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1.
Characterization of anaerobic sulfite reduction by Salmonella typhimurium and purification of the anaerobically induced sulfite reductase. 总被引:6,自引:5,他引:1
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Mutants of Salmonella typhimurium that lack the biosynthetic sulfite reductase (cysI and cysJ mutants) retain the ability to reduce sulfite for growth under anaerobic conditions (E. L. Barrett and G. W. Chang, J. Gen. Microbiol., 115:513-516, 1979). Here we report studies of sulfite reduction by a cysI mutant of S. typhimurium and purification of the associated anaerobic sulfite reductase. Sulfite reduction for anaerobic growth did not require a reducing atmosphere but was prevented by an argon atmosphere contaminated with air (less than 0.33%). It was also prevented by the presence of 0.1 mM nitrate, which argues against a strictly biosynthetic role for anaerobic sulfite reduction. Anaerobic growth in liquid minimal medium, but not on agar, was found to require additions of trace amounts (10(-7)M) of cysteine. Spontaneous mutants that grew under the argon contaminated with air also lost the requirement for 10(-7)M cysteine for anaerobic growth in liquid. A role for sulfite reduction in anaerobic energy generation was contraindicated by the findings that sulfite reduction did not improve cell yields, and anaerobic sulfite reductase activity was greatest during the stationary phase of growth. Sulfite reductase was purified from the cytoplasmic fraction of the anaerobically grown cysI mutant and was purified 190-fold. The most effective donor in crude extracts was NADH. NADPH and methyl viologen were, respectively, 40 and 30% as effective as NADH. Oxygen reversibly inhibited the enzyme. Two high-molecular-weight proteins separated by gel filtration (Mr 360,000 and 490,000, respectively) were required for maximal activity with NADH. Indirect evidence, including in vitro complementation experiments with a cysG mutant extract, suggested that the 360,000-Mr component contains siroheme and is the terminal reductase. This component was further purified to near homogeneity and was found to consist of a single subunit of molecular weight 67,500. The anaerobic sulfite reductase showed some resemblance to the biosynthetic sulfite reductase, but apparently it has a unique, as yet unidentified function. 相似文献
2.
Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida.
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Urocanase is inactivated in intact cells of Pseudomonas putida and photoactivated by brief exposure of the cells to the UV radiation in sunlight. The dark reversion (inactivation) in vitro is explained by the formation of a sulfite-NAD adduct. Our objective was to investigate the dark reversion in vivo. Various compounds were added to P. putida cells, and the reversion was measured, after sonication, by comparison of the activity before and after UV irradiation. Sulfite, cysteine sulfinate, and hypotaurine enhanced the reversion of urocanase in resting cells. The reversion was time and concentration dependent. Sulfite modified the purified enzyme, but cysteine sulfinate and hypotaurine could not, indicating that those two substances had to be metabolized to support the reversion. Both of those compounds yielded sulfite when they were incubated with cells. Transaminases form sulfite from cysteine sulfinate. P. putida extract contained a transaminase whose activity involved as alpha-keto acid and either cysteine sulfinate or hypotaurine for (i) production of sulfite, (ii) disappearance of substrates, (iii) formation of corresponding amino acids, and (iv) urocanase reversion. Porcine crystalline transaminase caused reversion of highly purified P. putida urocanase with cysteine sulfinate and alpha-ketoglutarate. We conclude that in P. putida cysteine sulfinate or hypotaurine is catabolized in vivo by a transaminase reaction to sulfite, which modifies urocanase to a form that can be photoactivated. We suggest that this photoregulatory process is natural because it occurs in cells with the aid of sunlight and cellular metabolism. 相似文献
3.
We have analyzed a step in cysteine biosynthesis in several strains of the pathogenic dimorphic fungus, Histoplasma capsulatum. Mycelial cells of all strains tested are prototrophic. However, the yeast phase cells of most stains do not grow in the absence of -SH-containing compounds due to the apparent lack of an active form of sulfite reductase, a crucial enzyme in the cysteine biosynthetic pathway. In contrast, the yeast phase cells of one strain (Downs) have been found to have an active sulfite reductase and can grow in the absence of cysteine if serine is added. A different metabolic block must thus exist in this strain. Sulfite reductase in the yeast form of Downs strain is completely repressed by growth on cysteine while the mycelial form seems to be constitutive. The yeast and mycelial phase extracts were analyzed on polyacrylamide gels. A distinct protein band appeared in extracts prepared from the yeast cells incubated in minimal or serine-containing media, but not in extracts from mycelia or from cysteine-grown yeast cells. 相似文献
4.
5.
