The action mechanism of prokaryotic fad synthetases as a potential drug target for the treatment of bacterial infections
- Autores: María Sebastián Valverde
- Directores de la Tesis: Milagros Medina Trullenque (dir. tes.)
- Lectura: En la Universidad de Zaragoza ( España ) en 2018
- Idioma: español
- Tribunal Calificador de la Tesis: Patricia Ferreira Neila (presid.), Susana Frago Moreno (secret.), Ángel Luis Pey Rodríguez (voc.)
- Programa de doctorado: Programa de Doctorado en Bioquímica y Biología Molecular por la Universidad de Zaragoza
- Materias:
- Enlaces
- Tesis en acceso abierto en: Zaguán
- Resumen
Bacterial FAD synthetases (FADS) are bifunctional and bimodular proteins that carry out the synthesis of the essential cofactors FMN and FAD from Riboflavin (RF,vitamin B2). These proteins are organized in two almost independent modules, each of which performs one of the activities that lead to FMN and FAD synthesis. The C-terminus module transforms RF into FMN through its riboflavin kinase activity (RFK), while the N-terminus module catalyzes the FMN adenylyltransferase activity (FMNAT) that transforms FMN into FAD. Having into account the essential character of the FMN and FAD flavinic cofactors, and the differences between prokaryotic FADSs and the corresponding human enzymes (especially between the FMNAT module of the bacterial proteins and mammalian enzymes that transform FMN into FAD), these enzymes appear as potential drug target. Thus, these bifunctional proteins are worthy of being studied in detail, with the objective of shedding light on their catalytic cycles, and on their regulatory strategies, focusing on the dissimilarities between different members of the family.
In that way, in the present thesis we have tackled three main projects: • The study of the structural determinants that modulate the oligomerization tendency of the FADS from the organism Corynebacterium ammoniagenes (CaFADS). During its catalytic cycles, CaFADS stabilizes different oligomeric assemblies, being of especial relevance the formation of a hexamer formed by a dimer of trimers. The head to tail disposition that the protomers adopt within such structure, suggests an active role of the dimer of trimers in channeling the FMN product from the RFK active site to the FMNAT one, to be subsequently transformed into FAD. To demostrate this hypothesis we produced and characterized point mutants in positions located in the interface between protomers. The studied residues apparently stabilized the dimer of trimers establishing salt bridges, Van der Waals contacts, or hydrophobic interactions with residues located at the other module of a neighboring protomer. Our results revealed that all the mutated variants were active and able to stabilize quaternary organizations, although many of them presented altered RFK and FMNAT activities, as well as ligand binding parameters and oligomerization tendency. Special attention requires the fact that mutations located at the RFK module altered the FMNAT activity and vice versa. These results only can be explained in the context of the dimer of trimers, where residues of different modules, which are very distant in the monomer, are in close contact within the trimer. These results validate our hypothesis about the formation of oligomeric structures during the catalytic cycles of CaFADS, where the protomers might adopt a head to tail disposition. Additionally, our study allows inferring the role that specific residues have in the RFK and FMNAT catalytic cycles of this enzyme.
• Comparative characterization of the RFK and FMNAT catalytic cycles of the FADSs from the organisms C. ammoniagenes and Streptococcus pneumoniae (CaFADS and SpnFADS, respectively). Despite the overall similarities between bacterial FADSs, enzymes from different organisms display important behavioral differences. The study of such differences might provide us with the suitable tools to design species-selective compounds, able to neutralize a specific organism through the selective inhibition of its FADS without affecting the flora of the host. That is the case of CaFADS and SpnFADS, which show great structural homology but important dissimilarities in their activities. Thus, we have addressed a first functional characterization of SpnFADS, identifying the three main differences of this protein regarding CaFADS. Thus, contrary to CaFADS, SpnFADS i) does not stabilize the dimer of trimers, ii) its FMNAT activity requires strong reducing conditions and iii) its RFK activity is not inhibited by excess of the RF substrate. Furthermore, a more detailed study of the RFK activity of both enzymes allowed us to identify the thermodynamic and kinetic determinants responsible of such differences. The integration of our data, and its interpretation in the context of the available crystallographic structures, bring to light the differential regulatory strategies adopted by these two enzymes.
• Development of new antibacterial drugs targeting bifunctional FADSs. Due to the emergence during the last decades of bacterial resistant strains, the need to find new antibacterial targets and drugs against them is a fact. In that way, because of their essential nature, and other good characteristics, we propose prokaryotic FADSs as exploitable targets for the development of new antibacterial drugs. In this work, we have optimized a protocol do identify potential inhibitors of these enzymes, using as model CaFADS, and we have utilized this methodology to identify broad-spectrum or species-selective compounds. Our methodology starts with the identification of potential inhibitors using an activity-based high-throughput screening protocol, and the subsequent identification of the affected activity. The best inhibitors of the FMNAT activity (they are more probable to have no effect on the mammal proteins) were further characterized, and their inhibition mechanisms identified. Concurrently, the effect of the hits of the screening was tested in different bacterial strains. In this project, we have identified promising compounds that could act either as selective drugs against FADS from a specific organism, or as broad-spectrum inhibitors.
