%), taurine (+56%) and branched-chain amino acids (+21-40%) increased (P < 0.05). On the basis of dietary arginine intake and estimated arginine utilization, the endogenous synthesis of arginine in the 28-kg pig provided >/=50.2% of total daily arginine requirement. Taken together, our results suggest an important role for endogenous synthesis of arginine in regulating arginine homeostasis in postweaning growing pigs, as previously shown in neonatal pigs.     

Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.     

Supplemental arginine and ornithine do not affect splenocyte proliferation in surgically treated rats

The present study was designed to determine whether arginine or ornithine supplementation enhanced immune responsiveness in surgically stressed rats. Young rats (130 to 150 g; n = 72) were fed one of three nonpurified diets: control, arginine-supplemented (30 g/kg of diet), or supplemented with ornithine on an equimolar basis to supplemental arginine. Control and ornithine-supplemented diets were made isonitrogenous to the arginine-supplemented diet with alanine. Food intake and body weight were monitored throughout the experimental period. Eight days after initiation of dietary treatments, 36 rats were given dorsal skin wounds. Rats were killed 7 days later. Blood was collected, spleen and thymus were weighed, and splenocytes were isolated to measure proliferation in response to mitogens and interleukin-2 production. Food intake, body weight gain, and thymus weight were lower in rats subjected to surgery than in controls rats (p < .01). Neither supplemental dietary arginine nor ornithine affected food intake, body weight gain, thymus weight, splenocyte proliferation, or splenocyte interleukin-2 production in any treatment group (p < .1). These data suggest that low-level dietary supplementation of arginine and ornithine did not ameliorate detrimental effects of minor surgery in rats.     

Ornithine carbamoyltransferase from the extreme thermophile Thermus thermophilus--analysis of the gene and characterisation of the protein

The ornithine carbamoyltransferase (OTC) gene from Thermus thermophilus was cloned from a lambda-ZAP genomic library. An ORF of 903 bp was found coding for a protein of Mr 33,200. The coding region has a very high overall G+C content of 68.0%. T. thermophilus OTC displays 38-48% amino acid identity with other OTC, the most closely related proteins being OTC from the archaeon Pyrococcus furiosus and from Bacillus subtilis. The enzyme was expressed in Escherichia coli and purified to homogeneity using a thermoshock followed by affinity chromatography on delta-N-phosphonoacetyl-L-ornithine-Sepharose. The native enzyme has an Mr of about 110,000, suggesting a trimeric structure, as for most anabolic OTC from various organisms. T. thermophilus OTC exhibits Michaelis-Menten kinetics for carbamoyl phosphate and ornithine with a Km(app) of 0.10 mM for both substrates. The pH optimum was dependent on ornithine concentration with an optimum at pH 8 for ornithine concentrations around Km values. Higher concentrations shift the optimum towards lower pH. The optimal temperature was above 65 degrees C and the activation energy 39.1 kJ/mol. The enzyme is highly thermostable. In the presence of its substrates the half-life time was several hours at 85 degrees C. Ionic and hydrophobic interactions contribute to the stability. The expression of T. thermophilus OTC was negatively regulated by arginine.     

Molecular physiology of carbamoylation under extreme conditions: what can we learn from extreme thermophilic microorganisms?

The importance of protein-protein interactions in the physiology of extreme thermophiles was investigated by analyzing the enzymes involved in biosynthetic carbamoylation in Thermus ZO5 and by comparing the results obtained with already available or as yet unpublished information concerning other thermophilic eu- and archaebacteria such as Thermotoga, Sulfolobus, and Pyrococcus. Salient observations were that (i) the highly thermolabile and reactive carbamoylphosphate molecule appears to be protected from thermodegradation by channelling towards the synthesis of citrulline and carbamoylaspartate, respectively precursors of arginine and the pyrimidines; (ii) Thermus ornithine carbamoyltransferase is clearly a thermophilic enzyme, intrinsically thermostable and showing a biphasic Arrhenius plot, whereas aspartate carbamoyltransferase is inherently unstable and is stabilized by its association with dihydroorotase, another enzyme encoded by the Thermus pyrimidine operon. Possible implications of these results are discussed.     

The ARG11 gene of Saccharomyces cerevisiae encodes a mitochondrial integral membrane protein required for arginine biosynthesis

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11. We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein. A deletion created in ARG11 causes the same arginine-leaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.     

