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1.
Fumarase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 130-fold under anoxic conditions. The native enzyme had an apparent molecular mass of 114 kDa and was composed of two subunits of 60 kDa. The enzyme exhibited maximum activity at pH 8.5 and approximately 54° C. The K m values for fumarate and l-malate were 0.25 mM and 2.38 mM, respectively. Fumarase was inactivated by oxygen, but the activity could be restored by addition of Fe2+ and β-mercaptoethanol under anoxic conditions. EPR spectroscopy of the purified enzyme revealed the presence of a [3Fe-4S] cluster. Under reducing conditions, only a trace amount of a [4Fe-4S] cluster was detected. Addition of fumarate resulted in a significant increase of this [4Fe-4S] signal. The N-terminal amino acid sequence showed similarity to the sequences of fumarase A and B of Escherichia coli (56%) and fumarase A of Salmonella typhimurium (63%). Received: 15 September 1995 / Accepted: 13 November 1995  相似文献   

2.
Abstract Malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 42-fold. The native enzyme had an apparent molecular mass of 68 kDa and consisted of two subunits of 35 kDa. The enzyme exhibited maximum activity with oxaloacetate at pH 8.5 and 60 °C. The K m for oxaloacetate was 50 μM and for NADH 30 μM. The K m values for l-malate and NAD were 4 and 1.1 mM, respectively. Substrate inhibition was found at oxaloacetate concentrations higher than 250 μM. The N-terminal amino acid sequence of the enzyme was similar to the sequences of a variety of other malate dehydrogenases from plants, animals and micro-organisms.  相似文献   

3.
Sulfate reduction by a syntrophic propionate-oxidizing bacterium   总被引:3,自引:0,他引:3  
The syntrophic propionate-oxidizing bacterium MPOB was able to grow in the absence of methanogens by coupling the oxidation of propionate to the reduction of sulfate. Growth on propionate plus sulfate was very slow (=0.024 day–1). An average growth yield was found of 1.5 g (dry weight) per mol of propionate. MPOB grew even slower than other sulfate-reducing syntrophic propionate-oxidizing bacteria. The growth rates and yields of strict sulfate-reducing bacteria (Desulfobulbus sp.) grown on propionate plus sulfate are considerably higher.  相似文献   

4.
The syntrophically glycolate-fermenting bacterium in the methanogenic binary coculture FlGlyM was isolated in pure culture (strain FlGlyR) with glyoxylate as sole substrate. This strain disproportionated 12 glyoxylate to 7 glycolate, 10 CO2, and 3 hydrogen. Glyoxylate was oxidized via the malyl-CoA pathway. All enzymes of this pathway, i.e. malyl-CoA lyase/malate: CoA ligase, malic enzyme, and pyruvate synthase, were demonstrated in cell-free extracts. Glycolate dehydrogenase, hydrogenase, and ATPase, as well as menaquinones as potential electron carriers, were present in the membranes. Everted membrane vesicles catalyzed hydrogen-dependent glyoxylate reduction to glycolate [86–207 nmol min-1 (mg protein)-1] coupled to ATP synthesis from ADP and Pi [38–82 nmol min-1 (mg protein)-1]. ATP synthesis was abolished entirely by protonophores or ATPase inhibitors (up to 98 and 94% inhibition, respectively) indicating the involvement of proton-motive force in an electron transport phosphorylation driven by a new glyoxylate respiration with hydrogen as electron donor. Measured reaction rates in vesicle preparations revealed a stoichiometry of ATP formation of 0.2–0.5 ATP per glyoxylate reduced.Abbreviations BES 2-Bromoethanesulfonate - CCCP Carbonylcyanide m-chlorophenylhydrazone - DCCD N,N-Dicyclohexylcarbodiimide - DCPIP 2,6-Dichlorophenolindophenol - DTE Dithioerythritol - TCS 3,5,4,5-Tetrachlorosalicylanilide - SF 6847 3,5-Di-tert-butyl-4-hydroxybenzylidenemalonitrile  相似文献   

