Fumarate reductase of Clostridium formicoaceticum

When Clostridium formicoaceticum was grown on fumarate or l-malate crude cell extracts contained a high fumarate reductase activity. Using reduced methyl viologen as electron donor the specific activity amounted to 2–3.5 U per mg of protein. Reduced benzyl viologen, FMNH₂ and NADH could also serve as electron donors but the specific activities were much lower. The NADH-dependent activity was strictly membrane-bound and rather labile. Its specific activity did not exceed 0.08 U per mg of particle protein. Fumarate reductase activity was also found in cells of C. formicoaceticum grown on fructose, gluconate, glutamate and some other substrates.The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions. The preparation of spheroplasts from cells of C. formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium. Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure. These results indicate a peripheral location of the fumarate reductase of C. formicoaceticum on the membrane.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - O.D optical density - DTE dithioerythritol     

Identification of lysine-78 as an essential residue in the Saccharomyces cerevisiae xylose reductase

Yeast xylose reductases are hypothesized as hybrid enzymes as their primary sequences contain elements of both the aldo-keto reductases (AKR) and short chain dehydrogenase/reductase (SDR) enzyme families. During catalysis by members of both enzyme families, an essential Lys residue H-bonds to a Tyr residue that donates proton to the aldehyde substrate. In the Saccharomyces cerevisiae xylose reductase, Tyr49 has been identified as the proton donor. However, the primary sequence of the enzyme contains two Lys residues, Lys53 and Lys78, corresponding to the conserved motifs for SDR and AKR enzyme families, respectively, that may H-bond to Tyr49. We used site-directed mutagenesis to substitute each of these Lys residues with Met. The activity of the K53M variant was slightly decreased as compared to the wild-type, while that of the K78M variant was negligible. The results suggest that Lys78 is the essential residue that H-bonds to Tyr49 during catalysis and indicate that the active site residues of yeast xylose reductases match those of the AKR, rather than SDR, enzymes. Intrinsic enzyme fluorescence spectroscopic analysis suggests that Lys78 may also contribute to the efficient binding of NADPH to the enzyme.     

Fumarate reductase is a soluble enzyme in anaerobically grown Shewanella putrefaciens MR-1

Abstract The expression and distribution of fumarate reductase activity was examined in Shewanella putrefaciens MR-1. Fumarate reductase was expressed at very low levels in aerobically grown cell and was markedly induced by growth under anaerobic conditions. Cells were fractionated into soluble and purified membrane components by four different methods. For all four methods used, and in marked contrast to the membrane-bound fumarate reductases of other bacteria, ≧ 98% of the fumarate reductase activity was localized in the soluble fraction. In cells subjected to osmotic shock or treated with lysozyme and EDTA to form spheroplasts, the specific activity of fumarate reductase was highest in the periplasmic fraction, while the majority of total fumarate reductase activity was in the cytoplasmic fraction.     

Characterization of the respiratory chain of Helicobacter pylori

The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous substrate for accepting electrons from NADH. The activity of NADH-FRD was inhibited by 2-thenoyltrifluoroacetone. A tentative scheme for the electron transfer pathway in H. pylori is proposed, which may be helpful in clarifying the pathogenesis of H. pylori and in opening new lines for chemotherapy against this bacterium.     

Characterization of E. coli MG1655 and frdA and sdhC mutants at various aerobiosis levels

Depending on the availability of oxygen, Escherichia coli is able to switch between aerobic respiratory metabolism and anaerobic mixed acid fermentation. An important, yet understudied, metabolic mode is the micro-aerobic metabolism at intermediate oxygen availabilities. The relationship between oxygen input, physiology and gene expression of E. coli MG1655 and two isogenic mutants lacking succinate dehydrogenase (SDH) and fumarate reductase (FRD) activities was analyzed at different aerobiosis levels. Growth rate and cell yield were very similar to the parent strain. By-product formation was altered in the sdhC mutant to higher acetic acid and glutamate production in batch cultures. In continuous cultures with defined oxygen input gene expression analysis revealed a dependency of many catabolic genes to aerobiosis. Acetate excretion was still detectable under aerobic conditions in the sdhC mutant; the frdA mutant lacked anaerobic succinate excretion. Anaerobic repression of the sdh operon was diminished in the frdA strain, possibly to allow SDH to partially replace FRD. The experiments illustrate the remarkable adaptability of E. coli physiology—to compensate for the absence of important metabolic genes by altering carbon flux and/or gene expression such that there are only minor changes in growth capability across the aerobiosis range.     

