Showing papers by "Manuela M. Pereira published in 2015"

Type-II NADH:quinone oxidoreductase from Staphylococcus aureus has two distinct binding sites and is rate limited by quinone reduction.

Abstract: A prerequisite for any rational drug design strategy is understanding the mode of protein-ligand interaction. This motivated us to explore protein-substrate interaction in Type-II NADH:quinone oxidoreductase (NDH-2) from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms. NDHs-2 are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, as these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We report fast kinetic analyses of the protein and detected a charge-transfer complex formed between NAD(+) and the reduced flavin, which is dissociated by the quinone. We observed that the quinone reduction is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor. We analyzed protein-substrate interactions by fluorescence and STD-NMR spectroscopies, which indicate that NADH and the quinone bind to different sites. In summary, our combined results show the presence of distinct binding sites for the two substrates, identified quinone reduction as the rate limiting step and indicate the establishment of a NAD(+)-protein complex, which is released by the quinone.

Expression, purification, crystallization and preliminary X‐ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus

Abstract: In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents In this work, the NDH-II enzyme from the Gram-positive human pathogen Staphylococcus aureus was recombinantly expressed in Escherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0873 A The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 818, b = 860, c = 2699 A, contained four monomers per asymmetric unit and diffracted to a resolution of 332 A A molecular-replacement solution was obtained and model building and refinement are currently under way