Günter A. Peschek 《Archives of microbiology》1978,119(3):313-322
Photosynthesis by Anacystis nidulans was studied in presence of reduced sulfur or nitrogen compounds, or of hydrogen. O2 evolution and CO2 fixation were depressed by sulfide, sulfite, cysteine, thioglycollate, hydroxylamine and hydrazine. Sulfite, cysteine and hydrazine inhibited O2 evolution much more strongly than CO2 fixation, indicating ability to supply electrons for CO2 photoreduction; DCMU suppressed these photoreductions. In contrast, some anoxygenic photosynthetic CO2 fixation insensitive to DCMU was found with sulfide, thiosulfate and hydrogen. Emerson enhancement studies confirmed that sulfite, cysteine and hydrazine acted on photosystem II, while photoreduction supported by sulfide, thiosulfate and hydrogen needed photosystem I only.Sulfite was photooxidized to sulfate, sulfide to elemental sulfur, and thiosulfate to sulfate plus elemental sulfur; the sulfur accumulated inside the cells. Results on the stoichiometries of the photoreductions were consistent with the photooxidation products determined. Inhibitor studies suggested photosynthetic CO2 fixation through the Calvin cycle.While photoreduction by all reductants used was found to be constitutive in Anacystis, the process was stimulated by anaerobic preincubation with the reductants only in the cases of hydrogen and thiosulfate; this adaptation was prevented by chloramphenicol and by O2. Anaerobic photoautotrophic growth of Anacystis was, however, not observed; the increase in dry weight with H2 and thiosulfate was not accompanied by cell multiplication or by an increase in chlorophyll content. Parallel short-term experiments with Chlorella did not reveal any constitutive photoreduction in this eukaryotic alga.Abbreviations CAP
chloramphenicol
- CCCP
carbonyl cyanide m-chlorophenylhydrazone
- DBMIB
dibromothymoquinone
- DCMU
dichlorophenyl dimethyl urea
- DSPD
disalicylidenepropane diamine-(1,3)
- EDAC
1-ethyl-3(3-dimethylaminopropyl-) carbodiimide 相似文献
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7.
Sulfite oxidizing activities are known since years in animals, microorganisms, and also plants. Among plants, the only enzyme well characterized on molecular and biochemical level is the molybdoenzyme sulfite oxidase (SO). It oxidizes sulfite using molecular oxygen as electron acceptor, leading to the production of sulfate and hydrogen peroxide. The latter reaction product seems to be the reason why plant SO is localized in peroxisomes, because peroxisomal catalase is able to decompose hydrogen peroxide. On the other hand, we have indications for an additional reaction taking place in peroxisomes: sulfite can be nonenzymatically oxidized by hydrogen peroxide. This will promote the detoxification of hydrogen peroxide especially in the case of high amounts of sulfite. Hence we assume that SO could possibly serve as "safety valve" for detoxifying excess amounts of sulfite and protecting the cell from sulfitolysis. Supportive evidence for this assumption comes from experiments where we fumigated transgenic poplar plants overexpressing ARABIDOPSIS SO with SO(2) gas. In this paper, we try to explain sulfite oxidation in its co-regulation with sulfate assimilation and summarize other sulfite oxidizing activities described in plants. Finally we discuss the importance of sulfite detoxification in plants. 相似文献
8.
Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3-)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor. 相似文献
9.
Eizo Saito Goro Tamura Sakae Shinano 《Bioscience, biotechnology, and biochemistry》2013,77(6):860-867
Sulfite reductase activity by algal extracts was investigated using reduced methylviologen as a hydrogen donor. Sulfite reductase appears to be widely distributed in various algae, but the enzymatic activity was not detected in the brown algae examined. The addition of phosphate buffer to the reaction mixture caused a marked decrease in activity. Sulfite reductase was partially purified from the autolysate of Porphyra tenera and some properties were studied. The optimal pH was 7.5 to 8.5 in Tris-HGl buffer system. The Km for sulfite was 6.65 × 10?4m. The enzymatic activity was completely inhibited by potassium cyanide at 5 × 10?4m. The enzyme catalyzed the reduction of sulfite to sulfide. Neither NADPH nor NADH acts as a hydrogen donor. However, it was revealed that ferredoxin can act as an electron carrier in sulfite reduction to sulfide in Porphyra extract. 相似文献
10.