5.
During growth with fumarate as the terminal electron transport acceptor and either formate or sulfide as the electron donor, Wolinella succinogenes induced a peri-plasmic protein (54 kDa) that reacted with an antiserum raised against the periplasmic fumarate reductase (Fcc) of Shewanella putrefaciens. However, the periplasmic cell fraction of W. succinogenes did not catalyze fumarate reduction with viologen radicals. W. succinogenes grown with polysulfide instead of fumarate contained much less (< 10%) of the 54-kDa antigen, and the antigen was not detectable in nitrate-grown bacteria. The antigen was most likely encoded by the fccA gene of W. succinogenes. The antigen was absent from a ΔfccABC mutant, and its size is close to that of the protein predicted by fccA. The fccA gene probably encodes a pre-protein carrying an N-terminal signal peptide. The sequence of the mature FccA (481 residues, 52.4 kDa) is similar (31% identity) to that of the C-terminal part (450 residues) of S. putrefaciens fumarate reductase. As indicated by Northern blot analysis, fccA is cotranscribed with fccB and fccC. The proteins predicted from the fccB and fccC gene sequences represent tetraheme cytochromes c. FccB is similar to the N-terminal part (150 residues) of S. putrefaciens fumarate reductase, while FccC resembles the tetraheme cytochromes c of the NirT/NapC family. The ΔfccABC mutant of W. succinogenes grew with fumarate and formate or sulfide, suggesting that the deleted proteins were not required for fumarate respiration with either electron donor. Received: 26 September 1997 / Accepted: 8 December  相似文献   

6.
The electron-transport chain catalyzing fumarate reduction by formate has recently been reconstituted from the formate dehydrogenase complex and the fumarate reductase complex from Vibro succinogenes, in a liposomal preparation containing vitamin K-1 (Unden, G. and Kröger, A. (1982) Biochim. Biophys. Acta 682, 258–263). We have now investigated the structural properties of this preparation. The preparation was found to consist of a homogeneous population of unilamellar proteoliposomes with an average diameter of about 100 nm and an internal volume of 2–4 ml / g phospholipid. The buoyant density (1.07 g / ml) was consistent with the protein / phospholipid ratio (0.2 g / g) of the preparation. Leakage of glucose from the internal spaces of the proteoliposomes was negligibly slow. Proteoliposomes prepared with either of the enzyme complexes showed peripheral projections mainly on the outer surface, when examined by electron microscopy after negative staining. The size, orientation and surface density of the projections were consistent with those of the enzymes. Most of the substrate and dye-reactive sites (70–90%) of the enzymes in the proteoliposomes were accessible to external non-permeant substrates. The proteoliposomes catalyzing electron transport were formed by freeze-thawing a mixture of liposomes and protein-phospholipid complexes which did not perform electron transport from formate to fumarate. Nearly the entire amount of the enzymes supplied (0.2 g protein / g phospholipid) was incorporated into the liposomes by this procedure. The transformation of liposomes into proteoliposomes was accompanied by exchange of the internal solutes with the external medium.  相似文献   

7.
We recently isolated an acetate-oxidizing rodshaped eubacterium (AOR) which was capable of oxidizing acetate to CO2 when grown in coculture with the hydrogenotrophic methanogen Methanobacterium sp. strain THF. The AOR was also capable of growing axenically on H2CO2 which it converted to acetate. Previous results for the acetate oxidizing coculture showed isotopic exchange between acetate and CO2, suggesting that the AOR was using a pathway for acetate oxidation resembling a reveral of the acetogenic (carbon monoxide) pathway. In this study, it was found that production of 14CO2 from 14CH3COO- by the coculture was inhibited by 200 M cyanide, while methanogenesis from H2–CO2 was unaffected, implying the involvement of carbon monoxide dehydrogenase (CODH) in acetate oxidation. CODH was present at 0.055 mol methyl viologen reduced min-1 mg-1 protein in extracts of Methanobacterium sp. strain THF, but was present in higher levels in the acetate oxidizing coculture and in the AOR grown axenically and on H2–CO2 (2.0 and 6.4 mol min-1 mg-1 protein respectively). Anaerobic activity stains for CODH in native polyacrylamide gels from the AOR coculture showed components co-migrating with bands from both organisms, as well as an additional band in extracts of the coculture. Formate dehydrogenase (FDH) was present in both the AOR coculture and monoculture but not in extracts of H2–CO2 grown cells of Methanobacterium sp. strain THF. Formyltetrahydrofolate (FTHF) synthetase was not detectable in extracts of the AOR monoculture or coculture, although it was found in high amounts in extracts of H2–CO2 grown cells of the thermophilic acetogen Acetogenium kivui. Extracts of H2–CO2 grown cells of the AOR showed a fluorescence spectrum typical of pterin derivatives. Bioassay for folates showed levels to be at anabolic rather than catabolic levels. It is possible that the AOR uses pterins distinct from folate for catabolism. Isocitrate dehydrogenase, a citric acid cycle enzyme, was also present in the AOR, but at anabolic levels and -ketoglutarate dehydrogenase was not detectable.Abbreviations (AOR) acetate-oxidizing rod - (CODH) carbon monoxide dehydrogenase - (FDH) formate dehydrogenase - (FTHF) formyltetrahydrofolate  相似文献   

