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Les gènes codant pour des protéines peuvent souvent être regroupés et intégrés en modules fonctionnels par rapport à un organelle. Ces modules peuvent avoir des composantes qui suivent une évolution corrélée pouvant être conditionnelle à un phénotype donné. Les gènes liés à la motilité possèdent cette caractéristique, car ils se suivent en cascade en réponse à des stimuli extérieurs. L’hyperthermophilie, d’autre part, est interreliée à la reverse gyrase, cependant aucun autre élément qui pourrait y être associé avec certitude n’est connu. Ceci peut être dû à un déplacement de gènes non orthologues encore non résolu. En utilisant une approche bio-informatique, une modélisation mathématique d’évolution conditionnelle corrélée pour trois gènes a été développée et appliquée sur des profils phylétiques d’archaea. Ceci a permis d’établir des théories quant à la fonction potentielle du gène du flagelle FlaD/E ainsi que l’histoire évolutive des gènes lui étant liés et ayant contribué à sa formation. De plus, une histoire évolutive théorique a été établie pour une ligase liée à l’hyperthermophilie. / Protein coding gene may often be grouped and integrated in functional modules with respect to an organelle. These modules may have constituents that follow a conditional correlated evolution to a given phenotype. Genes linked to motility posses this characteristic as they follow a cascade in response to external stimuli. Similarly, hyperthermophily is related to reverse gyrase, however no other element that could be associated with certainty is known. This may be caused by an unresolved case of non-orthologous gene displacement. Using a bioinformatic approach, a mathematical model for conditional correlated evolution for three genes has been developed and applied to the phyletic profiles of archaea. This has helped to develop theories about the potential functions of the flagellar gene FlaD/E and the evolutionary history of the genes that are linked to it and that may have contributed to its formation. In addition, a theoretical evolutionary history has been established for a ligase associated with hyperthermophily.

Les gènes codant pour des protéines peuvent souvent être regroupés et intégrés en modules fonctionnels par rapport à un organelle. Ces modules peuvent avoir des composantes qui suivent une évolution corrélée pouvant être conditionnelle à un phénotype donné. Les gènes liés à la motilité possèdent cette caractéristique, car ils se suivent en cascade en réponse à des stimuli extérieurs. L’hyperthermophilie, d’autre part, est interreliée à la reverse gyrase, cependant aucun autre élément qui pourrait y être associé avec certitude n’est connu. Ceci peut être dû à un déplacement de gènes non orthologues encore non résolu. En utilisant une approche bio-informatique, une modélisation mathématique d’évolution conditionnelle corrélée pour trois gènes a été développée et appliquée sur des profils phylétiques d’archaea. Ceci a permis d’établir des théories quant à la fonction potentielle du gène du flagelle FlaD/E ainsi que l’histoire évolutive des gènes lui étant liés et ayant contribué à sa formation. De plus, une histoire évolutive théorique a été établie pour une ligase liée à l’hyperthermophilie. / Protein coding gene may often be grouped and integrated in functional modules with respect to an organelle. These modules may have constituents that follow a conditional correlated evolution to a given phenotype. Genes linked to motility posses this characteristic as they follow a cascade in response to external stimuli. Similarly, hyperthermophily is related to reverse gyrase, however no other element that could be associated with certainty is known. This may be caused by an unresolved case of non-orthologous gene displacement. Using a bioinformatic approach, a mathematical model for conditional correlated evolution for three genes has been developed and applied to the phyletic profiles of archaea. This has helped to develop theories about the potential functions of the flagellar gene FlaD/E and the evolutionary history of the genes that are linked to it and that may have contributed to its formation. In addition, a theoretical evolutionary history has been established for a ligase associated with hyperthermophily.

The low molecular weight (L.M.W.) thiol redox couple formed by γ-L-glutamyl-L-cysteinyl glycine, also called glutathione (reduced and oxidized), is present in most eukaryotes and a few species of bacteria. Glutathione plays a role in numerous cellular processes by providing a means of shuttling electrons to different enzymatic systems. As a result, thiol-dependent redox metabolic processes are highly coupled. Due to tight coupling of redox reactions, it is difficult to understand how changes in the concentration of glutathione would affect a specific glutathione-dependent process. Interestingly, only a small subset of bacteria encode the canonical enzyme for the biosynthesis of glutathione, namely γ-glutamyl cysteine synthetase (gshA gene product). The mechanisms by which glutathione-dependent processes are carried out in bacteria which do not have the genes for biosynthesis of glutathione or other L.M.W. thiols is not well understood. A genetic selection to restore a glutathione-dependent phenotype in E. coli, lacking the gene involved in first step of glutathione biosynthesis (gshA), was used to address how bacteria lacking gshA might substitute for glutathione. Genetic and biochemical analyses of the E. coli mutants isolated in the selection revealed a de novo pathway for biosynthesis of γ-glutamyl cysteine, the product formed normally by GshA. Additionally we found that the unnatural analogue of glutathione, γ-glutamyl homocysteine could also be formed by this pathway. Bioinformatic analysis suggested that bacteria lacking gshA may use these de novo pathways for biosynthesis of γ-glutamyl cysteine or γ-glutamyl homocysteine, which could serve as potential substitutes for glutathione. The engineering of de novo biosynthetic pathways for γ-glutamyl cysteine and γ-glutamyl homocysteine provided us a strategy for engineering a pathway for biosynthesis of another unnatural analogue of glutathione, β-aspartyl cysteine. Both γ-glutamyl homocysteine and β-aspartyl cysteine could potentially be used as orthologus redox couples in E. coli operating in parallel to glutathione to shuttle electrons to specific pathways which may thus be decoupled from glutathione availability. Glutathione-dependent enzymes that can use orthologous redox couples instead are biochemically isolated from network of other redox reactions in the cell and could be used to direct metabolic fluxes to specific pathways with high efficiencies. Towards this end, we show that glutathione transferase, a glutathione-dependent enzyme, can be engineered to use analogous thiols like γ-glutamyl cysteine as cofactors. / text