棉花GhDHAR2基因克隆、功能序列分析及原核表达

通过RT-PCR方法从棉花纤维组织中克隆得到脱氢抗坏血酸还原酶基因GhDHAR2的cDNA,该基因开放阅读框为639 bp,编码212个氨基酸的蛋白质。同源性序列对比分析显示,GhDHAR2蛋白具有较高的保守性,具有典型的功能结构域,包括GST-N家族和GST-C-DHAR家族的功能结构域;进化树分析显示GhDHAR2和拟南芥AtDHAR2在进化关系上较近。将GhDHAR2基因连接到原核表达载体pET-28a中,将重组载体pET28a-GhDHAR2转入到表达菌株BL21(DE3)中,通过IPTG诱导表达出重组GhDHAR2蛋白,SDS-PAGE凝胶电泳分析显示重组蛋白大小约为28 kD,诱导表达的重组蛋白具有较高的DHAR活性。首次克隆了棉花GhDHAR2基因,通过结构域分析其可能的作用,并成功进行蛋白体外表达及酶活性分析。     

Activity assay of mammalian 2-cys peroxiredoxins using yeast thioredoxin reductase system

2-Cys peroxiredoxin (Prx) is a novel cellular peroxidase that reduces peroxides in the presence of thioredoxin, thioredoxin reductase, and nicotinamide adenine dinucleotide phosphate (NADPH) and that functions in H(2)O(2)-mediated signal transduction. Recent studies have shown that 2-cys Prx can be inactivated by cysteine overoxidation in conditions of oxidative stress. Therefore, peroxidase activity, rather than the protein level, of 2-cys Prx is the more important measure to predict its cellular function. Here, we introduce a modified activity assay method for mammalian 2-cys Prx based on yeast nonselenium thioredoxin reductase. Yeast thioredoxin reductase is expressed in Escherichia coli cells and purified at high yield (40 mg/L of culture broth) as an active flavoprotein by combined diethyl aminoethyl (DEAE) and phenyl hydrophobic chromatography. The optimal concentrations of yeast thioredoxin and thioredoxin reductase required to achieve maximum mammalian 2-cys Prx activity are 3.0 and 1.5 microM, respectively. This modified assay method is useful for measuring 2-cys Prx activity in cell lysates and can also be adapted for a 96-well plate reader for high-throughput screening of chemical compounds that target 2-cys Prx.     

Physiological role of soluble fumarate reductase in redox balancing during anaerobiosis in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, there are two isoenzymes of fumarate reductase (FRDS1 and FRDS2), encoded by the FRDS and OSM1 genes, respectively. Simultaneous disruption of these two genes results in a growth defect of the yeast under anaerobic conditions, while disruption of the OSM1 gene causes slow growth. However, the metabolic role of these isoenzymes has been unclear until now. In the present study, we found that the anaerobic growth of the strain disrupted for both the FRDS and OSM1 genes was fully restored by adding the oxidized form of methylene blue or phenazine methosulfate, which non-enzymatically oxidize cellular NADH to NAD(+). When methylene blue was added at growth-limiting concentrations, growth was completely arrested after exhaustion of oxidized methylene blue. In the double-disrupted strain, the accumulation of succinate in the supernatant was markedly decreased during anaerobic growth in the presence of methylene blue. These results suggest that fumarate reductase isoenzymes are required for the reoxidation of intracellular NADH under anaerobic conditions, but not aerobic conditions.     

The role of ntrA in the anaerobic metabolism of Salmonella typhimurium

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.     

Isolation of mitochondria from Plasmodium falciparum showing dihydroorotate dependent respiration.