Mycobacterium tuberculosis places an enormous burden on the welfare of humanity. Its ability to grow and its pathogenicity are linked to sulfur metabolism, which is considered a fertile area for the development of antibiotics, particularly because many of the sulfur acquisition steps in the bacterium are not found in the host. Sulfite reduction is one such mycobacterium-specific step and is the central focus of this paper. Sulfite reduction in Mycobacterium smegmatis was investigated using a combination of deletion mutagenesis, metabolite screening, complementation, and enzymology. The initial rate parameters for the purified sulfite reductase from M. tuberculosis were determined under strict anaerobic conditions [k(cat) = 1.0 (+/-0.1) electron consumed per second, and K(m(SO(3)(-2))) = 27 (+/-1) microM], and the enzyme exhibits no detectible turnover of nitrite, which need not be the case in the sulfite/nitrite reductase family. Deletion of sulfite reductase (sirA, originally misannotated nirA) reveals that it is essential for growth on sulfate or sulfite as the sole sulfur source and, further, that the nitrite-reducing activities of the cell are incapable of reducing sulfite at a rate sufficient to allow growth. Like their nitrite reductase counterparts, sulfite reductases require a siroheme cofactor for catalysis. Rv2393 (renamed che1) resides in the sulfur reduction operon and is shown for the first time to encode a ferrochelatase, a catalyst that inserts Fe(2+) into siroheme. Deletion of che1 causes cells to grow slowly on metabolites that require sulfite reductase activity. This slow-growth phenotype was ameliorated by optimizing growth conditions for nitrite assimilation, suggesting that nitrogen and sulfur assimilation overlap at the point of ferrochelatase synthesis and delivery. 相似文献
11.
Sulfite (SO(3)(2-)) has been widely used as preservative and antimicrobial in preventing browning of foods and beverages. SO(2), a common air pollutant, also is capable of producing sulfite and bisulfite depending on the pH of solutions. A molybdenum-dependent mitochondrial enzyme, sulfite oxidase, oxidizes sulfite to inorganic sulfate and prevents its toxic effects. In the present study, sulfite toxicity towards isolated rat hepatocytes was markedly increased by partial inhibition of cytochrome a/a(3) by cyanide or by putting rats on a high-tungsten/low-molybdenum diet, which result in inactivation of sulfite oxidase. Sulfite cytotoxicity was accompanied by a rapid disappearance of GSSG followed by a slow depletion of reduced glutathione (GSH). Depleting hepatocyte GSH beforehand increased cytotoxicity of sulfite. On the other hand, dithiothreitol (DTT), a thiol reductant, added even 1h after the addition of sulfite to hepatocytes, prevented cell death and restored hepatocyte GSH levels. Sulfite cytotoxicity was also accompanied by an increase of oxygen uptake, reactive oxygen species (ROS) formation and lipid peroxidation. Cytochrome P450 inhibitors, metyrapone and piperonyl butoxide also prevented sulfite-induced cytotoxicity and lipid peroxidation. Desferroxamine and antioxidants also protected the cells against sulfite toxicity. These findings suggest that cytotoxicity of sulfite is mediated by free radicals as ROS formation increases by sulfite and antioxidants prevent its toxicity. Reaction of sulfite or its free radical metabolite with disulfide bonds of GSSG and GSH results in the compromise of GSH/GSSG antioxidant system leaving the cell susceptible to oxidative stress. Restoring GSH content of the cell or protein-SH groups by DTT can prevent sulfite cytotoxicity. 相似文献
12.
Oktay Hasan Ozturk Suleyman Oktar Mehmet Aydin Vural Kucukatay 《Journal of physiology and biochemistry》2010,66(3):205-212
Sulfite and related chemical such as sulfite salts and sulfur dioxide has been used as a preservative in food and drugs. This
molecule has also been generated from the catabolism of sulfur-containing amino acids. Sulfite is a very reactive and potentially
toxic molecule and has to be detoxified by the enzyme sulfite oxidase (SOX). The aim of this study was to investigate the
effects of ingested sulfite on erythrocyte antioxidant status by measuring glucose-6-phosphate dehydrogenase (G-6-PD), superoxide
dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities and oxidant status by measuring thiobarbituric
acid reactive substances (TBARS) in normal and SOX-deficient rats. Rats were assigned to four groups (n = 10 rats/group) as follows; control (C), sulfite (CS), deficient (D), and deficient + sulfite (DS). SOX deficiency was established
by feeding rats a low molybdenum diet and adding to their drinking water 200 ppm tungsten (W). Sulfite (25 mg/kg) was administered
to the animals via their drinking water. At the end of 6 weeks, Erythrocyte G-6-PD, SOD, and GPx but not CAT activities were
found to be significantly increased with and without sulfite treatment in SOX-deficient groups. Sulfite treatment alone was
also significantly increased erythrocytes’ SOD activity in CS group compared to control. TBARS levels were found to be significantly
increased in CS and DS groups and decreased in D group. When SOX-deficient rats treated with sulfite, TBARS level was still
higher than other groups. In conclusion, these results suggested that erythrocyte antioxidant capacity, a defense mechanism
against the oxidative challenge, increased by endogenous and exogenous sulfite due to its oxidant nature. This increase was
also observed in CS and DS groups but it was insufficient to prevent lipid peroxidation. 相似文献
13.