Identification of the yeast ARG-11 gene as a mitochondrial ornithine carrier involved in arginine biosynthesis

The ARG-11 gene in Saccharomyces cerevisiae encodes a protein with the characteristic features of a family of 35 related membrane proteins that are encoded in the fungal genome. Some of them are known to transport various substrates and products across the inner membranes of mitochondria, but the functions of 29 members of the family are unknown. The yeast ARG-11 protein has been over-produced as inclusion bodies in Escherichia coli. It has been solubilized in the presence of sarkosyl, re-constituted into liposomes and shown to transport ornithine in exchange for protons. Its main physiological role is probably to take ornithine synthesized from glutamate in the mitochondrial matrix to the cytosol where it is converted to arginine.     

Unusual abundance of arginine and ornithine in porcine allantoic fluid

The concentrations of free arginine, ornithine, and glutamine in porcine allantoic and amniotic fluids were determined on Days 30,35, 40, and 45 of gestation. Arginine and ornithine were the most abundant amino acids in allantoic fluid on Days 35-40 and 45 of gestation, respectively. Arginine and ornithine nitrogen accounted for 40%, 50%, and 55% of the total free alpha-amino acid nitrogen in allantoic fluid on Days 35, 40, and 45 of gestation, respectively. Glutamine was the most abundant amino acid in amniotic fluid during early gestation and was also abundant in allantoic fluid. On Day 45 of gestation, glutamine nitrogen accounted for 41% of the total free alpha-amino acid nitrogen in amniotic fluid. The unusual abundance of arginine (2.5-4.1 mM) and ornithine (1.08-2.52 mM) in allantoic fluid on Days 35-40 of gestation has not been reported for any other biological fluid. These results are novel and interesting with respect to the role of these two basic amino acids in fetal-placental nutrition and metabolism.     

Use of Nessler''s reagent for recognition of lysine, ornithine, and arginine decomposition by gramnegative fermentative bacteria (author''s transl)

The reactions of lysine, ornithine and arginine decomposition are often difficult to read in Falkow's medium because either the decolorization of the indicator or the lack of sharp colour differences between positive and negative reactions. In such cases Nessler's reagent may be a useful aid. A volume of about 0.2 ml is added to the cultures after 4 days incubation through the mineral oil layer by means of a pipette. A positive reaction is indicated by an immediate white precipitation in case of lysine and ornithine decarboxylation, and a white or brownish precipitate which indicates arginine decomposition. A delayed opacity should be regarded as a negative reaction. Only unequivocal reactions should be considered. The specificity of the reactions was tested with pure substances of compounds which are formed by the decomposition of lysine, ornithine and arginine. Further studies of bacterial cultures in Falkow's medium and in a synthetic, amino acid containing medium without peptone gave identical results and showed that peptone derivates do not cause a false positive reaction with Nessler's reagent (Table 1). Comparative studies on 605 strains of Enterobacteriaceae and Vibrio in Falkow's medium with and without added Nessler's reagent gave corresponding results except some strains of Escherichia coli and Citrobacter freundii with different arginine reactions (Table 2). Strains of these species mostly decolorized the indicator thereby hindering the recognition of either a true positive or a true negative reaction. In these cases, however, the results obtained after addition of Nessler's reagent corresponded closely to the percentage of positive reactions cited in the literature.     

Phosphorylation of the periplasmic binding protein in two transport systems for arginine incorporation in Escherichia coli K-12 is unrelated to the function of the transport system

