Arginine deiminase from Halobacterium salinarium. Purification and properties.
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ABSTRACT:
SUBMITTER: Monstadt GM
PROVIDER: S-EPMC1149825 | biostudies-other | 1991 Feb
REPOSITORIES: biostudies-other
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Project description:Fermentative growth via the arginine deiminase pathway is mediated by the enzymes arginine deiminase, carbamate kinase, and catabolic ornithine transcarbamylase and by a membrane-bound arginine-ornithine antiporter. Recently we reported the characterization of catabolic ornithine transcarbamylase and the corresponding gene, arcB, from Halobacterium salinarium (formerly Halobacterium halobium). Upstream of the arcB gene, three additional open reading frames with halobacterial codon usage were found. They were identified as the arcC gene coding for carbamate kinase, the arcA gene coding for arginine deiminase, and a gene, tentatively termed arcR, coding for a putative regulatory protein. The identification of the arcC and arcA genes was verified, respectively, by heterologous expression of the enzyme in Haloferax volcanii and by protein isolation and N-terminal sequence determination of three peptides. The gene order arcRACB differs from the gene order arcDABC in Pseudomonas aeruginosa, the only other organism for which sequence information is available. Transcripts from H. salinarium cultures grown fermentatively or aerobically were characterized by Northern (RNA) blot and primer extension analyses. It was determined (i) that monocistronic transcripts corresponding to the four open reading frames exist and that there are three polycistronic transcripts, (ii) that the level of induction during fermentative growth differs for the various transcripts, and (iii) that upstream of the putative transcriptional start sites for the three structural genes there are sequences with similarities to the halobacterial consensus promoter. The data indicate that expression of the arc gene cluster and its regulation differ in H. salinarium and P. aeruginosa.
| S-EPMC178278 | biostudies-other
Project description:Halobacterium halobium (salinarium) is able to grow fermentatively via the arginine deiminase pathway, which is mediated by three enzymes and one membrane-bound arginine-ornithine antiporter. One of the enzymes, catabolic ornithine transcarbamylase (cOTCase), was purified from fermentatively grown cultures by gel filtration and ammonium sulfate-mediated hydrophobic chromatography. It consists of a single type of subunit with an apparent molecular mass of 41 kDa. As is common for proteins of halophilic Archaea, the cOTCase is unstable below 1 M salt. In contrast to the cOTCase from Pseudomonas aeruginosa, the halophilic enzyme exhibits Michaelis-Menten kinetics with both carbamylphosphate and ornithine as substrates with Km values of 0.4 and 8 mM, respectively. The N-terminal sequences of the protein and four peptides were determined, comprising about 30% of the polypeptide. The sequence information was used to clone and sequence the corresponding gene, argB. It codes for a polypeptide of 295 amino acids with a calculated molecular mass of 32 kDa and an amino acid composition which is typical of halophilic proteins. The native molecular mass was determined to be 200 kDa, and therefore the cOTCase is a hexamer of identical subunits. The deduced protein sequence was compared to the cOTCase of P. aeruginosa and 14 anabolic OTCases, and a phylogenetic tree was constructed. The halobacterial cOTCase is more distantly related to the cOTCase than to the anabolic OTCase of P. aeruginosa. It is found in a group with the anabolic OTCases of Bacillus subtilis, P. aeruginosa, and Mycobacterium bovis.
| S-EPMC176715 | biostudies-other
Project description:The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.
| S-EPMC134416 | biostudies-literature
Project description:The oral cavity is host to a complex microbial community whose maintenance depends on an array of cell-to-cell interactions and communication networks, with little known regarding the nature of the signals or mechanisms by which they are sensed and transmitted. Determining the signals that control attachment, biofilm development and outgrowth of oral pathogens is fundamental to understanding pathogenic biofilm development. We have previously identified a secreted arginine deiminase (ADI) produced by Streptococcus intermedius that inhibited biofilm development of the commensal pathogen Porphyromonas gingivalis through downregulation of genes encoding the major (fimA) and minor (mfa1) fimbriae, both of which are required for proper biofilm development. Here we report that this inhibitory effect is dependent on enzymic activity. We have successfully cloned, expressed and defined the conditions to ensure that ADI from S. intermedius is enzymically active. Along with the cloning of the wild-type allele, we have created a catalytic mutant (ADIC399S), in which the resulting protein is not able to catalyse the hydrolysis of l-arginine to l-citrulline. P. gingivalis is insensitive to the ADIC399S catalytic mutant, demonstrating that enzymic activity is required for the effects of ADI on biofilm formation. Biofilm formation is absent under l-arginine-deplete conditions, and can be recovered by the addition of the amino acid. Taken together, the results indicate that arginine is an important signal that directs biofilm formation by this anaerobe. Based on our findings, we postulate that ADI functions to reduce arginine levels and, by a yet to be identified mechanism, signals P. gingivalis to alter biofilm development. ADI release from the streptococcal cell and its cross-genera effects are important findings in understanding the nature of inter-bacterial signalling and biofilm-mediated diseases of the oral cavity.
