Role of the arginine deiminase system in protecting oral bacteria and an enzymatic basis for acid tolerance.

The arginine deiminase system was found to function in protecting bacterial cells against the damaging effects of acid environments. For example, as little as 2.9 mM arginine added to acidified suspensions of Streptococcus sanguis at a pH of 4.0 resulted in ammonia production and protection against killing. The arginine deiminase system was found to have unusual acid tolerance in a variety of lactic acid bacteria. For example, for Streptococcus rattus FA-1, the pH at which arginolysis was reduced to 10% of the maximum was between 2.1 and 2.6, or more than 1 full pH unit below the minimum for glycolysis (pH 3.7), and more than 2 units below the minimum for growth in complex medium (pH 4.7). The acid tolerance of the arginine deiminase system appeared to be primarily molecular and to depend on the tolerance of individual enzymes rather than on the membrane physiology of the bacteria; pH profiles for the activities of arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase in permeabilized cells showed that the enzymes were active at pHs of 3.1 or somewhat lower. Overall, it appeared that ammonia could be produced from arginine at low pH values, even by cells with damaged membranes, and that the ammonia could then protect the cells against acid damage until the environmental pH value rose sufficiently to allow for the reestablishment of a difference in pH (delta pH) across the cell membrane.     

Turning on and turning off the arginine deiminase system in oral streptococci

The arginine deiminase system in oral streptococci is highly regulated. It requires induction and is repressed by catabolites such as glucose or by aeration. A comparative study of regulation of the system in Streptococcus gordonii ATCC 10558, Streptococcus rattus FA-1, and Streptococcus sanguis NCTC 10904 showed an increase in activity of the system in S. sanguis of some 1467-fold associated with induction-depression of cells previously uninduced-repressed. The activity of the system was assayed in terms of levels of arginine deiminase, the signature enzyme of the system, in permeabilized cells. Increases in enzyme levels associated with induction-depression were less for the other two organisms, mainly because of less severe repression, especially for S. rattus FA-1, which was the least sensitive to catabolite repression or aeration. Regulation of the arginine deiminase system involving induction and catabolite repression was demonstrated also with monoorganism biofilms composed of cells of S. sanguis adherent to glass slides. Fully uninduced-repressed cells from suspension cultures or biofilms were compromised in their abilities to catabolize arginine to protect themselves against acid damage. However, it was found that the system can be rapidly turned on or turned off, although induction-depression did appear to require cell growth. Still, the system could respond rapidly to the availability of arginine to reestablish high capacity for alkali production.     

Arginine deiminase system and acid adaptation of oral streptococci.

Streptococcus rattus FA-1 and Streptococcus sanguis NCTC 10904 underwent phenotypic acid adaptation in batch cultures toward the end of sugar-fueled growth after the culture pH had dropped to triggering values. The bacteria could be derepressed or induced for arginine deiminase independently of acid adaptation, and arginolysis afforded protection against acid killing over and above that of acid adaptation.     

Role of arginine deiminase in growth of Mycoplasma hominis.

Arginine has been considered as the major energy source of nonglycolytic arginine-utilizing mycoplasmata. When three strains of Mycoplasma arginini, and one strain each of Mycoplasma arthritidis, Mycoplasma fermentans, Mycoplasma gallinarum, Mycoplasma gallisepticum and Mycoplasma hominis were grown in the medium with high arginine concentration (34 mM) compared with low arginine (4 mM), both the protein content of the organisms and the specific activity of arginine deiminase increased. M. fermentans, the one arginine-utilizing species included in the survey which is also glycolytic, showed an increase in protein content but no increase in specific activity of the enzyme. The glycolytic non-arginine-utilizing M. gallisepticum did not show an increase in either parameter. The Km for arginine deiminase from crude cell extracts was 1.66 X 10(-4)M. The enzyme demonstrated a hyperbolic activation curve subject to substrate inhibition and was not affected by the presence of L-histidine. When mycoplasmic protein and arginine deiminase were determined for M. hominis under aerobic and anaerobic conditions, aerobically grown cells exhibited no detectable enzymatic increases until late in log phase. Higher levels of arginine deiminase were observed earlier in the anaerobic growth cycle. The rate of 14CO2 evolution from [guanido-14C]arginine was not altered in arginine-supplemented cells compared with cells grown in low arginine. In addition, CO2 production did not parallel increased arginine deiminase activity. These observations argue that arginine is used only as an alternate energy source in these organisms.     