Purification and some properties of sulfite:ferric ion oxidoreductase from Thiobacillus ferrooxidans.
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Sulfite:ferric ion oxidoreductase in the plasma membrane of Thiobacillus ferrooxidans AP19-3 was purified to an electrophoretically homogeneous state. The enzyme had an apparent molecular weight of 650,000 and was composed of two subunits (M(rs), 61,000 and 59,000) as estimated by sodium sulfate-polyacrylamide gel electrophoresis. The Michaelis constants of sulfite:ferric ion oxidoreductase for Fe3+ and sulfite ions were 1.0 and 0.071 mM, respectively. Sulfite:ferric ion oxidoreductase suffered from end product inhibition by 1 mM Fe2+. 相似文献
14.
Since methionine and (or) cysteine are required by a large percentage of natural auxotrophic Micrococcus strains isolated from human skin, investigations were directed to determine the specific enzymes affected in sulfur amino acid biosynthesis. Known intermediates in the interrelated cysteine and methionine biosynthetic pathways were tested as growth stimulants. Based on these growth studies, sulfur amino acid auxotrophs were grouped into three cysteine classes and five methionine classes. Selected auxotrophs of M. luteus had deficiencies in ATP sulfurylase (EC 2.7.7.4) and adenosine-5-sulfatophosphate (APS) kinase (EC 2.7.1.25), sulfite reductase (EC 1.8.1.2), serine transacetylase (EC 2.3.1.30), or beta-cystathionase (EC 4.4.1.8) activity; auxotrophs of M. lylae had deficiencies in sulfite reductase and serine transacetylase, beta-cystathionase, or N5, N10-methyltetrahydrofolate reductase (EC 1.1.1.68) activity; all auxotrophs of M. sedentarius tested had deficiencies in N5,N10-methyltetrahydrofolate reductase activity; auxotrophs of M. nishinomiyaensis had deficiencies in adenosine-3-phosphate-5-sulfatophosphate (PAPS) reductase, sulfite reductase, serine transacetylase, or N5,N10-methyltetrahydrofolate reductase activity; auxotrophs of M. varians had deficiencies in APS kinase, PAPS reductase, sulfite reductase, homoserine omicron-transsuccinylase, beta-cystathionase, or N5,N10-methyltetrahydrofolate reductase activity; auxotrophs of M. kristinae had deficiencies in serine transacetylase or cystathionine-gamma-synthase (EC 4.2.99.9) activity; auxotrophs of M. roseus had deficiencies in PAPS reductase, sulfite reductase, or serine transacetylase activity. Results of studies with various mutagens suggested that sulfur amino acid auxotrophy was primarily the result of a single base substitution in usually one or two of the genes controlling biosynthesis. A preliminary study of the amino acid composition of sweat suggested that this important source of nutrients does not contain adequate amounts of cysteine for the growth of cysteine auxotrophs but contains methionine that may be utilized in place of cysteine. 相似文献
15.
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17.
Maria R. Uria-Nickelsen Edward R. Leadbetter Walter Godchaux III 《Archives of microbiology》1994,161(5):434-438
Selected biochemical features of sulfonate assimilation in Escherichia coli K-12 were studied in detail. Competition between sulfonate-sulfur and sulfur sources with different oxidation states, such as cysteine, sulfite and sulfate, was examined. The ability of the enzyme sulfite reductase to attack the C-S linkage of sulfonates was directly examined. Intact cells formed sulfite from sulfonate-sulfur. In cysteine-grown cells, when cysteine was present with either cysteate or sulfate, assimilation of both of the more oxidized sulfur sources was substantially inhibited. In contrast, none of three sulfonates had a competitive effect on sulfate assimilation. In studies of competition between different sulfonates, the presence of taurine resulted in a decrease in cysteate uptake by one-half, while in the presence of isethionate, cysteate uptake was almost completely inhibited. In sulfite-grown cells, sulfonates had no competitive effect on sulfite utilization. An E. coli mutant lacking sulfite reductase and unable to utilize isethionate as the sole source of sulfur formed significant amounts of sulfite from isethionate. In cell extracts, sulfite reductase itself did not utilize sulfonate-sulfur as an electron acceptor. These findings indicate that sulfonate utilization may share some intermediates (e.g. sulfite) and regulatory features (repression by cysteine) of the assimilatory sulfate reductive pathway, but sulfonates do not exert regulatory effects on sulfate utilization. Other results suggest that unrecognized aspects of sulfonate metabolism, such as specific transport mechanisms for sulfonates and different regulatory features, may exist. 相似文献
18.
