title: 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.
authors.affiliation: Instituto de Tecnologia Química e Biológica - António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.
authors.forename: Filipa V
authors.initials: FV
authors.lastname: Sena
authors.affiliation: Instituto de Tecnologia Química e Biológica - António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.
authors.forename: Ana P
authors.initials: AP
authors.lastname: Batista
authors.affiliation: Instituto de Tecnologia Química e Biológica - António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.|Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Teresa
authors.initials: T
authors.lastname: Catarino
authors.affiliation: Instituto de Tecnologia Química e Biológica - António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.
authors.forename: José A
authors.initials: JA
authors.lastname: Brito
authors.affiliation: Instituto de Tecnologia Química e Biológica - António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.
authors.forename: Margarida
authors.initials: M
authors.lastname: Archer
authors.affiliation: Center for Integrated Protein Science Munich (CIPSM) at the Department of Chemistry Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany.|Institute of Structural Biology, Helmholtz Zentrum München Neuherberg and Biomolecular NMR- Spectroscopy, Medical University of Graz, 8010, Graz, Austria.|Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010, Graz, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Viertler
authors.affiliation: Center for Integrated Protein Science Munich (CIPSM) at the Department of Chemistry Technische Universität München, Lichtenbergstr.4, 85747, Garching, Germany.|Institute of Structural Biology, Helmholtz Zentrum München Neuherberg and Biomolecular NMR- Spectroscopy, Medical University of Graz, 8010, Graz, Austria.|Institute of Molecular Biology & Biochemistry, Center of Molecular Medicine, Medical University of Graz, 8010, Graz, Austria.
authors.forename: Tobias
authors.initials: T
authors.lastname: Madl
authors.affiliation: REQUIMTE, UCIBIO, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516, Caparica, Portugal.
authors.forename: Eurico J
authors.initials: EJ
authors.lastname: Cabrita
authors.affiliation: Instituto de Tecnologia Química e Biológica - António Xavier, Universidade Nova de Lisboa, Av. da República EAN, 2780-157, Oeiras, Portugal.
authors.forename: Manuela M
authors.initials: MM
authors.lastname: Pereira
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D055808:Drug Discovery; D004579:Electron Transport; D006912:Hydroxyquinolines / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D010084:Oxidation-Reduction; D055503:Protein Multimerization; D011808:Quinone Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011809:Quinones / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006912:Hydroxyquinolines; D011809:Quinones; C001333:2-(n-heptyl)-4-hydroxyquinoline N-oxide; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases