Cloning, expression, and characterization of a thermostable l-arginase from Geobacillus thermodenitrificans NG80-2 for l-ornithine production.

Arginase (l-arginine amidinohydrolase, EC 3.5.3.1) can efficiently catalyze conversion of arginine to ornithine. Therefore, this enzyme can be used to produce l-ornithine from l-arginine. In this article, the l-arginase gene encoding the Geobacillus thermodenitrificans NG80-2 was cloned and overexpressed in Escherichia coli. The specific activity of the purified enzyme was 138.3 U/mg. The molecular mass of the l-arginase was approximately 33.0 kDa as estimated by SDS-PAGE and 192.0 kDa as determined by gel-filtration chromatography. Manganese ions were the optimum metal cofactor for activity, whereas the enzyme was slightly inhibited by Mg(2+) , Cu(2+) , Ba(2+) , Ca(2+) , and Zn(2+) . Activity was optimal at pH 9.0 and 80 °C, and the protein was stable at 40 and 50 °C. The recombinant enzyme was a uricotelic arginase. Using arginine as the substrate, the Michaelis-Menten constant (Km ) and catalytic efficiency (kcat /Km ) were measured to be 171.9 mM and 3.8 mM(-1)  s(-1) , respectively. Trp and His residues were directly involved in the l-arginase activity evaluated by inactivation agents. The biosynthesis yield of l-ornithine by the purified enzyme was 36.9 g/L, and the molar yield was 97.2%.

Purification and characterization of buffalo liver L-arginase and its kinetic properties with dihydropyrimidine and metal ions.

Arginase (L-arginine amidinohydrolase, EC.3.5.3.1) from animal tissues such as, liver and kidney has been partially characterized by many researchers. In this study, we purified arginase to homogeneity from buffalo liver with about ~2857 purification fold and a 20% recovery by chromatographic and spectroscopic analysis were obtained. The molecular mass determined by gel filtration and SDS-PAGE was found to be 118 kDa and 47 kDa, respectively. The optimal pH and temperature of the arginase was 9.5 and 40°C, respectively. Kinetic parameters (Km and Vmax) showed activation of arginase in the reaction medium with decrease in Km (7.14, 5.26, 4.0 and control 3.22 mM) and Vmax (0.05, 0.035, 0.027 and control 0.021 mg/mL/min), while co-factor activity of arginase was optimized using metal ions like Mn²âº and Mg²âº at 2 mM, which revealed an increase in Vmax values (0.011, 0.013, 0.015 and control 0.010 mg/mL/min) and a decrease in Km values (2.22, 2.12, 1.88 and control 1.66 mM). The kinetic data suggested that the arginase activity is enhanced in the presence of dihydropyrimidine derivative and metal ions, indicating essential mode of activation.

Construction of a highly efficient Bacillus subtilis 168 whole-cell biocatalyst and its application in the production of L-ornithine.

L-Ornithine, a non-protein amino acid, is usually extracted from hydrolyzed protein as well as produced by microbial fermentation. Here, we focus on a highly efficient whole-cell biocatalyst for the production of L-ornithine. The gene argI, encoding arginase, which catalyzes the hydrolysis of L-arginine to L-ornithine and urea, was cloned from Bacillus amyloliquefaciens B10-127 and expressed in GRAS strain Bacillus subtilis 168. The recombinant strain exhibited an arginase activity of 21.9 U/mg, which is 26.7 times that of wild B. subtilis 168. The optimal pH and temperature of the purified recombinant arginase were 10.0 and 40 °C, respectively. In addition, the recombinant arginase exhibited a strong Mn(2+) preference. When using whole-cell biocatalyst-based bioconversion, a hyper L-ornithine production of 356.9 g/L was achieved with a fed-batch strategy in a 5-L reactor within 12 h. This whole-cell bioconversion study demonstrates an environmentally friendly strategy for L-ornithine production in industry.

[Novel immobilization of arginase I via cellulose-binding domain and its application in producing of L-ornitine].

