Diabetes is closely associated with increased oxidative stress, especially originating from the mitochondria. A mechanism to reduce increased mitochondria superoxide production is to reduce the mitochondria membrane potential by releasing protons across the mitochondria membrane. This phenomenon is referred to as mitochondria uncoupling since oxygen is consumed independently of ATP being produced and can be mediated by Uncoupling Proteins (UCPs). However, increased oxygen consumption is potentially detrimental for the kidney since it can cause tissue hypoxia. Therefore, this thesis aimed to investigate the role of mitochondria uncoupling for development of diabetic nephropathy.      UCP-2 was demonstrated to be the only isoform expressed in the kidney, and localized to tubular segments performing the majority of tubular electrolyte transport. Streptozotocin-induced diabetes in rats increased UCP-2 protein expression and correlated to increased non-transport dependent oxygen consumption in isolated proximal tubular cells. These effects were prevented by intense insulin treatment to the diabetic animals demonstrating a pivotal role of hyperglycemia. Importantly, elevated UCP-2 protein expression increased mitochondria uncoupling in mitochondria isolated from diabetic kidneys. Mitochondria uncoupling and altered morphology was also evident in kidneys from db/db-mice, a model of type-2 diabetes, together with proteinuria and glomerular hyperfiltration which are both clinical manifestations of diabetic nephropathy. Treatment with the antioxidant coenzyme Q10 prevented mitochondria uncoupling as well as morphological and functional alterations in these kidneys. Acute knockdown of UCP-2 paradoxically increased mitochondria uncoupling in a mechanism involving the adenosine nucleotide transporter. Increased uncoupling via adenosine nucleotide transporter decreased mitochondria membrane potential and kidney oxidative stress but did not affect glomerular filtration rate, renal blood flow, total kidney oxygen consumption or intrarenal tissue oxygen tension.      The role of increased mitochondria oxygen consumption was investigated by administering the chemical uncoupler dinitrophenol to healthy rats. Importantly, increased mitochondria oxygen consumption resulted in kidney tissue hypoxia, proteinuria and increased staining of the tubular injury marker vimentin, demonstrating a crucial role of increased oxygen consumption per se and the resulting kidney tissue hypoxia for the development of nephropathy.      Taken together, the data presented in this thesis establishes an important role of mitochondria uncoupling for the development of diabetic nephropathy.

Reactive oxygen species (ROS) induce oxidative stress and although are primarily recognized for playing a deleterious biological role, they can be beneficial to cell systems. ROS are extremely short-lived and normally tightly regulated by antioxidant defence systems. Cells react to oxidative stress in different ways, which primarily depends on cell type, stress severity, or both. There is a general limitation in extrapolating to humans conclusions drawn from in vitro and animal studies because of important species-specific differences. Therefore, the general aim of this thesis was to study the influence of oxidative stress on human muscle and bone in vivo. In paper I we presented a one-step HPLC method optimized for the simultaneous determination of purine degradation products in small microdialysis samples. The clinical utility of the method was successfully tested in a patient with traumatic brain injury. In paper II we evaluated microdialysis as an in vivo method to characterize the relative kinetics of ROS-related metabolites in human skeletal muscle exposed to ischaemia-reperfusion. Results indicated that microdialysis was feasible and safe to use in monitoring metabolic events during tourniquet-assisted surgery. In paper III we investigated the association between an oxidative stress marker (urinary 8-iso-PGF2α) and bone mineral density (BMD) and whether α-tocopherol modified the association. The main finding was the negative association between 8-iso-PGF2α and BMD and that the association was further dependent on serum α-tocopherol level. In paper IV we performed a randomized controlled trial to evaluate the influence of Q10 supplementation on exercise performance and metabolites of muscular damage. We did not observe any effects on exercise capacity after 8 weeks of Q10 administration. Nor did we find a significant effect on serum markers related to oxidative stress. In conclusion we have studied the influence of oxidative stress on muscle and bone in vivo in humans. The oxidative stress was triggered by four different causes (trauma, ischemia-reperfusion, ageing, and exercise exhaustion).

