AbstractHumanity is facing an increasing health threat caused by a variety of multidrug resistant bacteria. Within this scenario, Staphylococcus aureus, in particular methicillin resistant S. aureus (MRSA), is responsible for a number of hospital-acquired bacterial infections. The emergence of microbial antibiotic resistance urgently requires the identification of new and innovative strategies to treat antibiotic resistant microorganisms. In this context, structure and function analysis of potential drug targets in metabolic pathways vital for bacteria endurance, such as the vitamin K₂ synthesis pathway, becomes interesting. We have solved and refined the crystal structure of the S. aureus DHNA thioesterase (SaDHNA), a key enzyme in the vitamin K₂ pathway. The crystallographic structure in combination with small angle X-ray solution scattering data revealed a functional tetramer of SaDHNA. Complementary activity assays of SaDHNA indicated a preference for hydrolysing long acyl chains. Site-directed mutagenesis of SaDHNA confirmed the functional importance of Asp16 and Glu31 for thioesterase activity and substrate binding at the putative active site, respectively. Docking studies were performed and rational designed peptides were synthesized and tested for SaDHNA inhibition activity. The high-resolution structure of SaDHNA and complementary information about substrate binding will support future drug discovery and design investigations to inhibit the vitamin K₂ synthesis pathway.