一株精氨酸脱亚胺酶产生菌的筛选及鉴定

精氨酸脱亚胺酶由于具有成为精氨酸营养缺陷型肿瘤如肝细胞瘤,黑素瘤治疗药物的潜力而被国内外多个科研机构进行研究。本文报道本研究室于无锡锡惠公园土样中筛选得到一株产精氨酸脱亚胺酶的菌株901,并对其进行了形态,生理生化特征分析及16S rRNA基因分析,该菌被鉴定为恶臭假单胞菌(Pseudomonas putida)。     

Arginine catabolism by sourdough lactic acid bacteria: purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1

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.     

Mapping of the arginine deiminase gene in Pseudomonas aeruginosa

A mutant of Pseudomonas aeruginosa PAO lacking arginine deiminase activity (arcA) was isolated by screening for a derivative of an arcB mutant (deficient in catabolic ornithine carbamoyltransferase) that did not excrete citrulline under conditions of limited aeration. The arcA mutation was highly cotransducible with arcB.     

Rational surface engineering of an arginine deiminase (an antitumor enzyme) for increased PEGylation efficiency

Arginine deiminase (ADI) is a therapeutic protein for cancer therapy of arginine-auxotrophic tumors. However, its application as anticancer drug is hampered by its poor stability under physiological conditions in the bloodstream. Commonly, random PEGylation is being used for increasing the stability of ADI and in turn the improved half-life. However, the traditional random PEGylation usually leads to poor PEGylation efficiency due to the limited number of Lys on the protein surface. To boost the PEGylation efficiency and enhance the stability of ADI further, surface engineering of PpADI (an ADI from Pseudomonas plecoglossicida) to increase the suitable PEGylation sites was carried out. A new in silico approach for increasing the PEGylation sites was developed. The validation of this approach was performed on previously identified PpADI variant M31 to increase potential PEGylation sites. Four Arg residues on the surface of PpADI M31 were selected through three criteria and subsequently substituted to Lys, aiming for providing primary amines for PEGylation. Two out of the four substitutions (R299K and R382K) enhanced the stability of PEGylated PpADI in human serum. The average numbers of PEGylation sites were increased from ~12 (tetrameric PpADI M31, starting point) to ~20 (tetrameric PpADI M36, final variant). Importantly, the PEGylated PpADI M36 after PEGylation exhibited significantly improved T_m values (M31: 40°C; M36: 40°C; polyethylene glycol [PEG]-M31: 54°C; PEG-M36: 64°C) and half-life in human serum (M31: 1.9 days; M36: 2.0 days; PEG-M31: 3.2 days; PEG-M36: 4.8 days). These proved that surface engineering is an effective approach to increase the PEGylation efficiency which therefore enhances the stability of therapeutic enzymes. Furthermore, the PEGylated PpADI M36 represents a highly attractive candidate for the treatment of arginine-auxotrophic tumors.     

Isolation and identification of an arginine deiminase producing strain Pseudomonas plecoglossicida CGMCC2039

Arginine deiminase (ADI), an arginine degrading enzyme, has been studied as a potential anti-cancer agent for arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas (HCCs). In this study, a strain SWP1 producing high activity of ADI was isolated from the Wuxi canal. Based on its morphological, biochemical characteristics and 16S rRNA gene sequence analysis, SWP1 was identified as Pseudomonas plecoglossicida and is now deposited at CGMCC (China General Microbiological Culture Collection Center) as P. plecoglossicida CGMCC2039. It is gram-negative, aerobe, rod-shaped, motile by one or several polar flagella. In vitro studies showed that HCC cell line HEPG2 was sensitive to ADI isolated from P. plecoglossicida CGMCC2039. Our study suggests that ADI from P. plecoglossicida CGMCC2039 could become a novel anti-tumor drug.     

Coordinate repression of arginine aminopeptidase and three enzymes of the arginine deiminase pathway in Streptococcus mitis

Streptococcus mitis contains two arginine aminopeptidases (I and II) as an arginine-supplying system and the arginine deiminase pathway as an arginine-utilizing system. The levels of arginine aminopeptidase I and three enzymes of the arginine deiminase pathway were suppressed by glucose in an apparently coordinate manner. Enzyme II appeared to be constitutive.     

Role of differential expression of streptococcal arginine deiminase in inhibition of fimA expression in Porphyromonas gingivalis

Streptococcus cristatus ArcA inhibits production of a major adhesin, FimA, in Porphyromonas gingivalis, a primary periodontal pathogen. In this study, we demonstrate the differential expression of arcA in two streptococcal species. The expression level of arcA in streptococci appears to be controlled by both cis and trans elements.     

