In order to address the main goal of the project, the assembly and the molecular mechanisms involved in the biosynthesis of S. enterica Hyd-5 have been studied. It has been demonstrated that HydH is essential for the biosynthesis of Hyd-5 under anaerobic and aerobic conditions. In addition, genetic approaches gave valuable information about the HydH interacting proteins that are required for ultimately transferring the precursor cofactor into the large structural subunit. Furthermore, this project has shown that HydD, the maturase of Hyd-5, can be partially replaced by HyaD, the maturase of Hyd-1, in the biosynthesis of the enzyme. In addition, both maturases were shown to form stable complexes with the large structural subunit (HydB). These interactions do not require the presence of the cleavable C-terminal extension of HydB but are dependent on the biosynthesis of a functional [NiFe] cofactor. Furthermore, the project has shown that accessory protein HydG is essential for the biosynthesis of the enzyme under anaerobic and aerobic conditions. On contrast, the essentiality of HydF is related with the levels of O2, being its role redundant under anaerobic conditions. Finally, the host group performed a bio-layer interferometry (BLI) screen and a set of compounds that could be precursors of novel inhibitors of the enzyme were identified. According to their ability to interact with Hyd-5, 28 compounds were selected and validated for their capacity to inhibit hydrogenase activity. However, finding a compound that could inhibit the function of one of the fastest known redox enzymes, with the smallest possible substrate molecule, resulted a challenge as, for instance, some compounds were required to be used at concentration as 100 µM to decrease a 50% the activity of the enzyme. Regarding the results obtained in this work, future work should be address to develop a BLI screen against auxiliary proteins to help to find compounds that inhibit their activity and as consequence, the assembly of Hyd-5. Maturation proteases, HyaD and HydD, might be good candidates as drug targets as they are involved in the biosynthesis of Hyd-1 and Hyd-5 being crucial for the infection process of S. enterica.