Cultivation to improve in vivo solubility of overexpressed arginine deiminases in Escherichia coli and the enzyme characteristics.
ABSTRACT: BACKGROUND: Overexpression of foreign genes in Escherichia coli cells is an efficient means to obtain recombinant proteins. The technique is, however, often hampered by misfolding, degradation, aggregation and formation in inclusion bodies of products. RESULTS: In this study, we reported that in vivo solubility of overexpressed arginine deiminases (ADI) improved by changing the cultivation conditions. ADI is enzymes that convert L-arginine to L-citrulline. After codon optimization, we synthesized the ADI gene of Pseudomonas putida and constructed it for overexpression in E. coli cells. The rADI products were mainly in inclusion body forms. We performed a series of optimization to enhance solubility of the protein. Co-expression with the GroES-GroEL chaperone team increased approximately 5-fold of the rADI activity. In addition the combination of L-arginine and D-glucose in the Luria-Bertani (LB) growth medium further increased the total activity to about 15 times. Separate L-arginine and D-glucose or the addition of other saccharides or amino acids had no such effects. The solubilization effects of the combination of L-arginine and D-glucose were further confirmed in the overexpression of another ADI from Listeria welshimeri. The enzymatic and conversion characteristics of the rADI products were further determined. CONCLUSIONS: Combined addition of L-arginine and D-glucose in the LB medium significantly improved in vivo solubility of rADI proteins. The present study suggested a new strategy to increase the solubilization of overexpressed recombinant proteins in E. coli cells.
Project description:Many malignant human tumors, including melanomas, are auxotrophic for arginine due to reduced expression of argininosuccinate synthetase-1 (ASS1), the rate-limiting enzyme for arginine biosynthesis. Pegylated arginine deiminase (ADI-PEG20), which degrades extracellular arginine, resulting in arginine deprivation, has shown favorable results in clinical trials for treating arginine-auxotrophic tumors. Drug resistance is the major obstacle for effective ADI-PEG20 usage. To elucidate mechanisms of resistance, we established several ADI-PEG20-resistant (ADI(R)) variants from A2058 and SK-Mel-2 melanoma cells. Compared with the parental lines, these ADI(R) variants showed the following characteristics: (i) all ADI(R) cell lines showed elevated ASS1 expression, resulting from the constitutive binding of the transcription factor c-Myc on the ASS1 promoter, suggesting that elevated ASS1 is the major mechanism of resistance; (ii) the ADI(R) cell lines exhibited enhanced AKT signaling and were preferentially sensitive to PI3K/AKT inhibitors, but reduced mTOR signaling, and were preferentially resistant to mTOR inhibitor; (iii) these variants showed enhanced expression of glucose transporter-1 and lactate dehydrogenase-A, reduced expression of pyruvate dehydrogenase, and elevated sensitivity to the glycolytic inhibitors 2-deoxy-glucose and 3-bromopyruvate, consistent with the enhanced glycolytic pathway (the Warburg effect); (iv) the resistant cells showed higher glutamine dehydrogenase and glutaminase expression and were preferentially vulnerable to glutamine inhibitors. We showed that c-Myc, not elevated ASS1 expression, is involved in upregulation of many of these enzymes because knockdown of c-Myc reduced their expression, whereas overexpressed ASS1 by transfection reduced their expression. This study identified multiple targets for overcoming ADI-PEG resistance in cancer chemotherapy using recombinant arginine-degrading enzymes.
Project description:Although Enteroccus faecalis is the paradigm for biochemical studies on the arginine deiminase (ADI) pathway of fermentative arginine catabolism, little genetic information exists on this pathway in this organism. We fill this important gap by characterizing, in an 8,228-bp region cloned from a lambdagt11 genomic library of E. faecalis, a five-gene cluster forming a transcriptional unit (revealed by Northern blots and primer extension in E. faecalis) that corresponds to the ADI operon. Four additional genes in the opposite DNA strand and one in the same DNA strand are also identified. Studies on the protein products, including heterologous expression and/or sequence comparisons, allow us to ascertain or propose functions for all but 1 of the 10 genes. The ADI operon genes, arcABCRD, encode, respectively, ADI, ornithine transcarbamylase, carbamate kinase, a putative Crp/Fnr-type regulator (ArcR), and a putative ornithine-arginine antiporter (ArcD). Arginine induces the expression of arcABCRD, most likely by means of two homologous ArgR/AhrC-type regulators encoded by two genes, argR1 and argR2, that precede arcABCRD in each DNA strand and that are transcribed monocistronically, their transcription being influenced differentially by glucose and arginine. Potential ArgR1/ArgR2 (double and single) binding sequences are found in the promoter regions of arcA and of argR1/argR2 themselves. In addition, putative binding sequences for ArcR and for CcpA are found, respectively, in the argR1/argR2 and arcA promoter regions. Of the three other genes identified, two form a transcriptional unit and encode a putative metal-sensitive transcriptional regulator (ArsR) and a cysteine protease.