Engineering an arginine catabolizing bioconjugate: Biochemical and pharmacological characterization of PEGylated derivatives of arginine deiminase from Mycoplasma arthritidis

Arginine is an important metabolite in the normal function of several biological systems, and arginine deprivation has been investigated in animal models and human clinical trials for its effects on inhibition of tumor growth, angiogenesis, or nitric oxide synthesis. In order to design an optimal arginine-catabolizing enzyme bioconjugate, a novel recombinant arginine deiminase (ADI) from Mycoplasma arthritidis was prepared, and multi-PEGylated derivatives were examined for enzymatic and biochemical properties in vitro, as well as pharmacokinetic and pharmacodynamic behavior in rats and mice. ADI bioconjugates constructed with 12 kDa or 20 kDa monomethoxy-poly(ethylene glycol) polymers with linear succinimidyl carbonate linkers were investigated via intravenous, intramuscular, or subcutaneous administration in rodents. The selected PEG-ADI compounds have 22 +/- 2 PEG strands per protein dimer, providing an additional molecular mass of about 0.2-0.5 x 10(6) Da and prolonging the plasma mean residence time of the enzyme over 30-fold in mice. Prolonged plasma arginine deprivation was demonstrated with each injection route for these bioconjugates. Pharmacokinetic analysis employed parallel measurement of enzyme activity in bioassays and enzyme assays and demonstrated a correlation with the pharmacodynamic analysis of plasma arginine concentrations. Either ADI bioconjugate depressed plasma arginine to undetectable levels for 10 days when administered intravenously at 5 IU per mouse, while the subcutaneous and intramuscular routes exhibited only slightly reduced potency. Both bioconjugates exhibited potent growth inhibition of several cultured tumor lines that are deficient in the anabolic enzyme, argininosuccinate synthetase. Investigations of structure-activity optimization for PEGylated ADI compounds revealed a benefit to constraining the PEG size and number of attachments to both conserve catabolic activity and streamline manufacturing of the experimental therapeutics. Specifically, ADI with either 12 kDa or 20 kDa PEG attachments on 33% of the primary amines retained about 60% or 48% of enzyme activity, respectively; the Km and pH profiles were nearly unchanged; IC50 values were diminished by less than 30%; while stability studies demonstrated full retention of activity at 4 degrees C for 5 months. A comparison of the enzymatic properties of a second ADI from Pseudomonas putida illustrated the superior characteristics of the M. arthritidis ADI enzyme.     

Role of arginine 180 and glutamic acid 177 of ricin toxin A chain in enzymatic inactivation of ribosomes.

The gene for ricin toxin A chain was modified by site-specific mutagenesis to change arginine 180 to alanine, glutamine, methionine, lysine, or histidine. Separately, glutamic acid 177 was changed to alanine and glutamic acid 208 was changed to aspartic acid. Both the wild-type and mutant proteins were expressed in Escherichia coli and, when soluble, purified and tested quantitatively for enzyme activity. A positive charge at position 180 was found necessary for solubility of the protein and for enzyme activity. Similarly, a negative charge with a proper geometry in the vicinity of position 177 was critical for ricin toxin A chain catalysis. When glutamic acid 177 was converted to alanine, nearby glutamic acid 208 could largely substitute for it. This observation provided valuable structural information concerning the nature of second-site mutations.     

精氨酸脱亚氨酶的表达、纯化和活性研究

目的：建立精氨酸脱亚氨酶的高效表达菌种和纯化工艺路线。方法：人工合成编码支原体精氨酸脱亚氨酶（arginine deiminase, ADI）的基因,构建pBV220-ADI原核表达载体,转染大肠杆菌DH5α中并诱导表达目的蛋白,离子交换层析和分子筛层析法纯化目标蛋白。采用体外精氨酸降解试验测定纯化产物活性。结果：成功构建了原核表达载体pBV220-ADI,基因侧序正确。转化大肠杆菌DH5α后筛选到高水平表达目的蛋白的菌株,目标蛋白以包含体形式存在于胞浆内,表达水平超过全菌体蛋白的35%。采用盐酸胍溶解包含体、低温条件下稀释和透析的方法进行复性。顺次采用阳离子交换和凝胶过滤层析对复性液进行纯化,最终获得纯度达到95%的活性产物。活性测定表明,纯化的ADI比活性为80IU/mg。结论：成功构建了ADI的高效表达菌种,建立了目标物质的分离纯化方法。