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Studies on energy-linked reactions. Energy-linked transhydrogenase reaction in Escherichia coli.

ABSTRACT: 1. Small particles from Escherichia coli catalyse an ATP-dependent reduction of NADP(+) by NADH. 2. The reaction was stimulated by Mg(2+) and only ATP and ITP could serve as energy donors. 3. Studies of the stoicheiometry of the reaction indicate that 1-2mol of ATP are utilized/mol of NADH produced. 4. The reaction is inhibited by uncoupling agents such as 2,4-dinitrophenol (200mum) and tetrachlorotrifluorobenzimidazole (2mum) but oligomycin does not inhibit. 5. The reaction is inhibited by relatively high concentrations (200mum) of piericidin A.

SUBMITTER: Sweetman AJ

PROVIDER: S-EPMC1176494 | biostudies-other | 1971 Jan

REPOSITORIES: biostudies-other

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The energy-linked transhydrogenase reaction in respiratory mutants of Escherichia coli K12.

Project description:1. Energy-linked and non-energy-linked transhydrogenase activities were assayed in membrane preparations from normal Escherichia coli K 12 and from various mutant strains. 2. The energy-linked transhydrogenase, which uses ATP as energy source, was dependent for activity on the presence of a functional Mg(2+)+Ca(2+)-stimulated adenosine triphosphatase. 3. Neither of the quinones formed by E. coli, namely ubiquinone-8 and menaquinone-8, was required for normal ATP-dependent energy-linked transhydrogenase activity. 4. The energy-linked transhydrogenase was inhibited by piericidin A at a site unrelated to the sites of inhibition of the electron-transport chain by piericidin A.

| S-EPMC1178083 | biostudies-other

Studies on energy-linked reactions. Energy-linked reduction of oxidized nicotinamide-adenine dinucleotide by succinate in Escherichia coli.

Project description:1. A method for the preparation of small particles from Escherichia coli is described. 2. These particles catalyse the ATP-driven reduction of NAD(+) by succinate. 3. ATP utilized/NADH produced ratios below 2.0 were measured and a stoicheiometry of 1 molecule of ATP utilized per molecule of NADH produced is proposed. 4. The reaction is not inhibited by 2,4-dinitrophenol or by oligomycin but is inhibited by other uncouplers such as pentabromophenol and dicoumarol. 5. The specificity of the energy source, the specificity of the electron acceptor, the effects of pH, Mg(2+), P(i) and temperature have been studied.

| S-EPMC1176493 | biostudies-other

Energy transfer between the nicotinamide nucleotide transhydrogenase and ATP synthase of Escherichia coli.

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A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency.

Project description:Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.

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The udhA gene of Escherichia coli encodes a soluble pyridine nucleotide transhydrogenase.

Project description:The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.

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Studies of energy-linked reactions. Inhibition of oxidative phosphorylation by DL-8-methyldihydrolipoate.

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Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase.

Project description:1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg(2+),Ca(2+)-stimulated adenosine triphosphatase. 2. The Mg(2+),Ca(2+)-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg(2+),Ca(2+)-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA(-)) could not be re-activated by the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris-HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg(2+),Ca(2+)-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA(-)).

| S-EPMC1177644 | biostudies-other

Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)-2-bromopropionate.

Project description:Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine-biosynthetic pathway converting pyruvate and L-aspartate-β-semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)-2-bromopropionate inactivates the enzyme in a pseudo-first-order process. An initial velocity pattern indicates that (S)-2-bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)-2-bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml-1 DHDPS. The crystals diffracted to 2.15 Å resolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active-site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half-reaction of the ping-pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)-2-bromopropionate indicates the covalent adduct formed from (S)-2-bromopropionate leads to a rotation of about 180° of the β-δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate.

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Studies of energy-linked reactions. Localization of the site of action of trialkyltin in yeast mitochondria.

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| S-EPMC1164640 | biostudies-other

The Escherichia coli transcriptome linked to growth fitness.

Project description:A series of Escherichia coli strains with varied genomic sequences were subjected to high-density microarray analyses to elucidate the fitness-correlated transcriptomes. Fitness, which is commonly evaluated by the growth rate during the exponential phase, is not only determined by the genome but is also linked to growth conditions, e.g., temperature. We previously reported genetic and environmental contributions to E. coli transcriptomes and evolutionary transcriptome changes in thermal adaptation. Here, we describe experimental details on how to prepare microarray samples that truly represent the growth fitness of the E. coli cells. A step-by-step record of sample preparation procedures that correspond to growing cells and transcriptome data sets that are deposited at the GEO database (GSE33212, GSE52770, GSE61739) are also provided for reference.

| S-EPMC4778597 | biostudies-literature

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