Sulfites, which are commonly used as preservatives, are continuously formed in the body during the metabolism of sulfur-containing amino acids. Sulfite oxidase (SOX) is an essential enzyme in the pathway of the oxidative degradation of sulfite to sulfate protecting cells from sulfite toxicity. This article investigated the effect of sulfite on total antioxidant capacity (TAC), total oxidant status, lipid hydroperoxide (LOOH), and total free sulfydryl groups (-SH) levels in normal and SOX-deficient male albino rat plasma. For this purpose, rats were divided into four groups: control, sulfite-treated, SOX-deficient, and sulfite-treated SOX-deficient groups. SOX deficiency was established by feeding rats a low molybdenum diet and adding to their drinking water 200 ppm tungsten. Sulfite (70 mg/kg) was administered to the animals via their drinking water. SOX deficiency together with sulfite treatment caused a significant increase in the plasma LOOH and total oxidant status levels. -SH content of rat plasma significantly decreased by both sulfite treatment and SOX deficiency compared to the control. There was also a significant decrease in plasma TAC level by sulfite treatment. In conclusion, sulfite treatment affects the antioxidant/oxidant balance of the plasma cells of the rats toward oxidants in SOX-deficient groups. 相似文献
19.
Kucukatay V Turgut S Kocamaz E Emmungil G Bor-Kucukatay M Turgut G Akca H Bagci H 《Biological trace element research》2006,114(1-3):185-195
Sulfite is a potentially toxic molecule that might enter the body via ingestion, inhalation, or injection. For cellular detoxification,
mammalians rely on sulfite oxidase to convert sulfite to sulfate. The purpose of this research was to determine the effect
of sulfite on zinc, iron, and copper levels in rat liver and kidney tissues. Forty normal and sulfite oxidase-deficient male
albino rats were divided into four groups that included untreated controls (group C), a sulfite-supplemented group that received
70 mg sodium metabisulfite per kilogram per day (group S), a sulfite oxidase-deficient group (group D), and a sulfite oxidase-deficient
group that was also given 70 mg sodium metabisulfite per kilogram per day (group DS). The iron and zinc levels in the liver
and kidney in groups S and DS were not affected by sulfite treatment compared to their respective controls (groups C and D).
Sulfite exposure led to an increase of kidney copper content in the S group when compared to untreated controls. The kidney
copper levels were significantly increased in the unexposed deficient rats, but it was not different than that of the deficient
rats that were given oral sulfite treatment. These results suggest that kidney copper levels might be affected by exogenous
or endogenous sulfite.
An erratum to this article is available at . 相似文献
20.
Sulfites are compounds commonly used as preservatives in foods, beverages and pharmaceuticals. Sulfite is also endogenously generated during the metabolism of sulfur-containing amino acids and drugs. It has been shown that sulfite is a highly toxic molecule. Many studies have examined the effects of sulfite toxicity, but the effect of ingested sulfite on the number of neurons in the hippocampus has not yet been reported. The present study was undertaken to investigate the effect of ingested sulfite on pyramidal neurons by counting cells in CA1 and CA3-2 subdivisions of the rat hippocampus. For this purpose, rats were assigned to one of four groups (6 rats per group): control (C), sulfite (S), deficient (D) and deficient+sulfite (DS). Sulfite oxidase deficiency was established by feeding rats a low molybdenum diet and adding 200ppm tungsten (W) to their drinking water. Sulfite (70mg/kg) was also administered to the animals via their drinking water. At the end of the experimental period, the rats were sacrificed by exsanguination under anesthesia, and their brains and livers quickly removed. The livers were used for a SOX activity assay, and the brains were used for neuronal counts in a known fraction of the CA1 and CA3-2 subdivisions of the left hippocampus using the optical fractionator method, which is a stereological method. The results showed that sulfite treatment caused a significant decrease in the total number of pyramidal neurons in three subdivisions of the hippocampus (CA1 and CA3-2) in the S, D and DS groups compared with the control group. It is concluded that exogenous administration of sulfite causes loss of pyramidal neurons in CA1 and CA3-2 subdivisions in both normal and SOX deficient rat hippocampus. This finding provides supporting evidence that sulfite is a neurotoxic molecule. 相似文献