8.
H. Mell  C. Wellnitz  A. Kr  ger 《BBA》1986,852(2-3):212-221
The electrochemical proton potential across the cytoplasmic membrane ( ) as well as the H+ / e ratio, which were brought about by the electron transport of Wolinella succinogenes, was measured with the aim of understanding the mechanism of electron-transport-coupled phosphorylation in this anaerobic bacterium. (1) Inverted vesicles derived from the bacterial membrane were found to take up protons from the external medium on initiation of fumarate reduction by H2. Proton uptake was dependent on the presence of K+ within the vesicles, was enhanced by the presence of valinomycin and DCCD and high internal buffer concentration, and was abolished by protonophores. The maximum H+ / e ratio slightly exceeded 1. (2) The vesicles accumulated thiocyanate in the steady state of fumarate reduction by H2. The concentration ratio (internal / external) was close to 1000 at an external thiocyanate concentration below 10 μM. Under the same conditions the uptake of methylamine was negligible. Thiocyanate uptake was abolished by the presence of a protonophore. (3) Cells of W. succinogenes accumulated tetraphenylphosphonium cation (TPP) in the steady state of fumarate reduction with H2 or formate. Under the same conditions the uptake of benzoic acid was negligible. From the amount of TPP taken up by the bacteria, the free internal concentration of TPP was evaluated according to the procedure of Zaritsky et al. (Zaritsky, A., Kihara, M. and MacNab, R.M. (1981) J. Membrane Biol. 63, 215–231). The concentration ratio (internal / external) was 700 in the absence and close to 1 in the presence of a protonophore or in the absence of external Na+. (4) The experimental results are consistent with the view that the energy transduction from electron transport to phosphorylation is done by means of the across the bacterial membrane.  相似文献   

9.
Conditions promoting maximal in vitro activity of the particulate NADH:fumarate reductase from Fibrobacter succinogenes were determined. This system showed a pH optimum of 6.0 in K+ MES buffer only when salt (NaCl or KCl) was present. Salt stimulated the activity eightfold at the optimal concentration of 150m M. This effect was due to stimulation of fumarate reductase activity as salt had little effect on NADH: decylubiquinone oxidoreductase (NADH dehydrogenase). The stimulation of fumarate reductase by salt at pH 6.0 was not due to removal of oxaloacetate from the enzyme. Kinetic parameters for several inhibitors were also measured. NADH dehydrogenase was inhibited by rotenone at a single site with a K i of 1 M. 2-Heptyl-4-hydroxyquinonline-N-oxide (HOQNO) inhibited NADH: fumarate reductase with a K i of 0.006 M, but NADH dehydrogenase exhibited two HOQNO inhibition constants of approximately 1 M and 24 M. Capsaicin and laurylgallate each inhibited NADH dehydrogenase by only 20% at 100 M. NADH dehydrogenase gave K m values of 1 M for NADH and 4 M for reduced hypoxanthine adenine dinucleotide.Published with the approval of the Director of the Agricultural Experiment Station, North Dakota State University, as journal article no. 2201  相似文献   