Project description:Arginine decarboxylase (encoded by adi) is induced under conditions of acidic pH, anaerobiosis, and rich medium. The DNA sequence of a 3-kb fragment of the Escherichia coli chromosome encoding biodegradative arginine decarboxylase was determined. This sequence encodes a protein of 755 amino acids with a molecular size of 84,420 daltons. The molecular weight and predicted Adi amino acid composition agree with those found in earlier work. The amino acid sequence of arginine decarboxylase showed homology to those of three other decarboxylases of E. coli: (i) CadA, encoding lysine decarboxylase; (ii) SpeC, encoding biosynthetic ornithine decarboxylase; and (iii) SpeF, encoding biodegradative ornithine decarboxylase and the lysine decarboxylase of Hafnia alvei. Unlike SpeC and SpeF, Adi is not similar to the biosynthetic arginine decarboxylase, SpeA. adi is also dissimilar to cadA and speF in that it does not appear to be part of an operon containing a metabolically related transport protein, indicating that it represents a new type of biodegradative decarboxylase regulation. Transcriptional fusions between fragments upstream of adi and lacZ, primer extension, and site-directed mutagenesis experiments defined the pH-regulated promoter. Deletion analysis of the upstream region and cloning of fragments to make adi::lacZ protein fusion implicated a region beyond an upstream SspI site in pH regulation. Induction of adi in the presence of sublethal concentrations of novobiocin or coumermycin A1, inhibitors of DNA gyrase, was dramatically decreased, indicating that DNA supercoiling is involved in adi expression. These results and those of promoter structure studies indicated that acid regulation of adi may involve a mechanism different from that of acid regulation of cad.
Project description:High concentrations of l-arginine or l-citrulline in the growth medium provided the wine bacterium Lactobacillus brevis with a significant growth advantage. The arginine deiminase pathway (ADI) arc gene cluster of Lactobacillus brevis contains three genes-arcD, arcE1, and arcE2-encoding putative l-arginine/l-ornithine exchangers. Uptake experiments with Lactococcus lactis cells expressing the genes showed that all three transported l-ornithine with affinities in the micromolar range. Similarly, ArcD and ArcE2 transported l-arginine, while ArcE1 transported l-citrulline, an intermediate of the ADI pathway. Chase experiments showed very efficient exchange of l-arginine and l-ornithine by ArcD and ArcE2 and of l-citrulline and l-ornithine by ArcE1. Low affinities (millimolar range) combined with low translocation rates were found for ArcD and ArcE2 with l-citrulline and for ArcE1 with l-arginine. Resting cells of Lactobacillus brevis grown in the presence of l-arginine and l-citrulline rapidly consumed l-arginine and l-citrulline, respectively, while producing ammonia and l-ornithine. About 10% of l-arginine degraded was excreted by the cells as l-citrulline. Degradation of l-arginine and l-citrulline was not subject to carbon catabolite repression by glucose in the medium. At a high medium pH, l-citrulline in the medium was required for induction of the l-citrulline degradation pathway. Pathways are proposed for the catabolic breakdown of l-arginine and l-citrulline that merge at the level of ornithine transcarbamylase in the ADI pathway. l-Arginine uptake is catalyzed by ArcD and/or ArcE2, l-citrulline by ArcE1. l-Citrulline excretion during l-arginine breakdown is proposed to be catalyzed by ArcD and/or ArcE2 through l-arginine/l-citrulline exchange.IMPORTANCELactobacillus brevis, a bacterium isolated from wine, as well as other food environments, expresses a catabolic pathway for the breakdown of l-citrulline in the medium that consists of the l-citrulline/l-ornithine exchanger ArcE1 and part of the catabolic arginine deiminase (ADI) pathway enzymes. The proposed pathways for l-arginine and l-citrulline breakdown provide a mechanism for l-citrulline accumulation in fermented food products that is the precursor of the carcinogen ethyl carbamate.
Project description:Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ?5.0 to ?5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ?ADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ?arcA1 (native) or ?arcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ?ADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.
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.