The recombinant Escherichia coli strain pET35b-ARG, which overexpresses arginase I fused to a cellulose-binding domain (CBD), was developed. After preparing cellulose microspheres, arginase I was immobilized via the CBD of the fusion protein. Under optimal reaction conditions (40 degrees C, pH 9.5, 1 mM of Mn2+, 30 microl/ml of immobilized enzyme, 30 g/l of L-Arg, and for I h), the conversion rate of L-Arg was 98.7%. After 7 reuses of 30 microl of immobilized enzyme in 1 ml of catalytic solution, 153 mg of L-Orn with 97.3% purity was obtained. This indicated that the immobilization method was effective, feasible and could be used for the industrial production of L-Orn in the future.

Overexpression of (His)6-tagged human arginase I in Saccharomyces cerevisiae and enzyme purification using metal affinity chromatography.

Arginase (EC 3.5.3.1; L-arginine amidinohydrolase) is a key enzyme of the urea cycle that catalyses the conversion of arginine to ornithine and urea, which is the final cytosolic reaction of urea formation in the mammalian liver. The recombinant strain of the yeast Saccharomyces cerevisiae that is capable of overproducing arginase I (rhARG1) from human liver under the control of the efficient copper-inducible promoter CUP1, was constructed. The (His)(6)-tagged rhARG1 was purified in one step from the cell-free extract of the recombinant strain by metal-affinity chromatography with Ni-NTA agarose. The maximal specific activity of the 40-fold purified enzyme was 1600 µmol min(-1) mg(-1) protein.

Oxidant-mediated modification of the cellular thiols is sufficient for arginase activation in cultured cells.

Increased arginase activity in the vasculature has been implicated in the regulation of nitric oxide (NO) homeostasis, leading to the development of vascular disease and the promotion of tumor cell growth. Recently, we showed that cysteine, in the presence of iron, promotes arginase activity by driving the Fenton reaction. In the present report, we showed that induction of oxidative stress in erythroleukemic cells with the thiol-specific oxidant, diamide, led to an increase in arginase activity by 42% (P = 0.02; vs. control). By using specific antibodies, it was demonstrated that this increase correlated with an increase in arginase-1 levels in the cells and with corresponding decreases in glutathione and protein thiol levels. Treatment of cells with aurothiomalate (ATM), a protein thiol-complexing agent, diminished the activity of arginase and arginase-1 levels by 19.5 and 35.2%, respectively (vs. control) and significantly decreased both glutathione and protein thiol levels, further implicating the thiol redox system in the cellular activation of arginase. Furthermore, diamide significantly altered the kinetics of arginase, resulting in the doubling of its V(max) (vs. control). Our presented data demonstrate, for the first time that the intracellular arginase activation is may be enhanced in part, via a cellular thiol-mediated mechanism.

Change in the structure of Shope papilloma virus-induced arginase associated with mutation of the virus.

The change in the state of the virus-induced enzyme associated with a mutation in the virus provides additional evidence that the enzyme is synthesized from virus rather than rabbit genetic information. This change in structure results in differences in stability of polymerization, degree of optical rotary dispersion (ORD) specific rotation, change in elution characteristics from carboxymethyl cellulose, and a reduction in specific activity of the arginase. Liver arginase differs markedly in ORD characteristics from the virus-induced enzyme. In contrast to the virus-induced enzyme, it showed no negative Cotton effect at 233 nm until it was activated with manganese. Manganese had no influence on the ORD spectrum of virus-induced arginase. In addition, liver arginase is denatured by 4 M urea, while the virus-induced enzyme requires 10 M urea for denaturation.

High-Throughput Fluorescence-Based Activity Assay for Arginase-1.

