Preparations of Terminal Oxidase Cytochrome bd-II Isolated from Escherichia coli Reveal Significant Hydrogen Peroxide Scavenging Activity

Biochemistry (Moscow) - Cytochrome bd-II is one of the three terminal quinol oxidases of the aerobic respiratory chain of Escherichia coli. Preparations of the detergent-solubilized untagged bd-II...     

Modified Recombinant Proteins Can Be Exported via the Sec Pathway in Escherichia coli

The correct folding of a protein is a pre-requirement for its proper posttranslational modification. The Escherichia coli Sec pathway, in which preproteins, in an unfolded, translocation-competent state, are rapidly secreted across the cytoplasmic membrane, is commonly assumed to be unfavorable for their modification in the cytosol. Whether posttranslationally modified recombinant preproteins can be efficiently transported via the Sec pathway, however, remains unclear. ACP and BCCP domain (BCCP87) are carrier proteins that can be converted into active phosphopantetheinylated ACP (holo-ACP) and biotinylated-BCCP (holo-BCCP) by AcpS and BirA, respectively. In the present study, we show that, when ACP or BCCP87 is fused to the C-terminus of secretory protein YebF or MBP, the resulting fusion protein preYebF-ACP, preYebF-BCCP87, preMBP-ACP or preMBP-BCCP87 can be modified and then secreted. Our data demonstrate that posttranslational modification of preYebF-ACP, preYebF-BCCP87 preMBP-ACP and preMBP-BCCP87 can take place in the cytosol prior to translocation, and the Sec machinery accommodates these previously modified fusion proteins. High levels of active holo-ACP and holo-BCCP87 are achieved when AcpS or BirA is co-expressed, especially when sodium azide is used to retard their translocation across the inner membrane. Our results also provide an alternative to achieve a high level of modified recombinant proteins expressed extracellularly.     

牛泡沫病毒长末端重复序列在大肠杆菌中的启动子功能

泡沫病毒具有复杂的基因组结构,包含典型反转录病毒共有的长末端重复序列(long terminal repeat,LTR)启动子,负责起始结构基因gag、pol、env的表达,并在env基因内部存在独有的内部启动子(internal promoter,IP)[13],用以起始下游反式作用因子Tas(在BFV中称Borf-1)的表达.有报道劳斯肉瘤病毒(rous sarcoma virus,RSV)和人免疫缺陷病毒(human immunodeficiency virus,HIV)LTR可在E.coli中起始基因表达[4,7].泡沫病毒至今未见报道.     

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.     

Effect of Temperature on the Activities of Cytochrome c Oxidase and Respiration in Cassava Root Mitochondria

Cosmid pR4Cl is a derivative of multicopy plasmid pIJ365 which has an insertion of the cos (cohesive end site) region of actinophage R4 [T. Morino, H. Takahashi and H. Saito, Mol. Gen. Genet., 198, 228 (1985)]. When the donor R4 phage was propagated in S. lividans carrying the plasmid, the phage lysate contained transducing particles which encapsulated head-to-tail concatemers of the plasmid DNA. These particles could mediate transfer of the plasmid at a high frequency. We examined conditions that gave a maximum transduction frequency of cosmid pR4Cl. Conditions which depress R4 phage propagation, such as incubation of recipient S. parvulus at a high temperature, improved the frequency. Obviously such conditions minimized the lethal effect of viable phage propogation. The highest transduction frequency obtained so far was around 3 × 10^-3 transductants per infected phage when S. lividans was used as the recipient. This was about 30 per cent of the cosmid transducing particles estimated from the cosmid DNA content in the transducing lysate. The significance of cosmid transduction for gene manipulation in Streptomyces strains is also discussed.     

Concentrations of Copper Thought To Be Toxic to Escherichia coli Can Induce the Viable but Nonculturable Condition

We have determined that concentrations of copper considered to be toxic can induce a fraction of a population of Escherichia coli to enter the viable but nonculturable (VBNC) condition. Copper-induced VBNC cells could be resuscitated for up to 2 weeks after entering the VBNC state.     

植物交替氧化酶(AOX)在大肠杆菌中优化表达

植物交替氧化酶(Alternative Oxidase,AOX)位于高等植物线粒体内膜,从细胞色素途径的辅酶Q分岔,催化4个电子还原氧分子形成水的另一终端氧化酶。分离纯化有活性的AOX比较困难。本文研究AOX原核表达,选择pFLAG-1分泌表达载体,用异丙基硫代-β-D-半乳糖苷(IPTG)诱导AOX优化表达,pFLAG-1-AOX大肠杆菌优化表达条件为:宿主DH5α、温度37℃、细胞密度OD600=0.6、IPTG浓度0.2mmol/L,诱导后60min收获细胞;获得少量可溶的细胞外周质AOX和大量不溶的AOX,为深入研究AOX打下基础,同时为研究膜蛋白原核表达提供依据。     

