Les abondances relatives naturelles des différents isotopes stables de l'azote (δ15N) varient selon les tissus au sein d'un individu et selon les individus au sein d'une population, et ces différences reflètent à la fois les caractéristiques de structure et de fonctionnement du métabolisme azoté et ses modulations en lien avec des variations des conditions nutritionnelles et physio-pathologiques. Cette thèse vise, à travers une approche couplée d'expérimentation et de modélisation, à mieux caractériser et comprendre les modulations des δ15N des différents pools azotés et à démontrer la capacité des δ15N à fournir des informations sur les flux azotés de l'organisme, leurs valeurs et modulations, qui sont encore mal connus. Nous avons, dans un premier temps, mesuré les δ15N dans plusieurs tissus (intestin, foie, plasma, muscles, rein, peau...) et dans différentes fractions azotées (acides aminés, protéines, urée, NH4) chez le rat, dans différentes conditions nutritionnelles (chez des rats nourris avec des P de qualité différente, les protéines de lait et de soja) ou physiopathologiques (chez des rats présentant ou non un syndrome métabolique, associant insulino-résistance et obésité, après avoir consommé un même régime potentiellement obésogène). Ces données expérimentales nous ont permis (i) de montrer que l'écart de δ15N entre les protéines tissulaires et le régime est plus important lorsque la qualité protéique est moindre, et (ii) de mettre en évidence que, lors de l'initiation précoce d'un syndrome métabolique associant insulino-résistance et obésité, les δ15N de certains pools métaboliques sont modulés et constituent des signatures isotopiques des modulations métaboliques associées. Par ailleurs, grâce à l'analyse par modélisation compartimentale des cinétiques de δ15N mesurées expérimentalement dans les fractions acides aminés et protéines de différents tissus après augmentation du δ15N du régime, nous avons pu estimer les taux de renouvellement protéique tissulaires et explorer la structure et le fonctionnement des échanges entre acides aminés et protéines des différents tissus et comparer leur degré de compartimentation. Enfin, nous avons développé un modèle multi-compartimental reproduisant l'ensemble des flux azotés inter- et intra-organes de l'organisme et rendant compte des variations de δ15N observées. Cette représentation globale du métabolisme azoté fournit une vision novatrice du fonctionnement intégré du métabolisme azoté dont les données éparses de la littérature ne donnaient auparavant qu'une vision parcellaire et fragmentée. Le modèle a permis de reconstituer les mécanismes qui conduisent à l'observation de différences de δ15N entre pools azotés, de mieux comprendre quelles modulations sont les plus susceptibles d'affecter les δ15N, avec quelle amplitude et dans quel sens, et finalement d'expliquer les variations de δ15N mises en évidence expérimentalement en terme de modulation des flux azotés. L'ensemble de nos résultats d'expérimentation et de modélisation démontre la capacité des δ15N à apporter des informations sur les flux métaboliques azotés et souligne l'intérêt prometteur de cette approche nouvelle pour acquérir une compréhension intégrée du système complexe du métabolisme azoté inter- et intra-organes et des processus homéostatiques qui le régulent et de ses dérégulations pré-pathologiques. / Natural abundances of stable nitrogen isotopes vary among tissues within an individual and among individuals within a population, and these differences are linked to the structural and functioning characteristics of the nitrogen metabolism and also to its modulations in response to variations in nutritional and physiological conditions. In this thesis, we developed an approach combining both experimentation and modeling, in order to better characterize and understand the modulations in the δ15N values of various nitrogen metabolic pools, and to show the capacity of the δ15N to provide information regarding the values and modulations of the body nitrogen fluxes, that are still poorly determined. We first measured the δ15N in various tissues (intestine, liver, plasma, muscle, kidney, skin …) and in various nitrogen fractions (amino acids, proteins, urea, NH4) in rats, under different nutritional (i.e. in rats fed with P of distinct quality, that were milk and soy P) or pathophysiological (i.e. in rats that had or not become obese and insulin resistant after being fed a high-fat diet for 10 weeks). From these experimental data, we showed (i) that the tissue nitrogen discrimination (i.e., the difference between tissue and diet δ15N) is higher when the P is of lesser quality, and (ii) that, during the onset of a metabolic syndrome, in the presence of both insulin resistance and obesity, the δ15N differed in some nitrogen pools and thus constitute isotopic signatures of the metabolic impact of such conditions. In this thesis, we also measured the δ15N kinetics in the amino acid and protein fractions of several tissues after a shift in the diet δ15N. The analysis of these kinetics, using a compartimental modeling approach, enabled us to estimate tissue fractional turnover rates and to investigate the structure and the functioning of the protein synthesis and breakdown exchanges in some tissues and their level of compartmentation. Lastly, we developed a multi-compartmental model that describes the various body nitrogen transfers between and within tissues and accounts for the observed δ15N variability. This model of the nitrogen metabolism provides a new and systemic insight of the interactions and modulations of the various nitrogen fluxes, as opposed to the fragmented information available from the literature data. This model enabled us to reconstruct the mechanisms that caused the observed δ15N differences between nitrogen pools, to better understand how they vary, depending on which metabolic modulation and with which amplitude, and finally to hypothesize which nitrogen fluxes alterations are the more likely to be responsible for the δ15N variations that we observed in our experimentations. In conclusion, our experimental and modelling results show that it is feasible to gain information from the δ15N values regarding the metabolic nitrogen fluxes and their modulations, and highlight the interest of this new approach to get an integrated insight into the complex nitrogen metabolic system and a better understanding of the way the various between and within tissues nitrogen fluxes are regulated and altered.

