Periodic Reporting for period 1 - SalHyd5 (Characterization of Salmonella enterica hydrogenase-5 biosynthesis for developing novel anti-infective compounds) Periodo di rendicontazione: 2015-09-01 al 2017-08-31 Sintesi del contesto e degli obiettivi generali del progetto Infections caused by bacteria that have become resistant to antibiotics are rising, making the development of novel anti-infective compounds necessary. In Salmonella enterica serovar Typhimurium the energy conserved by the respiratory oxidation of H2 is crucial for the infection process within the host. The S. enterica genome encodes three [NiFe]-hydrogenases that are involved in H2 oxidation and one of these, termed Hyd-5, is synthesized aerobically and oxides H2 in the presence of O2. S. enterica Hyd-5 cluster encodes three proteins that are only present in aerobic hydrogenase systems. HydH homologs have been proposed as a scaffolding protein for the transfer of the precursor cofactor into the large subunit protecting it against O2. HydF/HydG-like proteins have been reported to be involved in the assembly of the FeS clusters of the small subunit and also in the prevention of the export of the small subunit prior to dimerization with the large subunit. This project was conceived as a clinical need in advance in the knowledge of the mechanisms involved in the biosynthesis and assembly of S. enterica Hyd-5, an O2-tolerant [NiFe]-hydrogenase, as a starting point for the development of novel anti-infectives that could interfere with the virulence of S. enterica and other bacterial pathogens. In addition, understanding the molecular basis of O2-tolerant hydrogenase function has a particular interest in the bioenergy field, in H2 production or in the designing of enzyme-driven fuel cell. In order to circumvent these issues, a first strategy was to consider Hyd-5 as a credible target for the designing of inhibitors that prevent the function of the enzyme. A preliminary bio-layer interferometry (BLI) screen was performed and 28 compounds that interact with the enzyme were identified. One of the objectives of the action was to validate these compounds for their ability to inhibit the Hyd-5 activity. The identification of the interacting points between the enzyme and the most promising potential inhibitor by co-crystallization would gain insight the molecular mechanisms of H2 oxidation and would initiate the development of the anti-infective compounds. The second, and the most promising strategy, was to target the biosynthesis pathway for developing new inhibitors that prevent the assembly of the enzyme. This project emphasizes the importance of considering accessory proteins as target for the development of anti-infective compounds contributing to describe new observations in the field of metalloenzyme assembly. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti 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. Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) Understanding the molecular mechanisms and the functional role of accessory proteins, required for the assembly of S. enterica Hyd-5, have been considered necessary for the development of inhibitors that prevent the activity of the enzyme. HydH-like proteins have been proposed to function as a scaffolding protein for the assembly of the precursor cofactor prior its transfer to the large subunit. It is not well understood how the precursor cofactor is delivered to the hydrogenase apo-protein or to HydH-like proteins, in anaerobic or aerobic systems, respectively. In this work, genetic approaches have suggested that HypC might transfer the precursor cofactor to HydH in a ternary HypC-HypD-HydH complex. The project has revealed that HydG is essential for the biosynthesis of Hyd-5 under anaerobic and aerobic conditions whereas the role of HydF is redundant in the absence of O2. HydG-like proteins have been shown to be required for avoiding the translocation of the enzyme prior the assembly of the large subunit and a function in the biosynthesis of the small subunit FeS clusters has been reported. HydF-like proteins have been proposed to be involved in the protection of the FeS clusters containing the small subunit against the presence of O2 and the requirement of HydF homologs have been shown to be O2-dependent in other aerobic systems. This is consistent with the results obtained in this project in which the function of HydF is redundant under anaerobic conditions. This is of particular interest because would allow to define those genes that are essential and indispensable for the production of an active O2-tolerant hydrogenase in enzyme-driven fuel cells. Furthermore, this work contributes with new insights on the hydrogenase assembly research and describes an unexpected cross-talk between maturation proteases, HyaD and HydD, in the biosynthesis of Hyd-5. This project has advanced in the study of the molecular mechanisms for the biosynthesis of Hyd-5 and the functional role of some auxiliary proteins necessary for the assembly of the enzyme. In addition, this work emphasizes the importance of considering the accessory proteins as drug targets for the development of potential precursor of novel antiinfectives against S. enterica pathogenesis considering maturation proteases, HyaD and HydD, as good candidates as drug targets as they are required for the biosynthesis of Hyd-1 and Hyd-5, previously reported as essential for the infection process. Stable interactions between Hyd-5 large subunit HydB and maturation proteases HydD and HyaD Inhibitors of Salmonella nickel-dependent hydrogenase