Project description:Loss of argininosuccinate synthetase 1 (ASS1), a key enzyme for arginine synthesis, occurs in many cancers, making cells dependent on extracellular arginine and targetable by the arginine-degrading enzyme pegylated arginine deiminase (ADI-PEG 20). We evaluated ASS1 expression and effects of ASS1 loss in bladder cancer which, despite affecting >70,000 people in the United States annually, has limited therapies. ASS1 loss was identified in conventional and micropapillary urothelial carcinoma, small cell, and squamous cell carcinoma subtypes of invasive bladder cancer, as well as in T24, J82, and UM-UC-3 but not in 5637, RT112, and RT4 cell lines. ASS1-deficient cells showed preferential sensitivity to ADI-PEG 20, evidenced by decreased colony formation, reduced cell viability, and increased sub-G1 fractions. ADI-PEG 20 induced general control nonderepressible 2-dependent eukaryotic initiation factor 2α phosphorylation and activating transcription factor 4 and C/EBP homologous protein up-regulation, associated with caspase-independent apoptosis and autophagy. These effects were ablated with selective siRNA silencing of these proteins. ASS1 overexpression in UM-UC-3 or ASS1 silencing in RT112 cells reversed these effects. ADI-PEG 20 treatment of mice bearing contralateral flank UM-UC-3 and RT112 xenografts selectively arrested tumor growth in UM-UC-3 xenografts, which had reduced tumor size, reduced Ki-67, and increased terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. This suggests that ASS1 loss occurs in invasive bladder cancer and is targetable by ADI-PEG 20.
Project description:Analysis of mechanism underlying the metabolic effects of ADI-PEG20 at gene expression level. The hypothesis tested in the present study was that ASS1-deficient breast cancer cells would be arginine-auxotrophs and would therefore be sensitive to arginine starvation. Results provide important information of the response of ASS1-deficient breast cancer cells to ADI-PEG20-treatment, such as the suppression of mRNAs encoding mitochondrial respiratory chain proteins. Total RNA obtained from ADI-treated, or ADI- and L-arginine-treated 231 cells compared to untreated cells.
Project description:Enterococcus faecalis makes ATP from agmatine in three steps catalyzed by agmatine deiminase (AgDI), putrescine transcarbamylase (PTC), and carbamate kinase (CK). An antiporter exchanges putrescine for agmatine. We have cloned the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for agmatine catabolism, overexpressed them in Escherichia coli, purified the products, characterized them functionally as PTC and AgDI, and crystallized and X-ray diffracted them. The 1.65-Angstroms-resolution structure of AgDI forming a covalent adduct with an agmatine-derived amidine reactional intermediate is described. We provide definitive identification of the gene cluster for agmatine catabolism and confirm that ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to be trimeric) evolved from ornithine transcarbamylase. N-(Phosphonoacetyl)-putrescine was prepared and shown to strongly (K(i) = 10 nM) and selectively inhibit PTC and to improve PTC crystallization. We find that E. faecalis AgDI, which is committed to ATP generation, closely resembles the AgDIs involved in making polyamines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably supplied the genes for PTC and CK but not those for the agmatine/putrescine antiporter, and thus the AgDI and ADI pathways are not related by a single "en bloc" duplication event. The AgDI crystal structure reveals a tetramer with a five-blade propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of sequence identity, and explains substrate affinity, selectivity, and Cys357-mediated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks access to the active center.
Project description:We previously identified an operon involved in an arginine deiminase (ADI) pathway (arc operon) on a CTX-M-producing plasmid from an O102-ST405 strain of Escherichia coli As the ADI pathway was shown to be involved in the virulence of various Gram-positive bacteria, we tested whether the ADI pathway could be involved in the epidemiological success of extended-spectrum-?-lactamase (ESBL)-producing E. coli strains. We studied two collections of human E. coli isolated in France (n?=?493) and England (n?=?1,509) and show that the prevalence of the arc operon (i) is higher in ESBL-producing strains (12.1%) than in nonproducers (2.5%), (ii) is higher in CTX-M-producing strains (16%) than in other ESBL producers (3.5%), and (iii) increased over time in ESBL-producing strains from 0% before 2000 to 43.3% in 2011 to 2012. The arc operon, found in strains from various phylogenetic backgrounds, is carried by IncF plasmids (85%) or chromosomes (15%) in regions framed by numerous insertion sequences, indicating multiple arrivals. Competition experiments showed that the arc operon enhances fitness of the strain in vitro in lysogeny broth with arginine. In vivo competition experiments showed that the arc operon is advantageous for the strain in a mouse model of urinary tract infection (UTI), whereas it is a burden in a mouse model of intestinal colonization. In summary, we have identified a trait linked to CTX-M-producing strains that is responsible for a trade-off between two main E. coli lifestyles, UTI and gut commensalism. This trait alone cannot explain the wide spread of ESBLs in E. coli but merits epidemiological surveillance.