10.
11.
G. Unden  A. Kröger 《BBA》1982,682(2):258-263
Fumarate reduction by formate in Vibrio succinogenes is catalyzed by a membrane-bound electron-transport chain, and is coupled with the phosphorylation of ADP. The electron-transport chain was reconstituted in liposomes from the isolated components. The formate dehydrogenase complex (three different peptides), the fumarate reductase complex (three different peptides) and vitamin K-1 were required for the electron transport. The pathway of the electrons from formate to fumarate in the reconstituted chain was identical with that in the bacterial membrane. Each of the active enzyme complexes in the liposomes participated in the electron transport. This was valid for proteoliposomes with ratios of the contents of the two enzyme complexes ranging between 0.1 and 10. This indicates that vitamin K-1 forms a diffusible pool within the liposomal membrane that allows every quinone molecule to react with each molecule of the two enzyme complexes.  相似文献   

12.
Escherichia coli was grown anaerobically on sodium fumarate and molecular hydrogen or sodium formate in continuous culture. The maximal growth yield and the maintenance coefficient were determined. In a mineral medium a Y fum max value of 6.6 g dry weight per mol fumarate was found. This value increased to 7.5 when casamino acids were present in the medium. From these data and the corresponding Y ATP max values it could be calculated that per mol of fumarate reduced, 0.4 mol of ATP became available for growth. In batch culture a Yfum value of 4.8 g dry weight per mol fumarate was determined.  相似文献   

13.
The possible involvement of NtrA in the expression of several anaerobically induced genes in Salmonella typhimurium was investigated. Unlike Escherichia coli, where hydrogenase 3 is ntrA dependent, the introduction of a mutation in ntrA had virtually no effect on the hydrogenase activity, thought to be hydrogenase 3, of S. typhimurium LT7. Fumarate reductase and alcohol dehydrogenase activities were found to be diminished in ntrA mutant strains, but this may very well be indirect since fdhF mutant strains showed the same effect. These results suggest that in S. typhimurium NtrA is highly specific for the anaerobic expression of fdhF.  相似文献   

14.
Abstract In an ompF'-'lacZ fusion system carried by the open reading frame vector pORF1 in a supE mutant of Escherichia coli K12, read-through of an amber codon was decreased at temperatures higher than 40°C. This effect of temperature was dependent on the nucleotide sequence surrounding the amber codon, which was inserted into a site between the ompF and lacZ cistrons. Upon a temperature shift-up from 30 to 42°C, β-galactosidase synthesis directed by this fusion showed a transient arrest.  相似文献   

15.
Wolinella succinogenes was found to grow on H2S plus fumarate with the formation of elemental sulfur and succinate. The growth rate was 0.18 h-1 (t d=3.8 h) and the growth yield was estimated to be 6.0 g per mol fumarate used. Growth also occurred on formate plus elemental sulfur; the products formed were H2S and CO2. The growth rate and estimated growth yield were 0.58 h-1 (t d=1.2 h) and 3.5 g per mol formate used, respectively. These results suggest that certain chemotrophic anaerobes may be involved in both the formation and reduction of sulfur.  相似文献   

16.
Plasmodium falciparum is an apicomplexan parasite that causes the most severe malaria in humans. Due to a lack of effective vaccines and emerging of drug resistance parasites, development of drugs with novel mechanisms of action and few side effects are imperative. To this end, ideal drug targets are those essential to parasite viability as well as absent in their mammalian hosts. The mitochondrial electron transport chain (ETC) of P. falciparum is one source of such potential targets because enzymes, such as L-malate:quinone oxidoreductase (PfMQO), in this pathway are absent humans. PfMQO catalyzes the oxidation of L-malate to oxaloacetate and the simultaneous reduction of ubiquinone to ubiquinol. It is a membrane protein, involved in three pathways (ETC, the tricarboxylic acid cycle and the fumarate cycle) and has been shown to be essential for parasite survival, at least, in the intra-erythrocytic asexual stage. These findings indicate that PfMQO would be a valuable drug target for development of antimalarial with novel mechanism of action. Up to this point in time, difficulty in producing active recombinant mitochondrial MQO has hampered biochemical characterization and targeted drug discovery with MQO. Here we report for the first time recombinant PfMQO overexpressed in bacterial membrane and the first biochemical study. Furthermore, about 113 compounds, consisting of ubiquinone binding site inhibitors and antiparasitic agents, were screened resulting in the discovery of ferulenol as a potent PfMQO inhibitor. Finally, ferulenol was shown to inhibit parasite growth and showed strong synergism in combination with atovaquone, a well-described anti-malarial and bc1 complex inhibitor.  相似文献   