In Escherichia coli K-12, the accumulation of arginine is mediated by two distinct periplasmic binding protein-dependent transport systems, one common to arginine and ornithine (AO system) and one for lysine, arginine, and ornithine (LAO system). Each of these systems includes a specific periplasmic binding protein, the AO-binding protein for the AO system and the LAO-binding protein for the LAO system. The two systems include a common inner membrane transport protein which is able to hydrolyze ATP and also phosphorylate the two periplasmic binding proteins. Previously, a mutant resistant to the toxic effects of canavanine, with low levels of transport activities and reduced levels of phosphorylation of the two periplasmic binding proteins, was isolated and characterized (R. T. F. Celis, J. Biol. Chem. 265:1787-1793, 1990). The gene encoding the transport ATPase enzyme (argK) has been cloned and sequenced. The gene possesses an open reading frame with the capacity to encode 268 amino acids (mass of 29.370 Da). The amino acid sequence of the protein includes two short sequence motifs which constitute a well-defined nucleotide-binding fold (Walker sequences A and B) present in the ATP-binding subunits of many transporters. We report here the isolation of canavanine-sensitive derivatives of the previously characterized mutant. We describe the properties of these suppressor mutations in which the transport of arginine, ornithine, and lysine has been restored. In these mutants, the phosphorylation of the AO- and LAO-binding proteins remains at a low level. This information indicates that whereas hydrolysis of ATP by the transport ATPase is an obligatory requirement for the accumulation of these amino acids in E. coli K-12, the phosphorylation of the periplasmic binding protein is not related to the function of the transport system.     

Pediatric antifungal therapy

We have recently reported the synthesis of urea from ammonia, glutamine and arginine in enterocytes of postweaning pigs. The present study was conducted to determine the compartmentation and kinetics of urea cycle enzymes in these cells. Carbamoyl phosphate synthase I (CPS I) and ornithine carbamoyltransferase (OCT) were located exclusively in mitochondria, whereas argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were found in the cytosol. Arginase isozymes were present in both the cytosol and mitochondria of enterocytes, and differed in their sensitivity to heat inactivation. Except for OCT, Vmax values of urea cycle enzymes were much lower in enterocytes than in the liver of pigs, and vice versa for their Km values. Because of a low rate of ureagenesis in enterocytes compared with the liver, intestinal urea cycle enzymes may function primarily to synthesize citrulline. The co-localization of CPS I and OCT and a high activity of OCT in enterocyte mitochondria favors the intestinal synthesis of citrulline from ammonia, HCO3- and ornithine. Low activities of cytosolic ASS and ASL minimize the conversion of citrulline into arginine and therefore, the recycling of citrulline into ornithine via arginase in postweaning-pig enterocytes. These kinetic properties of intestinal urea cycle enzymes maximize the net synthesis of citrulline from glutamine and explain the release of large amounts of citrulline by the pig small intestine. The two compartmentally separated arginase isozymes in enterocytes may play an important role in regulating the intestinal metabolism of proline, nitric oxide and polyamines.     

The active-site arginine of S-adenosylmethionine synthetase orients the reaction intermediate

S-Adenosylmethionine (AdoMet) synthetase catalyzes the formation of AdoMet and tripolyphosphate (PPPi) from ATP and L-methionine and the subsequent hydrolysis of the PPPi to PPi and Pi before product release. Little is known about the roles of active-site residues involved in catalysis of the two sequential reactions that occur at opposite ends of the polyphosphate chain. Crystallographic studies of Escherichia coli AdoMet synthetase showed that arginine-244 is the only arginine near the polyphosphate-binding site. Arginine-244 is embedded as the seventh residue in the conserved sequence DxGxTxxKxI which is also found at the active site of inorganic pyrophosphatases, suggesting a potential pyrophosphate-binding motif. Chemical modification of AdoMet synthetase by the arginine-specific reagents phenylglyoxal or p-hydroxyphenylglyoxal inactivates the enzyme. ATP and PPPi protect the enzyme from inactivation, consistent with the presence of an important arginine residue in the vicinity of the polyphosphate-binding site. Site-specific mutagenesis has been used to change the conserved arginine-244 to either leucine (R244L) or histidine (R244H). In the overall reaction, the R244L mutant has the kcat reduced approximately 10(3)-fold, with a 7 to 10-fold increase in substrate Km values; the R244H mutant has an approximately 10(5)-fold decrease in kcat. In contrast, the kcat values for hydrolysis of added PPPi by the R244L and R244H mutants have been reduced by less than 2 orders of magnitude. In contrast to the wild-type enzyme in which 98% of the Pi formed originates as the gamma-phosphoryl group of ATP, in the R244L mutant the orientation of the PPPi intermediate equilibrates at the active site yielding equal amounts of Pi from the alpha- and gamma-phosphoryl groups of ATP. Thus, the active-site arginine has a profound role in the cleavage of PPPi from ATP during AdoMet formation and in maintaining the orientation of PPPi in the active site, while playing a lesser role in the subsequent PPPi hydrolytic reaction.