One‐step purification of the NADH dehydrogenase fragment of the <i>Escherichia coli</i> complex I by means of <i>Strep</i>‐tag affinity chromatography

Bungert, Stefanie; Krafft, Bianca; Schlesinger, Ramona; Friedrich, Thorsten

doi:10.1016/s0014-5793(99)01341-1

Cited by 28 publications

(41 citation statements)

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“…EPR signals observed subsequently were essentially identical and, following the initial study, were matched to N4 also (12,18,19). Similarly, although N4 was identified, EPR spectra of the NADH dehydrogenase fragment of E. coli complex I (NuoE, F, and G) (33,34) reveal no intensity in the region of the true N4 g z signal (2.09-2.11, Table 2). Further evidence to support the assignment of N4 to NuoG is scant and comprises only the results of mutating the cluster ligands.…”

Section: Resultsmentioning

confidence: 67%

Reevaluating the relationship between EPR spectra and enzyme structure for the iron–sulfur clusters in NADH:quinone oxidoreductase

Yakovlev

Reda

Hirst

2007

Proc. Natl. Acad. Sci. U.S.A.

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Section: Resultsmentioning

confidence: 67%

Reevaluating the relationship between EPR spectra and enzyme structure for the iron–sulfur clusters in NADH:quinone oxidoreductase

Yakovlev

Reda

Hirst

2007

Proc. Natl. Acad. Sci. U.S.A.

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“…Expression of NuoEFG NADH: ubiquinone oxidoreductase soluble fragments was performed using a pET11a/nuoB-G/NuoFc expression vector that added a Streptag II to the C-terminus of NuoF, kindly supplied by Professor Thorsten Friedrich (13). Details are given in SI Text.…”

Section: Methodsmentioning

confidence: 99%

“…The overexpression of NuoBCDEFG leads to a properly assembled soluble fragment (NuoEFG) that has an NADH:ubiquinone oxidoreductase activity that can transfer electrons from NADH to ferricyanide, an artificial electron acceptor, with the same efficiency as the entire complex (12). Bungert et al developed a simple and fast method for overexpressing and purifying an active soluble NADH:ubiquinone oxidoreductase fragment using direct affinity chromatography, based on engineered Nuo subunits (13). Using the same strategy, NuoEFG-native and NuoEF*G-evolved soluble fragments were successfully produced and purified.…”

Section: Identification Of Genetic Targets Responsible For E Coli Admentioning

confidence: 99%

Stress-induced evolution of Escherichia coli points to original concepts in respiratory cofactor selectivity

Auriol

Bestel-Corre

Claude

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial metabolism is characterized by a remarkable capacity to rapidly adapt to environmental changes. We restructured the central metabolic network in Escherichia coli to force a higher production of NADPH, and then grew this strain in conditions favoring adaptive evolution. A six-fold increase in growth capacity was attained that could be attributed in multiple clones, after whole genome mutation mapping, to a specific single mutation. Each clone had an evolved NuoF*(E183A) enzyme in the respiratory complex I that can now oxidize both NADH and NADPH. When a further strain was constructed with an even higher degree of NADPH stress such that growth was impossible on glucose mineral medium, a solid-state screening for mutations restoring growth, led to two different types of NuoF mutations in strains having recovered growth capacity. In addition to the previously seen E183A mutation other clones showed a E183G mutation, both having NADH and NADPH oxidizing ability. These results demonstrate the unique solution used by E. coli to overcome the NADPH stress problem. This solution creates a new function for NADPH that is no longer restricted to anabolic synthesis reactions but can now be also used to directly produce catabolic energy. adaptive evolution | NADPH metabolism | complex I E volutionary microbiology under environmental or metabolic constraints is an emergent field of research. Although it is known that growing microorganisms are not static over either short or long periods, the evolutionary process called adaptive evolution developed to adapt to constraints is poorly understood at the genetic, biochemical, and metabolic levels.Experiments of adaptive evolution have been conducted in the laboratory to understand the behavior of Escherichia coli in response to different environmental or metabolic constraints: (i) wide serial subculturing for over 20 years (more than 40,000 generations) on glucose-limited mineral medium (1), (ii) growth for more than 700 generations on glycerol, a maladapted substrate although a complete metabolic pathway for this substrate was already present (2), and (iii) culture over 600-800 generations of single gene deletion mutants affected in metabolic key branch points [phosphoglucose isomerase (Δpgi), phosphoenolpyruvate carboxylase (Δppc), triose phosphoisomerase (Δtpi), phosphotransacetylase (Δpta)] to evaluate the effect of targeted metabolic perturbations on the structure of the metabolic network (3). The main conclusions of this work highlighted the strong robustness of the genetic and metabolic networks of E. coli and indicated that evolution never invented a new solution such as a reassignment of enzyme function to adapt to the environmental or metabolic constraints. Instead, the main feature of evolution was an increased in the capacity of already active pathways or the activation of latent pathways.The objective of the present work was to study the ability of E. coli to adapt to and overcome a strong unbalance and an imposed rigidity of the redox state of cellular metabo...

