Molecular and dynamic mechanisms of prokaryotic and eukaryotic flavoenzymes: insights into their implication in human metabolism and health

Las flavoenzimas y flavoproteínas son biomoléculas versátiles y diversas que están implicadas en el metabolismo energético y otros procesos celulares como la transducción de señales, la síntesis de nucleótidos, el plegamiento de proteínas o la defensa frente al estrés oxidativo. Estas proteínas tienen como cofactores los derivados de riboflavina (RF, vitamina B2), mononucleótido de flavina (FMN) y/o dinucleótido de flavina y adenina (FAD), que les confieren propiedades únicas y versátiles. Todos los organismos contienen flavoproteínas y flavoenzimas clave, y muchas de ellas se han convertido en interesantes dianas terapéuticas o herramientas biotecnológicas. En esta tesis, se ha indagado en los mecanismos moleculares de flavoenzimas y flavoproteínas con funciones metabólicas clave en procariotas y eucariotas, como las enzimas humanas RF quinasa (Publicación I), FAD sintetasa (FADS) (Publicación II) o NAD(P)H:quinona oxidorreductasa 1 (Publicación III), o las FADS procariotas (Publicaciones IV y V). La caracterización detallada de estas enzimas contribuye a la mejor comprensión de sus patologías asociadas, y sienta las bases de nuevas estrategias terapéuticas y del diseño de compuestos dirigidos a estas dianas. Por ejemplo, aquí presentamos una primera aproximación a la búsqueda de inhibidores de las FADS procariotas que puede contribuir a su explotación farmacológica como potencial agentes antimicrobianos (Publicaciones IV y V).Esta Tesis Doctoral, presentada en la modalidad de compendio de publicaciones, incluye las siguientes publicaciones:− Publicación I. Anoz-Carbonell E, Ribero M, Polo V, Velázquez-Campoy A, Medina M. 2020. Human riboflavin kinase: species-specific traits in thebiosynthesis of the FMN cofactor. The FASEB Journal, 34:10871–10886.JCR Impact Factor 2019: 4.966. Rank: Q1 (57/297) Biochemistry and Molecular Biology; D1 (9/93) Biology; Q2 (58/195) Cell Biology.− Publicación II. Leone P, Galluccio M, Quarta S, Anoz-Carbonell E, Medina M, Indiveri C, Barile M. 2019. Mutation of aspartate 238 in FADsynthase isoform 6 increases the specific activity by weakening the FAD binding. International Journal of Molecular Sciences, 20(24):6203.JCR Impact Factor 2019: 4.556. Rank: Q1 (74/297) Biochemistry and Molecular Biology; Q2 (48/177) Chemistry (multidisciplinary). − Publicación III. Anoz-Carbonell E, Timson DJ, Pey AL, Medina M. 2020. The catalytic cycle of the antioxidant and cancer-associated human NQO1enzyme: hydride transfer, conformational dynamics and functional cooperativity. Antioxidants, 9(9):E772. JCR Impact Factor 2019: 5.014. Rank: Q1 (56/297) Biochemistry and Molecular Biology; Q1 (7/61) Medicinal Chemistry; D1 (10/139) Food Science & Technology.− Publicación IV. Sebastián M, Anoz-Carbonell E, Gracia B, Cossio P, Aínsa JA, Lans I, Medina M. 2018. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases. Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 241-254. JCR Impact Factor 2018: 4.027. Rank: Q1 (10/61) Medicinal Chemistry; Q2 (84/299) Biochemistry and Molecular Biology.− Publicación V. Lans I, Anoz-Carbonell E, Palacio-Rodríguez K, Aínsa JA, Medina M, Cossio P. 2020. In silico discovery and biological validation ofligands FAD synthase, a promising new antimicrobial target. PLOS Computational Biology, 16(8):e1007898. JCR Impact Factor 2019: 4.700. Rank: Q1 (9/77) Biochemical Research Methods; Q1 (6/59) Mathematical & Computational Biology.Flavoenzymes and flavoproteins are versatile and diverse biomolecules that are implicated in the energetic metabolism and other cellular processes such as signalling, nucleotide synthesis, protein folding or defense against oxidative stress. These proteins have as cofactors the riboflavin (RF, vitamin B2) derivatives flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which confer them their unique and versatile properties. All organisms contain key flavoproteins and flavoenzymes, and many of them are becoming interesting as therapeutic targets or biotechnological tools. In the present thesis, we have delved into the molecular mechanisms of flavoenzymes with key metabolic functions in prokaryotes and eukaryotes, such as the human RF kinase (Publication I) and FAD synthase (FADS) (Publication II), human NAD(P)H:quinone oxidoreductase 1 (Publication III), and prokaryotic FADS (Publications IV and V). The detailed characterization of these enzymes contributes to the better understanding of their associated pathologies, and provides a framework to novel therapeutic strategies and to the design of compounds targeting them. For instance, here we show a first approximation for identification of inhibitors of the prokaryotic FADS that might contribute to exploit them as pharmacological antimicrobial drugs (Publications IV and V). This Doctoral Thesis, presented in the form of a compendium of publications, comprises the following publications: − Anoz-Carbonell E, Ribero M, Polo V, Velázquez-Campoy A, Medina M. 2020. Human riboflavin kinase: species-specific traits in the biosynthesis of the FMN cofactor. The FASEB Journal, 34:10871–10886. − Leone P, Galluccio M, Quarta S, Anoz-Carbonell E, Medina M, Indiveri C, Barile M. 2019. Mutation of aspartate 238 in FAD synthase isoform 6 increases the specific activity by weakening the FAD binding. International Journal of Molecular Sciences, 20(24):6203. − Anoz-Carbonell E, Timson DJ, Pey AL, Medina M. 2020. The catalytic cycle of the antioxidant and cancer-associated human NQO1 enzyme: hydride transfer, conformational dynamics and functional cooperativity. Antioxidants, 9(9):E772. − Sebastián M, Anoz-Carbonell E, Gracia B, Cossio P, Aínsa JA, Lans I, Medina M. 2018. Discovery of antimicrobial compounds targeting bacterial type FAD synthetases. Journal of Enzyme Inhibition and Medicinal Chemistry, 33:1, 241-254. − Lans I, Anoz-Carbonell E, Palacio-Rodríguez K, Aínsa JA, Medina M, Cossio P. 2020. In silico discovery and biological validation of ligands FAD synthase, a promising new antimicrobial target. PLOS Computational Biology, 16(8):e1007898.<br /