Arginase-1, which converts the amino acid L-arginine into L-ornithine and urea, is a promising new drug target for cancer immunotherapy, as it has a role in the regulation of T-cell immunity in the tumor microenvironment. To enable the discovery of small-molecule Arginase-1 inhibitors by high-throughput screening, we developed a novel homogeneous (mix-and-measure) fluorescence-based activity assay. The assay measures the conversion of L-arginine into L-ornithine by a decrease in fluorescent signal due to quenching of a fluorescent probe, Arginase Gold. This way, inhibition of Arginase-1 results in a gain of signal when compared with the uninhibited enzyme. Side-by-side profiling of reference inhibitors in the fluorescence-based assay and a colorimetric urea formation assay revealed similar potencies and the same potency rank order among the two assay formats. The fluorescence-based assay was successfully automated for high-throughput screening of a small-molecule library in 384-well format with a good Z'-factor and hit confirmation rate. Finally, we show that the assay can be used to study the binding kinetics of inhibitors.

Identification of an Arginase II Inhibitor via RapidFire Mass Spectrometry Combined with Hydrophilic Interaction Chromatography.

Peripheral arterial disease (PAD) is an occlusive disease that can lead to atherosclerosis. The involvement of arginase II (Arg II) in PAD progression has been proposed. However, no promising drugs targeting Arg II have been developed to date for the treatment of PAD. In this study, we established a method for detecting the activity of Arg II via high-throughput label-free RapidFire mass spectrometry using hydrophilic interaction chromatography, which enables the direct measurement of l-ornithine produced by Arg II. This approach facilitated a robust high-concentration screening of fragment compounds and the identification of a fragment that inhibits the activity of Arg II. We further confirmed binding of the fragment to the potential allosteric site of Arg II using a surface plasmon resonance assay. We concluded that the identified fragment is a promising compound that may lead to novel drugs to treat PAD, and our method for detecting the activity of Arg II can be applied to large-scale high-throughput screening to identify other structural types of Arg II inhibitors.

Leveraging Rational Protein Engineering to Improve mRNA Therapeutics.

Messenger RNA (mRNA) is a promising new class of therapeutics that has potential for treatment of diseases in fields such as immunology, oncology, vaccines, and inborn errors of metabolism. mRNA therapy has several advantages over DNA-based gene therapy, including the lack of the need for nuclear import and transcription, as well as limited possibility of genomic integration. One drawback of mRNA therapy, especially in cases such as metabolic disorders where repeated dosing will be necessary, is the relatively short in vivo half-life of mRNA (∼6-12 h). We hypothesize that protein engineering designed to improve translation, yielding longer-lasting protein, or modifications that would increase enzymatic activity would be helpful in alleviating this issue. In this study, we present two examples where sequence engineering improved the expression and duration, as well as enzymatic activity of target proteins in vitro. We then confirmed these findings in wild-type mice. This work shows that rational engineering of proteins can lead to improved therapies in vivo.

A liver-derived immunosuppressive factor is an arginase: identification and mechanism of immunosuppression.

We found a substance in culture medium of neonatal pig liver fragments, which suppresses an immune response monitored by (3)H-thymidine incorporation using phytohemagglutinin (PHA)-stimulated lymphocytes. We named it as an immunosuppressive factor (ISF). To purify ISF, ammonium sulfate fractionation, DE52, SP-Sephadex, hydroxyapatite, blue Sepharose, heparin Sepharose and Superdex gel filtration columns were used. Using these purification procedures, ISF was purified 1,254-fold, with 9.2% recovery, from the culture medium of neonatal pig liver fragments, and was identified as arginase by its biochemical characteristics including molecular size, amino acid sequences of digested peptides and expression of arginase activity. The addition of ISF caused to decrease in arginine concentration in culture medium and at the same time DNA synthesis was suppressed dose-dependently, both of which were recovered by the addition of NOHA (N(G)-hydroxy-L-arginine), an arginase inhibitor. In addition, the depletion of arginine in culture medium also led to the inhibition of DNA synthesis. These results led us to the conclusion that immunosuppressive effect of ISF was due to arginase activity that decreased arginine concentration in culture medium, not to another function of ISF.

Arginase purified from endophytic Pseudomonas aeruginosa IH2: Induce apoptosis through both cell cycle arrest and MMP loss in human leukemic HL-60 cells.

