[SCAN Highlight] Manuela Pereira

SCAN Paper highlight

Title: Exploring the energy supply in Staphylococcus aureus: investigation of its respiratory NADH:quinone oxidoreductase.

Speaker: Manuela Pereira

Affiliation: Biological Energy Transduction Lab, ITQB

Paper Highlight

Filipa V. Sena, Ana P. Batista, Teresa Catarino, José A. Brito, Margarida Archer, Martin Viertler, Tobias Madl, Eurico J. Cabrita and Manuela M. Pereira

Type-II NADH:quinone oxidoreductases (NDHs-2) are involved in respiratory chains and recognized as suitable targets for novel antimicrobial therapies, since these are the only enzymes with NADH:quinone oxidoreductase activity expressed in many pathogenic organisms. This motivated us to explore NDH-2 from Staphylococcus aureus, a worldwide problem in clinical medicine due to its multiple drug resistant forms.

We obtained crystal and solution structures of NDH-2 from S. aureus, showing that it is a dimer in solution. We addressed the discussion on the number of substrate binding sites by analyzing protein-substrate interactions using fluorescence and STD-NMR spectroscopies, which revealed the two substrates, NADH and the quinone, bind to different sites. We investigated the catalytic mechanism of the reaction by steady-state and time resolved kinetic analyses and we observed the establishment of charge-transfer complex formed between NAD+ and the reduced cofactor, FAD, of the enzyme. The charge-transfer complex was only dissociated by the quinone, the second substrate, which indicates that a ternary complex is formed during catalysis. In addition we determined the FAD oxidation is the rate limiting step and also the only half-reaction affected by the presence of HQNO, an inhibitor.

In summary, our combined results show the presence of distinct binding sites for the two substrates, identified FAD oxidation as the rate limiting step and indicated the establishment of a NAD+-protein complex, which is released by the quinone.