Cofactors and pathogens: Flavin mononucleotide and flavin adenine dinucleotide (FAD) biosynthesis by the FAD synthase from Brucella ovis

The biosynthesis of the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), cofactors used by 2% of proteins, occurs through the sequential action of two ubiquitous activities: a riboflavinkinase (RFK) that phosphorylates the riboflavin (RF) precursor to FMN, and a FMN:adenylyltransferase (FMNAT) that transforms FMN into FAD. In most mammals two different monofunctional enzymes have each of these activities, but in prokaryotes a single bifunctional enzyme, FAD synthase (FADS), holds them. Differential structural and functional traits for RFK and FMNAT catalysis between bacteria and mammals, as well as within the few bacterial FADSs so far characterized, has envisaged the potentiality of FADSs from pathogens as targets for the development of species-specific inhibitors. Here, we particularly characterize the FADS from the ovine pathogen Brucella ovis (BoFADS), causative agent of brucellosis. We show that BoFADS has RFK activity independently of the media redox status, but its FMNAT activity (in both forward and reverse senses) only occurs under strong reducing conditions. Moreover, kinetics for flavin and adenine nucleotides binding to the RFK site show that BoFADS binds preferentially the substrates of the RFK reaction over the products and that the adenine nucleotide must bind prior to flavin entrapment. These results, together with multiple sequence alignments and phylogenetic analysis, point to variability in the less conserved regions as contributing to the species-specific features in prokaryotic FADSs, including those from pathogens, that allow them to adopt alternative strategies in FMN and FAD biosynthesis and overall flavin homeostasis

Mutation of aspartate 238 in FAD synthase isoform 6 increases the specific activity by weakening the FAD binding