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“…Complex I from wild type was prepared with the same procedure. The NADH dehydrogenase fragment of the complex was isolated as described (15). For electrochemistry, the preparations were concentrated by ultrafiltration (Centricon 100, Amicon) to ϳ0.2 mM.…”

Section: Materials and Strains-e Coli Strains Dh5␣ (22) An387 (23)mentioning

confidence: 99%

Involvement of Tyrosines 114 and 139 of Subunit NuoB in the Proton Pathway around Cluster N2 in Escherichia coliNADH:Ubiquinone Oxidoreductase

Flemming

Hellwig²,

Friedrich³

2003

Journal of Biological Chemistry

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The proton-pumping NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Electron transfer is accomplished by FMN and a series of iron-sulfur clusters. Its coupling with proton translocation is not yet understood. Here, we report that the redox reaction of the FeS cluster N2 located on subunit NuoB of the Escherichia coli complex I induces a protonation/deprotonation of tyrosine side chains. Electrochemically induced FT-IR difference spectra revealed characteristic tyrosine signals at 1,515 and 1,498 cm ؊1 for the protonated and deprotonated form, respectively. Mutants of three conserved tyrosines on NuoB were generated by complementing a chromosomal in-frame deletion strain with nuoB on a plasmid. Though the single mutations did not alter the electron transport activity of complex I, the EPR signal of cluster N2 was slightly shifted. The tyrosine signals detected by FT-IR spectroscopy were roughly halved in the mutants Y114C and Y139C while only minor changes were detected in the Y154H mutant. The enzymatic activity of the Y114C/Y139F double mutant was 80% reduced, and FT-IR difference spectra of the double mutant revealed a complete loss the modes characteristic for protonation reactions of tyrosines. Therefore, we propose that tyrosines 114 and 139 on NuoB were protonated upon reduction of cluster N2 and were thus involved in the proton-transfer reaction coupled with its redox reaction.The NADH:ubiquinone oxidoreductase, also known as respiratory complex I, links the electron transfer from NADH to ubiquinone with the translocation of protons across the membrane. By this means, complex I establishes a proton motive force required for energy consuming processes (1-3). Homologues of the complex are present in archaea, bacteria, and eukaryotes (4, 5). Complex I from bacteria generally consists of 14 different subunits (4 -6). Seven of these are peripheral proteins including those subunits that bear all known redox groups of complex I, namely one FMN and up to nine ironsulfur (FeS) 1 clusters. The remaining 7 subunits are hydrophobic proteins predicted to fold into 54 ␣-helices across the membrane (1, 2). They are most likely involved in ubiquinone reduction and proton translocation. The mitochondrial complex I of eukaryotes contains at least 29 extra proteins in addition to the homologues of the 14 prokaryotic complex I subunits (1, 2). The genes of the Escherichia coli complex I are named nuoA to nuoN (7). nuoC and D are fused in E. coli giving rise to 13 different subunits assembling the complex (8). The preparation of the E. coli complex I contains one non-covalently bound FMN, two binuclear (N1a and N1b), and five tetranuclear (N2, N3, N4, N6a, and N6b) FeS clusters (Refs. 9, 10, and 11).2 The tetranuclear cluster N5 present in complex I from other species has not yet been detected in this preparation. However, it should be present because of the preservation of the corresponding binding motif (7,12,13). The ...

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One‐step purification of the NADH dehydrogenase fragment of the Escherichia coli complex I by means of Strep‐tag affinity chromatography

Cited by 28 publications

References 16 publications

Reevaluating the relationship between EPR spectra and enzyme structure for the iron–sulfur clusters in NADH:quinone oxidoreductase

Reevaluating the relationship between EPR spectra and enzyme structure for the iron–sulfur clusters in NADH:quinone oxidoreductase

Stress-induced evolution of Escherichia coli points to original concepts in respiratory cofactor selectivity

Involvement of Tyrosines 114 and 139 of Subunit NuoB in the Proton Pathway around Cluster N2 in Escherichia coliNADH:Ubiquinone Oxidoreductase

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