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The Action Mechanism of Prokaryotic FAD Synthetases As a Potential Drug Target for the Treatment of Bacterial Infections

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The Action Mechanism of Prokaryotic FAD Synthetases As a Potential Drug Target for the Treatment of Bacterial Infections 2018 112 María Sebastián Valverde The action mechanism of prokaryotic FAD synthetases as a potential drug target for the treatment of bacterial infections Departamento Bioquímica y Biología Molecular y Celular Director/es MEDINA TRULLENQUE, MILAGROS Reconocimiento – NoComercial – © Universidad de Zaragoza SinObraDerivada (by-nc-nd): No se Servicio de Publicaciones permite un uso comercial de la obra original ni la generación de obras derivadas. ISSN 2254-7606 Tesis Doctoral THE ACTION MECHANISM OF PROKARYOTIC FAD SYNTHETASES AS A POTENTIAL DRUG TARGET FOR THE TREATMENT OF BACTERIAL INFECTIONS Autor María Sebastián Valverde Director/es MEDINA TRULLENQUE, MILAGROS UNIVERSIDAD DE ZARAGOZA Bioquímica y Biología Molecular y Celular 2018 Repositorio de la Universidad de Zaragoza – Zaguan http://zaguan.unizar.es Abstract 1 Resumen 7 Chapter I: Introduction 13 1. The need of new antibacterial targets 15 1.1. From the beginnings to the golden era in the development of antibiotics 15 1.2. The resistance era put in check our effectiveness against pathogens 16 1.3. What makes a drug target good? 17 1.4. Proteins involved in flavins synthesis are promising drug targets 18 2. Flavins and flavoproteins: past, present and properties 20 2.1. A brief history of flavins and flavoproteins 20 2.2. Flavins are heterocyclic molecules 21 2.3. Flavins exhibit versatile redox properties 23 2.4. Flavins show useful spectroscopic properties 24 2.4.1. Absorption in the UV depends on the redox state 24 2.4.2. Flavins are fluorescent compounds 26 2.5. Biosynthesis of FMN and FAD in different organisms 26 3. FMN and FAD biosynthesis in different types of organisms 27 3.1. Prokaryotes own bifunctional FAD Synthetases 30 3.2. The FAD Synthetase from Corynebacterium ammoniagenes is the best characterized member of the family 32 3.2.1. Kinetics of CaFADS and interaction with substrates and products 33 3.2.2. The CaFADS is organized in two independent modules 35 3.2.3. Notable conformational changes occur during the CaFADS RFK catalytic cycle 42 3.2.4. CaFADS can be organized into a dimer of trimers 46 3.2.5. Binding of ligands induces oligomerization in CaFADS 49 3.3. Other prokaryotic FADSs 51 3.3.1. The FADS from Streptococcus pneumoniae: SpnFADS 51 3.3.2. The FADSs from Listeria monocytogenes and Mycobacterium tuberculosis 54 3.4. Bacterial diseases needing new efficient treatments 56 3.4.1. Streptococcus pneumoniae and pneumonia 56 3.4.2. Mycobacterium tuberculosis and tuberculosis 60 I María Sebastián Valverde 3.5. CaFADS, SpnFADS and MtFADS as drug targets 65 4. Objectives 67 Chapter II: Materials and Methods 69 1. General techniques in Molecular Biology 71 1.1. Production of thermo-competent cells 71 1.2. Transformation of thermo-competent cells through heat shock 71 1.3. Isolation of plasmid DNA 72 2. Production of the recombinant CaFADS and its variants 72 2.1. Over-expression of CaFADS and its variants 72 2.2. Purification of CaFADS and its variants 73 2.2.1. Preparation of the crude extract 73 2.2.2. Fractionation with ammonium sulphate 73 2.2.3. Phenyl-Sepharose affinity chromatography 73 2.2.4. DEAE-Cellulose anionic interchange chromatography 74 2.2.5. Size exclusion chromatography 74 3. Production of Δ (1-182) CaFADS 75 3.1. Purification of Δ(1-182) CaFADS 75 4. Production of SpnFADS 75 4.1. Cloning and over-expression of SpnFADS 75 4.2. Purification of SpnFADS 76 4.2.1. Preparation of the crude extract 76 4.2.2. His-trap affinity chromatography 76 4.2.3. Cleavage of the His-Tag and His-trap/GS-trap chromatography 76 5. Biophysical characterization of proteins 77 5.1. Spectroscopic characterization 77 5.1.1. Absorption spectroscopy in the UV-visible 77 5.1.2. Circular dichroism 77 5.1.3. Analysis of the formation of flavin-FADS complexes through difference spectroscopy 78 5.2. Analysis of the formation of quaternary organizations 79 5.2.1. Native electrophoresis 79 5.2.2. Size exclusion chromatography 80 II 5.2.3. Evaluation of the effect of substrates and products on the monomer-oligomer equilibrium 81 5.3. Steady-state kinetic analysis of the RFK and the FMNAT activities 82 5.3.1. Qualitative study of the RFK, the FMNAT and the FADpp activities through Thin Layer Chromatography 82 5.3.2. Quantitative analysis of the CaFADS riboflavin kinase (RFK) activity and determination of kinetic parameters through High Performance Liquid Chromatography (HPLC) 83 5.3.3. Fluorometric characterization of the FMN adenylyl-transferase activity (FMNAT) of CaFADSs 87 5.3.4. Analysis of the RFK and FMNAT activities of SpnFADS through HPLC 89 5.3.5. Analysis of the FMNAT activity of SpnFADS using