Arginase is a therapeutic enzyme for arginine-auxotrophic cancers but their low anticancer activity, less proteolytic tolerance and shorter serum half-life are the major shortcomings. In this study, arginase from Pseudomonas aeruginosa IH2 was purified to homogeneity and estimated as 75 kDa on native-PAGE and 37 kDa on SDS-PAGE. Arginase showed optimum activity at pH 8 and temperature 35 °C. Mn2+ and Mg2+ ions enhanced arginase activity while, Li+, Cu2+, and Al3+ ions reduced arginase activity. In-vitro serum half-life of arginase was 36 h and proteolytic half-life against trypsin and proteinase-K was 25 and 29 min, respectively. Anticancer activity of arginase was evaluated against colon, breast, leukemia, and prostate cancer cell lines and lowest IC50 (0.8 IU ml-1) was found against leukemia cell line HL-60. Microscopic studies and flow cytometric analysis of Annexin V/PI staining of HL-60 cells revealed that arginase induced apoptosis in dose-dependent manner. Cell cycle analysis suggested that arginase induced cell cycle arrest in G0/G1 phase. The increasing level of MMP loss, ROS generation and decreasing level of SOD, CAT, GPx and GSH suggested that arginase treatment triggered dysfunctioning of mitochondria. The cleavage of caspase-3, PARP-1, activations of caspase-8, 9 and high expression of proapoptotic protein Bax, low expression of anti-apoptotic protein Bcl-2 indicated that arginase treatment activates mitochondrial pathway of apoptosis. Purified arginase did not exert cytotoxic effects on human noncancer cells. Our study strongly supports that arginase could be used as potent anticancer agent but further studies are required which are underway in our lab.

Arginase-flotillin interaction brings arginase to red blood cell membrane.

Flotillin-1 and arginase are both up-regulated in red blood cell membrane of type 2 diabetic patients. For studying why the soluble arginase can bind to the membrane and whether such binding would modify arginase activity, the arginase1 and related proteins were cloned and expressed. The results showed that flotillin-1 can interact with arginase1, and hence arginase activity was up-regulated by 26.8%. It was estimated that about 61% of arginase1 is bound to the membrane mediated by flotillin-1. The arginase activity in diabetic patients was significantly higher than that of the controls (752.4+/-38.5 U/mg protein vs 486.7+/-28.7 U/mg protein).

Characterisation of arginase from the extreme thermophile 'Bacillus caldovelox'.

A thermostable arginase (L-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile 'Bacillus caldovelox' (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor. The purified enzyme is a hexamer with a subunit Mr of 31,000 +/- 2000 and contains greater than or equal to 1 Mn atom per subunit. Maximum activation on incubation with Mn2+ is 29%. Activity is optimal at pH 9 and at 60 degrees C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM. Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60 degrees C (t1/2 (half life) = 4.5 min) and individually 0.1 mM Mn2+ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce greater than 1000-fold increase and a t1/2 = 105 min at 95 degrees C. Aspartic acid and other species that bind Mn2+ can replace BSA, and it is suggested that arginase can be inactivated by free Mn2+. A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5. Reassociation occurs at pH 9 and is unusual in that it does not require Mn2+.

Purification, affinity to anti-human arginase immunoglobulin-Sepharose 4B and subunit molecular weights of mammalian arginases.

The affinities of anti-human liver arginase antibodies raised in rabbits to liver arginases from man, bovine, pig, dog, guinea pig, rat and mouse were investigated by Scatchard analysis of the binding of the arginases from crude liver extracts to Sepharose-bound immunoglobulins. All arginases bound with good affinity, but the binding capacities of the immunosorbent for the enzymes from various species decreased with decreasing phylogenetic relationship of the species. Arginase from murine peritoneal macrophages did not bind to the immunosorbent at all. A simple two-step purification method for the liver arginases of all species mentioned above is given. All arginases were purified to electrophoretical homogeneity. The molecular weights of their subunits were estimated.