FAD synthase (FADS, or FMN:ATP adenylyl transferase) coded by the FLAD1 gene is the last enzyme in the pathway of FAD synthesis. The mitochondrial isoform 1 and the cytosolic isoform 2 are characterized by the following two domains: the C-terminal PAPS domain (FADSy) performing FAD synthesis and pyrophosphorolysis; the N-terminal molybdopterin-binding domain (FADHy) performing a Co++ /K+-dependent FAD hydrolysis. Mutations in FLAD1 gene are responsible for riboflavin responsive and non-responsive multiple acyl-CoA dehydrogenases and combined respiratory chain deficiency. In patients harboring frameshift mutations, a shorter isoform (hFADS6) containing the sole FADSy domain is produced representing an emergency protein. With the aim to ameliorate its function we planned to obtain an engineered more efficient hFADS6. Thus, the D238A mutant, resembling the D181A FMNAT “supermutant” of C. glabrata, was overproduced and purified. Kinetic analysis of this enzyme highlighted a general increase of Km, while the kcat was two-fold higher than that of WT. The data suggest that the FAD synthesis rate can be increased. Additional modifications could be performed to further improve the synthesis of FAD. These results correlate with previous data produced in our laboratory, and point towards the following proposals (i) FAD release is the rate limiting step of the catalytic cycle and (ii) ATP and FMN binding sites are synergistically connected

A Natural Chimeric Pseudomonas Bacteriocin with Novel Pore-Forming Activity Parasitizes the Ferrichrome Transporter

Modular bacteriocins represent a major group of secreted protein toxins with a narrow spectrum of activity, involved in interference competition between Gram-negative bacteria. These antibacterial proteins include a domain for binding to the target cell and a toxin module at the carboxy terminus. Self-inhibition of producers is provided by coexpression of linked immunity genes that transiently inhibit the toxin''s activity through formation of bacteriocin-immunity complexes or by insertion in the inner membrane, depending on the type of toxin module. We demonstrate strain-specific inhibitory activity for PmnH, a Pseudomonas bacteriocin with an unprecedented dual-toxin architecture, hosting both a colicin M domain, potentially interfering with peptidoglycan synthesis, and a novel colicin N-type domain, a pore-forming module distinct from the colicin Ia-type domain in Pseudomonas aeruginosa pyocin S5. A downstream-linked gene product confers PmnH immunity upon susceptible strains. This protein, ImnH, has a transmembrane topology similar to that of Pseudomonas colicin M-like and pore-forming immunity proteins, although homology with either of these is essentially absent. The enhanced killing activity of PmnH under iron-limited growth conditions reflects parasitism of the ferrichrome-type transporter for entry into target cells, a strategy shown here to be used as well by monodomain colicin M-like bacteriocins from pseudomonads. The integration of a second type of toxin module in a bacteriocin gene could offer a competitive advantage against bacteria displaying immunity against only one of both toxic activities

Mycobacterial Aminoglycoside Acetyltransferases: A Little of Drug Resistance, and a Lot of Other Roles

Aminoglycoside acetyltransferases are important determinants of resistance to aminoglycoside antibiotics in most bacterial genera. In mycobacteria, however, aminoglycoside acetyltransferases contribute only partially to aminoglycoside susceptibility since they are related with low level resistance to these antibiotics (while high level aminoglycoside resistance is due to mutations in the ribosome). Instead, aminoglycoside acetyltransferases contribute to other bacterial functions, and this can explain its widespread presence along species of genus Mycobacterium. This review is focused on two mycobacterial aminoglycoside acetyltransferase enzymes. First, the aminoglycoside 2′-N-acetyltransferase [AAC(2′)], which was identified as a determinant of weak aminoglycoside resistance in M. fortuitum, and later found to be widespread in most mycobacterial species; AAC(2′) enzymes have been associated with resistance to cell wall degradative enzymes, and bactericidal mode of action of aminoglycosides. Second, the Eis aminoglycoside acetyltransferase, which was identified originally as a virulence determinant in M. tuberculosis (enhanced intracellular survival); Eis protein in fact controls production of pro-inflammatory cytokines and other pathways. The relation of Eis with aminoglycoside susceptibility was found after the years, and reaches clinical significance only in M. tuberculosis isolates resistant to the second-line drug kanamycin. Given the role of AAC(2′) and Eis proteins in mycobacterial biology, inhibitory molecules have been identified, more abundantly in case of Eis. In conclusion, AAC(2′) and Eis have evolved from a marginal role as potential drug resistance mechanisms into a promising future as drug targets

Counterintuitive structural and functional effects due to naturally occurring mutations targeting the active site of the disease-associated NQO1 enzyme*