Metabolism of D-amino acids is of considerable interest due to their key importance in cellular functions. The enzymes D-serine dehydratase (DSD) and D-cysteine desulfhydrase (DCyD) are involved in the degradation of D-Ser and D-Cys, respectively. We determined the crystal structure of Salmonella typhimurium DSD (StDSD) by multiple anomalous dispersion method of phasing using selenomethione incorporated protein crystals. The structure revealed a fold typical of fold type II PLP-dependent enzymes. Although holoenzyme was used for crystallization of both wild type StDSD (WtDSD) and selenomethionine labeled StDSD (SeMetDSD), significant electron density was not observed for the co-factor, indicating that the enzyme has a low affinity for the cofactor under crystallization conditions. Interestingly, unexpected conformational differences were observed between the two structures. The WtDSD was in an open conformation while SeMetDSD, crystallized in the presence of isoserine, was in a closed conformation suggesting that the enzyme is likely to undergo conformational changes upon binding of substrate as observed in other fold type II PLP-dependent enzymes. Electron density corresponding to a plausible sodium ion was found near the active site of the closed but not in the open state of the enzyme. Examination of the active site and substrate modeling suggested that Thr166 may be involved in abstraction of proton from the Cα atom of the substrate. Apart from the physiological reaction, StDSD catalyses α, β-elimination of D-Thr, D-Allothr and L-Ser to the corresponding α-keto acids and ammonia. The structure of StDSD provides a molecular framework necessary for understanding differences in the rate of reaction with these substrates. Salmonella typhimurium DCyD (StDCyD) is a fold type II PLP-dependent enzyme that catalyzes the degradation of D-Cys to H2S and pyruvate. We determined the crystal structure of StDCyD using molecular replacement method in two different crystal forms. The better diffracting crystal form obtained in presence of benzamidine illustrated the influence a small molecule in altering protein interfaces and crystal packing. The polypeptide fold of StDCyD consists of a small domain (residues 48-161) and a large domain (residues 1-47 and 162-328) which resemble other fold type II PLP-dependent enzymes. X-ray crystal structures of StDCyD were also obtained in the presence of substrates, D-Cys and βCDA, and substrate analogs, ACC, D-Ser, L-Ser, D-cycloserine (DCS) and L-cycloserine (LCS). The structures obtained in the presence of D-Cys and βCDA show the product, pyruvate, bound at a site 4.0-6.0 Å away from the active site. ACC forms an external aldimine complex while D and L-Ser bind non-covalently suggesting that the reaction with these ligands is arrested at Cα proton abstraction and transimination steps, respectively. In the active site of StDCyD cocrystallized with DCS or LCS, electron density for a pyridoxamine phosphate (PMP) was observed. Crystals soaked in cocktail containing these ligands show density for PLP-cycloserine. Spectroscopic observations also suggested formation of PMP by the hydrolysis of cycloserines. Mutational studies suggested that Ser78 and Gln77 are key determinants of enzyme specificity and the phenolate of Tyr287 is responsible for Cα proton abstraction from D-Cys. Based on these studies, we proposed a probable mechanism for the degradation of D-Cys by StDCyD. The acid-induced arginine decarboxylase (ADC) is part of an enzymatic system in Salmonella typhimurium that contributes to making this organism acid resistant. ADC is a PLP-dependent enzyme that is active at acidic pH. It consumes a proton in the decarboxylation of arginine to agmatine, and by working in tandem with an arginine-agmatine antiporter, this enzymatic cycle protects the organism by preventing the accumulation of protons inside the cell. We have determined the structure of the acid-induced StADC to 3.1 Å resolution. StADC structure revealed an 800 kDa decamer composed as a pentamer of five homodimers. Each homodimer has an abundance of acidic surface residues, which at neutral pH prevent inactive homodimers from associating into active decamers. Conversely, acidic conditions favor the assembly of active decamers. Therefore, the structure of arginine decarboxylase presents a mechanism by which its activity is modulated by external pH.