photorreduced FMN 90 5.4 Kinetic study of the inhibition of the RFK activity of CaFADS and SpnFADS by the products of the reaction 90 5.5. Pre-steady state kinetics: Stopped-flow spectrophotometry 92 5.5.1. Basis of the technique 92 5.5.2. Determination of observed rate constants (kobs) for different processes involving flavins and FADS 93 5.5.3. Analysis of the data 94 5.6. Determination of thermodynamic parameters for ligand binding to FADS through Isothermal Titration Calorimetry (ITC) 95 5.6.1. Basis of the technique 95 5.6.2. Titration of FADS with a single ligand 96 5.6.3. Titration of pre-formed binary FADS:ligand complexes 98 6. Discovery of antibacterial drugs 100 6.1. Activity-based high-throughput screening for CaFADS 100 6.1.2. Identification of the activity inhibited by each of the primary HTS hits 101 6.1.3. Determination of the potency of FMNAT hits 102 6.1.4. Determination of the inhibition mechanism of selected in the FMNAT activity 102 6.1.5 Thermodynamic characterization of binding of the selected FMNAT hits to CaFADS through ITC 103 6.1.6. In-vivo determination of the antibacterial effect of the hits 104 III María Sebastián Valverde 7. Statistical analysis of the data 105 8. AFM measurements 105 9. Crystallographic structures 105 10. Culture of pathogenic microorganisms 105 11. Software 105 Chapter III: Quaternary organization in a bifunctional prokaryotic FAD synthetase: Involvement of an arginine at its adenylyltransferase module on the riboflavin kinase activity 107 1. Expression, purification and distribution profile of quaternary organizations for R66 CaFADS variants 111 2. Spectral properties of the R66 CaFADS variants 115 3. Effects of the mutations in the catalytic activities of CaFADS 117 4. Interaction parameters for CaFADS with flavins and ATP 119 5. Crystal structure of the CaFADS variants 125 6. Discussion 126 Chapter IV: The trimer interface in the quaternary structure of the bifunctional prokaryotic FAD synthetase from Corynebacterium ammoniagenes 133 1. Spectral properties of CaFADS variants 137 2. Size distribution of CaFADS variants 140 3. Effects of the mutations in the catalytic activities of CaFADS variants 143 4. CaFADS variants interacting with flavins and ATP 145 7. Crystal Structure of the CaFADS variants 154 6. Discussion 157 Chapter V: Kinetics and thermodynamics in the protein-ligand interactions in the riboflavin kinase activity of FAD synthetase from Corynebacterium ammoniagenes 161 1. The RFK activity shows inhibition by the RF substrate as well as by the products of the reaction. 163 IV 2. Changes in flavin fluorescence upon binding allow studying the association and dissociation kinetics of flavinic ligands to the RFK module. 166 3. The thermodynamic diagram for the RFK-ligand interactions 170 4. Adenine and flavin nucleotide ligands cooperate in their binding to the RFK module 175 5. Discussion 177 Chapter VI: The FAD synthetase from the human pathogen Streptococcus pneumoniae: a bifunctional enzyme exhibiting activity-dependent redox requirements 183 1. SpnFADS is purified free of flavin ligands 187 2. SpnFADS catalyzes the RFK, FMNAT and FADpp activities. 189 3. The reduced state of flavins is critical for the FMNAT activity but not for the RFK one. 189 4. SpnFADS does not bind oxidized flavins by itself 193 5. SpnFADS exists mainly as a monomer but other oligomeric states are populated during catalysis 196 6. Discussion 200 Chapter VII: The RFK catalytic cycle of the FADS from Streptococcus pneumoniae 207 2. Fast kinetics in the pre-steady state RFK reaction of SpnFADS 213 3. Thermodynamics of the interactions of SpnFADS with its ligands 217 4. FLV and ANP ligands show cooperative effects in their binding to SpnFADS 221 5. Discussion 223 Chapter VIII: Discovery of antimicrobial compounds targeting bifunctional FAD synthetases 231 1. Identification of Potential Inhibitors of the CaFADS activities through HTS 235 2. Effect of the HTS hits on RFK and FMNAT activities of CaFADS 235 3. The Inhibition Mechanisms of C24, C27 and C31 239 4. ITC constants for the CaFADS interaction with C24, C27 and C31 245 5. Effect of the HTS hits on the RFK and FMNAT activities of SpnFADS 247 V María Sebastián Valverde 6. Effect of selected HTS hits on different bacterial cells 248 7. Discussion 249 Chapter IX: General discussion 255 1. Quaternary organizations of bifunctional prokaryotic FAD synthetases 257 2. Catalytic cycles of CaFADS and SpnFADS 259 3. Discovery of antibacterial drugs targeting bifunctional FADSs 261 Chapter X: Conclusions 265 1. The FADS from C. ammoniagenes 267 2. The FADS from S. pneumoniae 268 3. Discovery of antibacterial compounds 269 Capítulo X: Conclusiones 271 1. La FADS de C. ammoniagenes 273 2. La FADS de S. pneumoniae 274 3. Desarrollo de nuevos compuestos antibacterianos 275 Bibliography 277 Abbreviations 293 VI Abstract 1 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).
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