Our knowledge on the genetic diversity of the human genome is exponentially growing. However, our capacity to establish genotype–phenotype correlations on a large scale requires a combination of detailed experimental and computational work. This is a remarkable task in human proteins which are typically multifunctional and structurally complex. In addition, mutations often prevent the determination of mutant high-resolution structures by X-ray crystallography. We have characterized here the effects of five mutations in the active site of the disease-associated NQO1 protein, which are found either in cancer cell lines or in massive exome sequencing analysis in human population. Using a combination of H/D exchange, rapid-flow enzyme kinetics, binding energetics and conformational stability, we show that mutations in both sets may cause counterintuitive functional effects that are explained well by their effects on local stability regarding different functional features. Importantly, mutations predicted to be highly deleterious (even those affecting the same protein residue) may cause mild to catastrophic effects on protein function. These functional effects are not well explained by current predictive bioinformatic tools and evolutionary models that account for site conservation and physicochemical changes upon mutation. Our study also reinforces the notion that naturally occurring mutations not identified as disease-associated can be highly deleterious. Our approach, combining protein biophysics and structural biology tools, is readily accessible to broadly increase our understanding of genotype–phenotype correlations and to improve predictive computational tools aimed at distinguishing disease-prone against neutral missense variants in the human genome

Different phenotypic outcome due to site-specific phosphorylation in the cancer-associated NQO1 enzyme studied by phosphomimetic mutations

Protein phosphorylation is a common phenomenon in human flavoproteins although the functional consequences of this site-specific modification are largely unknown. Here, we evaluated the effects of site-specific phosphorylation (using phosphomimetic mutations at sites S40, S82 and T128) on multiple functional aspects as well as in the structural stability of the antioxidant and disease-associated human flavoprotein NQO1 using biophysical and biochemical methods. In vitro biophysical studies revealed effects of phosphorylation at different sites such as decreased binding affinity for FAD and structural stability of its binding site (S82), conformational stability (S40 and S82) and reduced catalytic efficiency and functional cooperativity (T128). Local stability measurements by H/D exchange in different ligation states provided structural insight into these effects. Transfection of eukaryotic cells showed that phosphorylation at sites S40 and S82 may reduce steady-levels of NQO1 protein by enhanced proteasome-induced degradation. We show that site-specific phosphorylation of human NQO1 may cause pleiotropic and counterintuitive effects on this multifunctional protein with potential implications for its relationships with human disease. Our approach allows to establish relationships between site-specific phosphorylation, functional and structural stability effects in vitro and inside cells paving the way for more detailed analyses of phosphorylation at the flavoproteome scale

Allosteric Communication in the Multifunctional and Redox NQO1 Protein Studied by Cavity-Making Mutations

Allosterism is a common phenomenon in protein biochemistry that allows rapid regulation of protein stability; dynamics and function. However, the mechanisms by which allosterism occurs (by mutations or post-translational modifications (PTMs)) may be complex, particularly due to long-range propagation of the perturbation across protein structures. In this work, we have investigated allosteric communication in the multifunctional, cancer-related and antioxidant protein NQO1 by mutating several fully buried leucine residues (L7, L10 and L30) to smaller residues (V, A and G) at sites in the N-terminal domain. In almost all cases, mutated residues were not close to the FAD or the active site. Mutations L\u2192G strongly compromised conformational stability and solubility, and L30A and L30V also notably decreased solubility. The mutation L10A, closer to the FAD binding site, severely decreased FAD binding affinity ( 4820 fold vs. WT) through long-range and context-dependent effects. Using a combination of experimental and computational analyses, we show that most of the effects are found in the apo state of the protein, in contrast to other common polymorphisms and PTMs previously characterized in NQO1. The integrated study presented here is a first step towards a detailed structural-functional mapping of the mutational landscape of NQO1, a multifunctional and redox signaling protein of high biomedical relevance

Mécanismes moléculaires et dynamiques des flavoenzymes prokaryotes et eucaryotes : éclairages sur leur implication dans le métabolisme humain et la santé

