Functionality of the Na+-translocating NADH:quinone oxidoreductase and quinol:fumarate reductase from Prevotella bryantii inferred from homology modeling.

Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiota. In Prevotella bryantii, the Na+-translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction. P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. To elucidate the possible mode of function, we present 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking reveals the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR. Our findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR.

The low mutational flexibility of the EPSP synthase in Bacillus subtilis is due to a higher demand for shikimate pathway intermediates.

Glyphosate (GS) inhibits the 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase that is required for aromatic amino acid, folate and quinone biosynthesis in Bacillus subtilis and Escherichia coli. The inhibition of the EPSP synthase by GS depletes the cell of these metabolites, resulting in cell death. Here, we show that like the laboratory B. subtilis strains also environmental and undomesticated isolates adapt to GS by reducing herbicide uptake. Although B. subtilis possesses a GS-insensitive EPSP synthase, the enzyme is strongly inhibited by GS in the native environment. Moreover, the B. subtilis EPSP synthase mutant was only viable in rich medium containing menaquinone, indicating that the bacteria require a catalytically efficient EPSP synthase under nutrient-poor conditions. The dependency of B. subtilis on the EPSP synthase probably limits its evolvability. In contrast, E. coli rapidly acquires GS resistance by target modification. However, the evolution of a GS-resistant EPSP synthase under non-selective growth conditions indicates that GS resistance causes fitness costs. Therefore, in both model organisms, the proper function of the EPSP synthase is critical for the cellular viability. This study also revealed that the uptake systems for folate precursors, phenylalanine and tyrosine need to be identified and characterized in B. subtilis.

Conformational coupling of redox-driven Na+-translocation in Vibrio cholerae NADH:quinone oxidoreductase.

In the respiratory chain, NADH oxidation is coupled to ion translocation across the membrane to build up an electrochemical gradient. In the human pathogen Vibrio cholerae, the sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) generates a sodium gradient by a so far unknown mechanism. Here we show that ion pumping in Na+-NQR is driven by large conformational changes coupling electron transfer to ion translocation. We have determined a series of cryo-EM and X-ray structures of the Na+-NQR that represent snapshots of the catalytic cycle. The six subunits NqrA, B, C, D, E, and F of Na+-NQR harbor a unique set of cofactors that shuttle the electrons from NADH twice across the membrane to quinone. The redox state of a unique intramembranous [2Fe-2S] cluster orchestrates the movements of subunit NqrC, which acts as an electron transfer switch. We propose that this switching movement controls the release of Na+ from a binding site localized in subunit NqrB.

Fast IMAC purification of non-tagged S100A8/A9 (calprotectin) from Homo sapiens and Sus scrofa.

S100A8/A9 (calprotectin) is a damage-associated molecular pattern molecule (DAMP) that plays a key role in the innate immune response of mammalia. S100A8/A9 is therefore widely used as a biomarker in human and veterinary medicine, but diagnostic tools for the detection of S100A8/A9 are rarely optimised for the specific organism, since the corresponding S100A8/A9 is often not available. There is need for an easy, reliable protocol for the production of recombinant, highly pure S100A8/A9 from various mammalia. Here we describe the expression and purification of recombinant human and porcine S100A8/A9 by immobilized metal affinity chromatography (IMAC), which takes advantage of the intrinsic, high-affinity binding of native un-tagged S100A8/A9 to metal ions. Highly pure S100A8/A9 is obtained by a combination of IMAC, ion exchange and size exclusion chromatographic steps. Considering the high sequence homology and conservation of the metal ion coordinating residues of S100A8/A9 metal binding sites, the protocol is presumably applicable to S100A8/A9 of various mammalia.