17.
Fumarase-free electron particles from Propionibacterium freudenreichii and P. pentosaceum were prepared by discontinuous sucrose gradient centrifugation, and the influence of 2-n-heptyl-4-hydroxy-quinoline-N-oxide (HQNO) and ultraviolet irradiation on the reduction of menaquinone and cytochrome b with l-lactate or glycerol-3-phosphate and the reoxidation by fumarate was studied. In the presence of HQNO the steady state reduction level of menaquinone during fumarate reduction was increased whereas the steady state reduction level of cytochrome b was decreased as compared with the reduction levels measured in the absence of HQNO. The steady state reduction level of menaquinone during electron transport to fumarate was not influenced by ultraviolet irradiation and the steady state reduction level of cytochrome b was decreased at increasing irradiation times. The data indicate that cytochrome b is involved in the electron transport to fumarate.Abbreviations HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NQNO 2-n-nonyl-4-hydroxyquinoline-N-oxide Visiting Professor at the Biological Laboratory  相似文献   

18.
The effect of respiration on the positive phototactic movement of swarming agar colonies of the facultative phototroph Rhodospirillum centenum was studied. When the electron flow was blocked at the bc 1 complex level by myxothiazol, the oriented movement of the colonies was totally blocked. Conversely, inhibition of respiration via the cytochrome c oxidase stimulated the phototactic response. No phototaxis was observed in a photosynthesis deficient mutant (YB707) lacking bacteriochlorophylls. Analyses of the respiratory activities as monitored by a oxygen microelectrode in single agar colonies during light/dark transitions showed a close functional correlation between the photosynthetic and respiratory apparatuses. The respiratory chain of Rsp. centenum was formed by two oxidative pathways: one branch leading to a cytochrome c oxidase inhibited by low cyanide concentrations and a second pathway formed by an oxidase less-sensitive to cyanide that also catalyzes the light-driven respiration. These results were interpreted to indicate that (1) there is a cyclic electron transport, and (2) photoinduced cyclic electron flow is required for the phototactic response of Rsp. centenum. Furthermore, under oxic conditions in the light, reducing equivalents may switch from photosynthetic to respiratory components so as to reduce both the membrane potential and the rate of locomotion. Received: 25 September 1996 / Accepted: 11 November 1996  相似文献   

19.
刘鹏程  朱雯雯  肖翔 《微生物学通报》2015,42(11):2238-2244
以模式菌株Shewanella oneidensis MR-1为代表的Shewanella菌属产电微生物广泛分布于自然水体环境中。作为兼性厌氧菌,Shewanella菌除了能进行有氧呼吸外,还能利用多种电子受体进行厌氧呼吸。通过多种细胞色素所组成的复杂电子传递网络,Shewanella菌不仅能利用渗入到周质空间的可溶性电子受体进行厌氧呼吸,更为特殊的是其能够借助电子的跨膜传递实现对胞外不溶性电子受体的异化还原代谢。本文概述了近年来Shewanella菌厌氧代谢途径的研究进展,探讨电子传递网络对Shewanella菌呼吸多样性及环境适应性的影响。  相似文献   

20.
The effects of organic nitrogen on the metabolism of Clostridium acetobutylicum were investigated in batch fermentations. For this study, amino acids were added to a chemically defined medium in groups from the same biosynthetic pathways. In all cases the addition of amino acids shifted the solvent ratio to higher butanol production at the expense of that of acetone (except for the glutamic acid group) and ethanol (except for histidine). Highest biomass production was obtained from media containing aromatic amino acids and histidine (4.57 g · l−1 and 5.4 g · l−1, respectively). However, the solvent production (ca. 20 g · l−1) and the solvent yield (ca. 33%) in both cases, were similar to those obtained from the synthetic medium. Lower values were obtained from fermentations carried out with other families of amino acids. The strongest inhibition of cell growth (1.13 g · l−1) which related to the lowest solvent production (3.15 g · l−1) was observed on a medium complemented with amino acids of the pyruvic acid group. During the second phase of fermentation, amino acids-complemented media caused a less efficient remetabolization of acetic and butyric acids. Highest production of acids was obtained with the aspartic acid group (7.4 g · l−1). These observations suggest that amino acids can be used as a competitive nitrogen source and also modify the level of enzyme activities involved in acid and solvent production.  相似文献   

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