Control of Respiration by Cytochrome c Oxidase in Intact Cells: ROLE OF THE MEMBRANE POTENTIAL*

Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Δψ) and chemical (ΔpH) component of the mitochondrial transmembrane proton electrochemical gradient (Δμ_H⁺) was investigated. The overall O₂ consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Δψ and ΔpH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (C_IV = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (C_IV = 0.85) was also measured in the presence of nigericin, i.e. when Δψ is maximal, and in the presence of nigericin and valinomycin (C_IV = 0.77), when Δμ_H⁺ is abolished. In contrast, CcOX displayed a markedly lower control coefficient (C_IV = 0.30) upon addition of valinomycin, when Δψ is converted into ΔpH. These results show that Δψ is responsible for the tight control of CcOX over respiration in actively phosphorylating cells.     

菠菜乙醇酸氧化酶基因的克隆及表达

采用RT-PCR技术从菠菜总RNA中分离扩增了乙醇酸氧化酶（GO）基因的cDNA序列,首先克隆到质粒pMD18T,进行了测序。然后将乙醇酸氧化酶的cDNA分别亚克隆至质粒pThioHisC、 pTIGTrx、pBV220和pET-2b(+),分别转化大肠杆菌DH5α和BL21（DE3）,并对重组乙醇酸氧化酶在大肠杆菌中的表达进行了研究。SDSPAGE和酶活分析表明,菠菜乙醇酸氧化酶在E.coli BL21 (DE3) （pTIGTrxGO）和E.coli BL21(DE3) （pET-22b(+)GO）里得到了高水平的表达,其中E.coli BL21(DE3) （pET-22b(+)GO）的乙醇酸氧化酶活性较高。     

Loss of Function of Phosphodiesterase 11A4 Shows that Recent and Remote Long-Term Memories Can Be Uncoupled

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Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H₂O₂ was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H₂O₂, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.     

The Dinuclear Center of Cytochrome bo 3 from Escherichia coli

For the study of the dinuclear center of heme-copper oxidases cytochrome bo ₃ from Escherichia coli offers several advantages over the extensively charactererized bovine cytochrome c oxidase. The availability of strains with enhanced levels of expression allows purification of the significant amounts of enzyme required for detailed spectroscopic studies. Cytochrome bo ₃ is readily prepared as the fast form, with a homogeneous dinuclear center which gives rise to characteristic broad EPR signals not seen in CcO. The absence of Cu_A and the incorporation of protohemes allows for a detailed interpretation of the MCD spectra arising from the dinuclear center heme o ₃. Careful analysis allows us to distinguish between small molecules that bind to heme o ₃, those which are ligands of Cu_B, and those which react to yield higher oxidation states of heme o ₃. Here we review results from our studies of the reactions of fast cytochrome bo ₃ with formate, fluoride, chloride, azide, cyanide, NO, and H₂O₂.     

Evolution of the Cytochrome c Oxidase Proton Pump

The superfamily of quinol and cytochrome c terminal oxidase complexes is related by a homologous subunit containing six positionally conserved histidines that ligate a low-spin heme and a heme–copper dioxygen activating and reduction center. On the basis of the structural similarities of these enzymes, it has been postulated that all members of this superfamily catalyze proton translocation by similar mechanisms and that the Cu_A center found in most cytochrome c oxidase complexes serves merely as an electron conduit shuttling electrons from ferrocytochrome c into the hydrophobic core of the enzyme. The recent characterization of cytochrome c oxidase complexes and structurally similar cytochrome c:nitric oxide oxidoreductase complexes without Cu_A centers has strengthened this view. However, recent experimental evidence has shown that there are two ubiquinone(ol) binding sites on the Escherichia coli cytochrome bo ₃ complex in dynamic equilibrium with the ubiquinone(ol) pool, thereby strengthening the argument for a Q(H₂)-loop mechanism of proton translocation [Musser SM et al. (1997) Biochemistry 36:894–902]. In addition, a number of reports suggest that a Q(H₂)-loop or another alternate proton translocation mechanism distinct from the mitochondrial aa ₃ -type proton pump functions in Sulfolobus acidocaldarius terminal oxidase complexes. The possibility that a primitive quinol oxidase complex evolved to yield two separate complexes, the cytochrome bc ₁ and cytochrome c oxidase complexes, is explored here. This idea is the basis for an evolutionary tree constructed using the notion that respiratory complexity and efficiency progressively increased throughout the evolutionary process. The analysis suggests that oxygenic respiration is quite an old process and, in fact, predates nitrogenic respiration as well as reaction-center photosynthesis. Received: 11 June 1997 / Accepted: 30 October 1997