| S-EPMC3709564 | biostudies-literature
Project description:Protein arginine deimination leading to the non-coded amino acid citrulline remains a key question in the field of post-translational modifications ever since its discovery by Rogers and Simmonds in 1958. Citrullination is catalyzed by a family of enzymes called peptidyl arginine deiminases (PADIs). Initially, increased citrullination was associated with autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, as well as other neurological disorders and multiple types of cancer. During the last decade, research efforts have focused on how citrullination contributes to disease pathogenesis by modulating epigenetic events, pluripotency, immunity and transcriptional regulation. However, our knowledge regarding the functional implications of citrullination remains quite limited, so we still do not completely understand its role in physiological and pathological conditions. Here, we review the recently discovered functions of PADI2-mediated citrullination of the C-terminal domain of RNA polymerase II in transcriptional regulation in breast cancer cells and the proposed mechanisms to reshape the transcription regulatory network that promotes cancer progression.
| S-EPMC7072959 | biostudies-literature
Project description:Protein arginine deiminases (PADs) play an important role in the pathogenesis of various diseases, including rheumatoid arthritis, multiple sclerosis, lupus, ulcerative colitis, and breast cancer. Therefore, the development of PAD inhibitors has drawn significant research interest in recent years. Herein, we describe the development of the first photoswitchable PAD inhibitors. These compounds possess an azobenzene photoswitch to optically control PAD activity. Screening of a series of inhibitors structurally similar to BB-Cl-amidine afforded compounds 1 and 2 as the most promising candidates for the light-controlled inhibition of PAD2; the cis isomer of 1 is 10-fold more potent than its trans isomer, whereas the trans isomer of 2 is 45-fold more potent than the corresponding cis isomer. The altered inhibitory potency upon photoisomerization has been confirmed in a competitive activity-based protein profiling (ABPP) assay. Further investigations indicate that the trans isomer of 2 is an irreversible inhibitor, whereas the cis isomer acts as a competitive inhibitor. In cells, the trans isomer of compound 1 is completely inactive, whereas the cis isomer inhibits histone H3-citrullination in a dose-dependent manner. Taken together, 1 serves as the foundation for developing photopharmaceuticals that can be activated at the desired tissue, using light, to treat diseases where PAD activity is dysregulated.
| S-EPMC5910237 | biostudies-literature
Project description:The arginine dihydrolase (ADH) pathway has an analogous function to the urea cycle in mitochondria-containing cells, by removing nitrogen from amino acids and generating ATP. Subcellular localization of the ADH pathway enzymes in Trichomonas vaginalis revealed that arginine deiminase (ADI) localizes to the hydrogenosome, a mitochondrion-like organelle of anaerobic protists. However the other enzymes of the ADH pathway, ornithine carbamyltransferase and carbamate kinase localize to the cytosol. Three gene sequences of T. vaginalis ADI (ADI 1-3) were identified in the T. vaginalis genome, all having putative mitochondrial targeting sequences. The ADI sequences were cloned and used to probe T. vaginalis using a carboxyterminal di-hemogglutinin epitope tag which demonstrated co-localization with malic enzyme confirming the hydrogenosome localization of this enzyme.
| S-EPMC3026898 | biostudies-literature
Project description:1. Halobacterium cutirubrum L-alanine dehydrogenase was purified approx. 100-fold. 2. It has a mol. wt. of 72 500, about one-third that of two well-studied alanine dehydrogenases from non-halophiles. 3. The activity of the enzyme increases with temperature up to 70 degrees C, but the protein itself is not thermostable. 4. In the reductive amination reaction, the enzyme is fully active in the presence of high concentrations of K+, Na+ or NH4+ and partially active with Cs+ or Li+, but for oxidative deamination it has an absolute requirement for K+.
| S-EPMC1164509 | biostudies-other
Project description:Protein citrullination is a post-translational modification catalyzed by the protein arginine deiminases (PADs). This modification plays a crucial role in the pathophysiology of numerous autoimmune disorders including RA. Recently, there has been a growing interest in investigating physiological regulators of PAD activity to understand the primary cause of the associated disorders. Apart from calcium, it is well-documented that a reducing environment activates the PADs. Although the concentration of thioredoxin (hTRX), an oxidoreductase that maintains the cellular reducing environment, is elevated in RA patients, its contribution toward RA progression or PAD activity has not been explored. Herein, we demonstrate that hTRX activates PAD4. Kinetic characterization of PAD4 using hTRX as the reducing agent yielded parameters that are comparable to those obtained with a routinely used non-physiological reducing agent, e.g., DTT, suggesting the importance of hTRX in PAD regulation under physiological conditions. Furthermore, we show that various hTRX mutants, including redox inactive hTRX variants, are capable of activating PAD4. This indicates a mechanism that does not require oxidoreductase activity. Indeed, we observed non-covalent interactions between PAD4 and hTRX variants, and propose that these redox-independent interactions are sufficient for hTRX-mediated PAD4 activation.
| S-EPMC6396667 | biostudies-literature
Project description:Protein arginine deiminase 4 (PAD4) facilitates the post-translational citrullination of the core histones H3 and H4. While the precise epigenetic function of this modification has not been resolved, it has been shown to associate with general chromatin decompaction and compete with arginine methylation. Recently, we found that histones are subjected to methylglyoxal (MGO)-induced glycation on nucleophilic side chains, particularly arginines, under metabolic stress conditions. These non-enzymatic adducts change chromatin architecture and the epigenetic landscape by competing with enzymatic modifications, as well as changing the overall biophysical properties of the fiber. Here, we report that PAD4 antagonizes histone MGO-glycation by protecting the reactive arginine sites, as well as by converting already-glycated arginine residues into citrulline. Moreover, we show that similar to the deglycase DJ-1, PAD4 is overexpressed and histone citrullination is upregulated in breast cancer tumors, suggesting an additional mechanistic link to PAD4's oncogenic properties.
| S-EPMC7319962 | biostudies-literature