Using N₂ cavitation, we established a protocol to prepare the active mitochondria from Plasmodium falciparum showing a higher succinate dehydrogenase activity than previously reported and a dihydroorotate-dependent respiration. The fact that fumarate partially inhibited the dihydroorotate dependent respiration suggests that complex II (succinate–ubiquinone reductase/quinol–fumarate reductase) in the erythrocytic stage cells of P. falciparum functions as a quinol–fumarate reductase.     

On the catalytic role of the active site residue E121 of E. colil-aspartate oxidase

l-aspartate oxidase (LASPO) is a flavoenzyme catalyzing the first step in the de novo biosynthesis of NAD⁺. The enzyme oxidizes l-aspartate both under aerobic and anaerobic conditions using oxygen as well as fumarate as electron acceptor.     

A sodium-stimulated membrane-bound fumarate reductase system in Bacteroides amylophilus

Membrane vesicles derived from whole cells of the strictly anaerobic rumen bacterium Bacteroides amylophilus exhibited fumarate reductase activity with NADH, FADH₂, FMNH₂, or reduced viologens as electron donors. The fumarate reductase system is most likely localized on the cytoplasmic side of the plasma membrane. Cytochromes and menaquinone were not detectable.The NADH-dependent activity was inactivated by oxygen, an endogenous protease, and by irradiation at 254 nm. The electron transport inhibitor HpHOQnO and Zn²⁺ were identified as strong inhibitors of the fumarate reductase reaction. Two types of functional SH-groups might be operative in this system as probed by ClHgSO₃H. The oxidation of NADH by fumarate was stimulated by low concentrations of Na⁺.Concentrations of Na⁺ in the range of 4 to 30 mM had a pronounced influence on growth rate and cell yield of B. amylophilus. In the presence of 1 mM NaCl growth was observed only after a lag-period of 15 h.Abbreviations ClHgSO₃H 4-chloromercuriphenylsulfonate - DTE dithioerythritol - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - HpHOQnO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NoHOQnO 2-n-nonyl-4-hydroxyquinoline-N-oxide - PMSF phenylmethylsulfonylfluoride - Tris tris(hydroxymethyl)-aminomethaneDedicated to Prof. N. Pfennig on the occasion of his 60th birthdayPresent address: Heinz-Georg Wetzstein, Bayer AG, EP-AQ-QL, E39, D-5090 Leverkusen, FRG     

人成骨蛋白成熟肽基因在毕氏酵母(Pichia Yeast)中的高效表达

采用PCR方法,根据文献报道的人成骨蛋白（osteogenic protein-1,OP-1）成熟肽基因序列,设计并合成一对引物,从构建的含人成骨蛋白基因的质粒中扩增获得大小为420bp的DNA片段,连接到pGEM-T载体进行测序,证明获得人成骨蛋白成熟肽基因片段,并以pPIC9K为表达载体构建重组表达质粒,转化大肠杆菌细胞,经鉴定的阳性重组质粒并线形化,电转化毕氏酵母细胞GS115,于30℃进行甲醇诱导分泌表达,表达产物存在于培养基中,占分泌蛋白的10％.重组表达产物进行Western Blot可以检测到重组表达产物,ELISA检测其具有特异性结合活性.     

Solvent treatment for β-d-galactosidase release from yeast cells

A method is presented for the release of β-d-galactosidase (β-d-galactoside galactohydrolase, EC 3.2.1.23) from yeast cells. Enzyme release is attained by mixing yeast cells with concentrated solvents (20 to 95%) and subsequently suspending and agitating the cells in buffer. Many solvents, including isopropanol, ethanol and methanol, were found to be effective. Enzyme release into buffer was relatively slow: 10–20 h was required for maximum yields. The release of protease and β-d-galactosidase was monitored. β-d-Galactosidase solubilization was achieved in high yield: 90% of the intracellular enzyme was released into the buffer. Because this method exhibits resistance to yield loss due to microbial degradation and is not sensitive to small changes in solvent in buffer concentration or treatment time, it is particularly suited to industrial-scale enzyme recoveries.