Flavoenzymes and flavoproteins are versatile and diverse biomolecules that are implicated in the energetic metabolism and other cellular processes such as signalling, nucleotide synthesis, protein folding or defense against oxidative stress. These proteins have as cofactors the riboflavin (RF, vitamin B2) derivatives flavin mononucleotide (FMN) and/or flavin adenine dinucleotide (FAD), which confer them their unique and versatile properties. All organisms contain key flavoproteins and flavoenzymes, and many of them are becoming interesting as therapeutic targets or biotechnological tools.In the present thesis, we have delved into the molecular mechanisms of flavoenzymes with key metabolic functions in prokaryotes and eukaryotes, such as the human RF kinase (Publication I) and FAD synthase (FADS) (Publication II), human NAD(P)H:quinone oxidoreductase 1 (Publication III), and prokaryotic FADS (Publications IV and V). The detailed characterization of these enzymes contributes to the better understanding of their associated pathologies, and provides a framework to novel therapeutic strategies and to the design of compounds targeting them. For instance, here we show a first approximation for identification of inhibitors of the prokaryotic FADS that might contribute to exploit them as pharmacological antimicrobial drugs (Publications IV and V).Les flavoenzymes et les flavoprotéines sont des biomolécules polyvalentes et diverses impliquées dans le métabolisme énergétique et d'autres processus cellulaires tels que la signalisation, la synthèse des nucléotides, le repliement des protéines ou la défense contre le stress oxydatif. Ces protéines ont comme cofacteurs les dérivés de la riboflavine (RF, vitamine B2) flavin mononucléotide (FMN) et/ou flavin adénine dinucléotide (FAD), qui leur confèrent leurs propriétés uniques et polyvalentes. Tous les organismes contiennent des flavoprotéines et des flavoenzymes clés, et beaucoup d'entre eux deviennent intéressants en tant que cibles thérapeutiques ou outils biotechnologiques.Dans la présente thèse, nous avons exploré les mécanismes moléculaires des flavoenzymes ayant des fonctions métaboliques clés chez les procaryotes et les eucaryotes, tels que la kinase RF humaine (Publication I) et la synthase de FAD (FADS) (Publication II), la NAD(P)H:quinone oxydoréductase 1 humaine (Publication III), et la FADS procaryotique (Publications IV et V). La caractérisation détaillée de ces enzymes contribue à une meilleure compréhension de leurs pathologies associées et fournit un cadre pour de nouvelles stratégies thérapeutiques et la conception de composés les ciblant. Par exemple, nous présentons ici une première approximation pour l'identification d'inhibiteurs de la FADS procaryotique qui pourraient contribuer à les exploiter en tant que médicaments antimicrobiens pharmacologiques (Publications IV et V)

Reactivity of ClDaf and Pyr with different metalic salts

&lt;p&gt;Evaluation of the reactivity of ClDaf and Pyridomycin when mixed with 10 equivalents of different metalic salts.&nbsp;&lt;/p&gt; &lt;p&gt;Samples were analyzed by analytical UPLC.&nbsp;&lt;/p&gt; &lt;p&gt;Analytical (non-high resolution) spectrometry analysis (UHPLC-MS) were performed on an Ultimate 3000 UHPLC&nbsp;system, coupled with a LCQ Fleet Ion Trap Mass Spectrometer (Thermo Scientific). Chromatographic separation&nbsp;was achieved using an Acquity UPLC Peptide BEH C18 Column (300&Aring;, 1.7 &micro;m, 2.1 mm X 100 mm). Mobile phase&nbsp;system was composed of solvent A (H2O, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid), typically run&nbsp;through a linear gradient from 0% to 100% of solvent B in 10 min. Elution of compounds was monitored by UV&nbsp;absorbance at 215 nm and 254 nm, and by mass spectrometry by electrospray ionization.&lt;/p&gt; &lt;p&gt;Data published in: Caradec T, Anoz-Carbonell E, Petrov R, Billamboz M, Antraygues K, Cantrelle FX, Boll E, Beury D, Hot D, Drobecq H, Trivelli X, Hartkoorn RC. A Novel Natural Siderophore Antibiotic Conjugate Reveals a Chemical Approach to Macromolecule Coupling. ACS Cent Sci. 2023 Nov 10;9(11):2138-2149. doi: 10.1021/acscentsci.3c00965. PMID: 38033789; PMCID: PMC10683483.&lt;/p&gt; &lt;div&gt;&nbsp;&lt;/div&gt; &lt;p&gt;&nbsp;&lt;/p&gt; &lt;p&gt;&nbsp;&lt;/p&gt