Approximately one third of the genes for the completely sequenced bacterial genomes have an unknown, uncertain, or incorrect functional annotation. Approximately 11,000 putative proteins identified from the fully-sequenced microbial genomes are members of the catalytically diverse Amidohydrolase Superfamily. Members of the Amidohydrolase Superfamily separate into 24 Clusters of Orthologous Groups (cogs). Cog3653 includes proteins annotated as N-acyl-D-amino acid deacetylases (DAAs), and proteins within cog2355 are homologues to the human renal dipeptidase. The substrate profiles of three DAAs (Bb3285, Gox1177 and Sco4986) and six microbial dipeptidase (Sco3058, Gox2272, Cc2746, LmoDP, Rsp0802 and Bh2271) were examined with N-acyl-L-, N-acyl-D-, L-Xaa-L-Xaa, L-Xaa-D-Xaa and D-Xaa-L-Xaa substrate libraries. The rates of hydrolysis of the library components were determined by separating the amino acids by HPLC and quantitating the products. Gox1177 and Sco4986 hydrolyzed several N-acyl-D-amino acids, especially those where the amino acid was a hydrophobic residue. Gox1177 hydrolyzed L-Xaa-D-Xaa and N-acetyl-D-amino acids with similar catalytic efficiencies (~10⁴ M⁻¹s⁻¹). The best substrates identified for Gox1177 and Sco4986 were N-acetyl-D-Trp and N-acetyl-D-Phe, respectively. Conversely, Bb3285 hydrolyzed N-acyl-D-Glu substrates (kcat/Km ⁹́⁸ 5 x 10⁶M⁻¹s⁻¹) and, to a lesser extent, L-Xaa-D-Glu dipeptides. The structure of a DAA from A. faecalis did not help explain the substrate specificity of Bb3285. N-methylphosphonate derivatives of D-amino acids were inhibitors of the DAAs examined. The structure of Bb3285 was solved in complex with the N-methylphosphonate derivative of D-Glu or acetate/formate. The specificity of Bb3285 was due to an arginine located on a loop which varied in conformation from the A. faecalis enzyme. In a similar manner, six microbial renal dipeptidase-like proteins were screened with 55 dipeptide libraries. These enzymes hydrolyzed many dipeptides but favored L-D dipeptides. Respectable substrates were identified for proteins Bh2271 (L-Leu-D-Ala, kcat/Km = 7.4 x 10⁴ M⁻¹s⁻¹), Sco3058 (L-Arg-D-Asp, kcat/Km = 7.6 x 10⁵ M⁻¹s⁻¹), Gox2272 (L-Asn-D-Glu, kcat/Km = 4.7 x 10⁵ M⁻¹s⁻¹), Cc2746 (L-Met-D-Leu, kcat/Km = 4.6 x 10⁵ M⁻¹s⁻¹), LmoDP (L-Leu-D-Ala, kcat/Km = 1.1 x 10⁵ M⁻¹s⁻¹), Rsp0802 (L-Met-D-Leu, kcat/Km = 1.1 x 10⁵ M⁻¹s⁻¹). Phosphinate mimics of dipeptides were inhibitors of the dipeptidases. The structures of Sco3058, LmoDP and Rsp0802 were solved in complex with the pseudodipeptide mimics of L-Ala-D-Asp, L-Leu-D-Ala and L-Ala-D-Ala, respectively. The structures were used to assist in the identification of the structural determinants of substrate specificity.

The sensitivity, speed, and reproducibility of modern mass spectrometers enable in-depth new functional looks into the cellular proteome. Thousands of proteins can be detected in a single sample. In Co-Fractionation Mass Spectrometry (CF-MS) method, the input sample is fractionated by any biochemical method of choice. The reduced complexity of each fractionated sample leads to better proteome coverage. The separation profiles provide functional information on the proteins. This application has been used to predict organelle localization based on co-purification with marker proteins. More recently, CF-MS is being used to measure the apparent masses and determine the localization of soluble or membrane-associated protein complexes. This Ph.D. dissertation focuses on the extension of the boundary of CF-MS application to learn how protein complex evolution and protein complex composition have been accomplished. In the first part of this dissertation, the data will be presented on the degree to which variation in protein oligomerization across plant species is present, how proteomics in phylogenetic analysis (phyloproteomics/evolutionary proteomics) helps understand the evolutionary changes, and how oligomerization drives neofunctionalization during plant evolution. The latter part will describe that CF-MS coupled with multiple orthogonal chromatographic separations increases the resolving power of the profiling technique, enabling the composition of protein complexes to be predicted in the subaleurone layers of rice endosperm. Lots of novel protein complexes involved in RNA binding protein, translation, and the tissue-species metabolism will be discussed.

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