Intracellular localization of arginase in chick kidney.

Although chickens are uricotelic and do not have significant urea-ornithine cycle in any tissue, the kidneys contain a high concentration of arginase which apparently functions to regulate degradation of dietary arginine. A series of investigations has been made to determine the intracellular localization of this arginase in chicken kidney. Tissue fractionation using sucrose density gradients and differential centrifugation showed as association of arginase activity with certain marker enzymes and with fractions identified as mitochondria by electron microscopy. This is consistent with the localization of the arginase in the mitochondrial matrix of chicken kidney cells. Such a finding has significance in understanding the regulation of arginine degradation in chickens.

Urea cycle of Fasciola gigantica: purification and characterization of arginase.

The ornithine-urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase:canavanase ratio (>10). The enzyme exhibited a maximal activity at pH 9.5 and Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine>isoleucine>ornithine>valine>leucine>proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.

Partial purification of a liver-derived tumor cell growth inhibitor that differentially inhibits poorly-liver metastasizing cell lines: identification as an active subunit of arginase.

Organ specific tumor metastasis is thought in part to require the ability of metastatic cells to respond to target-organ-associated growth factors or to avoid the effects of target organ associated growth inhibitors. We previously found that murine and rat liver-conditioned media inhibited the growth of the poorly-liver metastasizing murine RAW117-P large-cell lymphoma cells more than their highly liver-metastasizing RAW117-H10 counterparts. Using a six step chromatographic procedure, the major RAW117-P cell proliferation inhibitor from a rat liver extract was purified. The factor displayed a Mr of approximately 35,000 and an isoelectric point > 8.5. This material inhibited the growth of many cells at high concentration; however, in dose-response studies it displayed a higher IC50 for highly-liver metastatic murine RAW117-H10 lymphoma and human KM12SM colon carcinoma cells than for their poorly-liver metastatic counterparts. Attempts to identify the growth inhibitor led to the supplementation of tissue culture inhibitor assays with various components, including excess amino acids, and this was found to completely abrogate the factor's activity. Specifically, the addition of excess arginine resulted in the complete cellular recovery from inhibitor exposure. This tentatively identified the liver growth inhibitor as the enzyme arginase, a Mr approximately 10,000 multisubunit protein. A microtiter plate-based assay for arginase was developed and the purification repeated using human liver as a source of activity and the human KM12C colon carcinoma line as a target. The growth inhibitory and arginase activities were found to co-purify, identifying the factor as arginase. Highly-metastatic cells displayed no ability to preferentially inactivate or inhibit the activity of arginase, but they did they display slightly greater amounts of intracellular arginine. The liver is a major site of arginase localization as the enzyme is required for the functioning of the urea cycle. The results indicate that certain liver-colonizing tumor cells can escape, to a degree, the proliferation-damping effects of arginine depletion.

Purification and properties of rat small intestinal arginase.

Arginase [L-arginine amidinhydrolase EC 3.5.3.1] from rat small intestine was purified about 2,200-fold and its properties were compared with those of the rat liver and kidney enzymes. Intestinal arginase was extremely labile on storage either at -10 degrees or 4 degrees and lost activity during purification unless 25 mM L-valine was present. The purified enzyme appeared to be homogeneous by disc electrophoresis and its molecular weight was estimated to be 120,000 by Sephadex G-100 filtration...

Purification of arginase from Aspergillus nidulans.

Arginase (EC 3.5.3.1) of Aspergillus nidulans, the enzyme which enables the fungus to use arginine as the sole nitrogen source was purified to homogeneity. Molecular mass of the purified arginase subunit is 40 kDa and is similar to that reported for the Neurospora crassa (38.3 kDa) and Saccharomyces cerevisiae (39 kDa) enzymes. The native molecular mass of arginase is 125 kDa. The subunit/native molecular mass ratio suggests a trimeric form of the protein. The arginase protein was cleaved and partially sequenced. Two out of the six polypeptides sequenced show a high degree of homology to conserved domains in arginases from other species.