Le coenzyme Q (ubiquinone ou Q) est une molécule organique lipophile composée d'une benzoquinone substituée et d'une chaîne polyisoprényle contenant 6 unités chez Saccharomyces cerevisiae (Q6), 8 chez Escherichia coli (Q8) et 10 chez l'homme (Q10). Q a un rôle bien connu de transporteur d'électrons dans les chaînes respiratoires et fonctionne également comme un antioxydant membranaire. La déficience primaire en Q10 a maintenant été attribuée à des mutations dans 6 gènes de la biosynthèse de Q10 et cause des pathologies sévères. La biosynthèse de Q6 est mitochondriale et nécessite au moins 9 protéines organisées au sein d'un complexe multiprotéique chez la levure (Coq1-Coq9). L'acide 4-hydroxybenzoique (4-HB) et l'acide para-aminobenzoique (pABA) sont les deux précurseurs connus du noyau aromatique de Q6. Malgré de nombreuses recherches et l'importance cruciale de Q dans le métabolisme eucaryote, certaines étapes de la voie de biosynthèse de Q ne sont pas connues. L'étude présentée dans ce manuscrit a permis de montrer l'implication de la protéine Coq6, proposée comme étant une mono-oxygénase à flavine, dans une seule des trois réactions d'hydroxylation que compte la voie de biosynthèse de Q6: l'hydroxylation en C5. De plus, notre étude sur Coq8, une protéine kinase dont sa surexpression stabilise le complexe multiprotéique, nous a permis de confirmer les fonctions de certaines protéines Coq (Coq5, Coq7), de découvrir la fonction de Coq6 et d'éclaircir le rôle des autres (Coq4, Coq9). Nous rapportons également que des analogues hydroxylés ou méthoxylés de 4-HB et du pABA peuvent court-circuiter des étapes déficientes des mutants particuliers conduisant ainsi à la synthèse du coenzyme Q6 dans ces derniers. Ce résultat ouvre de nouvelles perspectives pour traiter les déficiences en coenzyme Q10 qui jusqu'à présent sont traitées par supplémentation en Q. Finalement, la réaction de déamination, essentielle à la biosynthèse de Q6 à partir du pABA, reste incomprise mais nos résultats suggèrent fortement l'implication de Coq6 dans cette étape.

A hiperglicemia crônica favorece a ocorrência da nefropatia induzida por contraste iodado (NIC). Diabetes Mellitus (DM) e NIC compartilham mecanismos de lesão oxidativa e indução de enzimas de proteção e adaptação celular como a coenzima Q-10 (COQ-10). O objetivo deste estudo foi avaliar o papel da COQ-10 na função e hemodinâmica renal, perfil oxidativo e histologia renal em ratos diabéticos submetidos ao modelo de NIC. Métodos: Ratos Wistar, machos, 250 a 290 g, foram randomizados nos grupos: Citrato: animais que receberam tampão citrato 0,01M, (veículo da estreptozotocina), 0,4 ml intravenoso (i.v), 1 vez; Tween 80: animais que receberam Tween 80, 1%, (veículo da COQ-10), 0,5 ml, intraperitoneal (i.p.), 1 vez; DM: animais que receberam estreptozotocina (65 mg/kg), i.v., 1 vez, no 1º dia do protocolo; DM+CI: animais DM que no 26º dia de protocolo receberam contraste iodado (CI, 6 ml/kg), i.p., 1 vez; DM+CI+COQ-10: animais DM com pré-condicionamento com COQ-10 (10 mg/kg), 1 vez por 6 dias a partir do 22º dia de protocolo, e o tratamento com CI. O protocolo de todos os grupos teve duração de 4 semanas. Foram avaliados parâmetros fisiológicos (ingestão de ração e água, peso, glicemia, razão peso do rim e peso do animal), a função renal (clearance de inulina), a hemodinâmica renal (fluxo sanguíneo renal e resistência vascular renal), o perfil oxidativo (peróxidos, óxido nítrico e substâncias reativas ao ácido tiobarbitúrico na urina, tióis no tecido renal) e análise histológica renal. Resultados: Animais DM apresentaram hiperglicemia, polidipsia, poliúria, polifagia, perda de peso e aumento da relação peso rim/animal, com redução da função renal, além de redução do fluxo sanguíneo renal, elevação da resistência vascular renal, com aumento na excreção de metabólitos oxidativos e consumo de reserva antioxidante endógena. O grupo DM+CI demonstrou redução adicional na função, alterações na hemodinâmica renal e aumento nos parâmetros de estresse oxidativo. A administração de COQ-10 atenuou a redução da função renal, preveniu alterações hemodinâmicas renais e reduziu o estresse oxidativo no grupo DM+CI. As alterações histológicas no DM e DM+CI foram discretas e o tratamento com COQ-10 previniu a progressão de danos histológicos mais extensos nos animais que receberam CI. Conclusão: O tratamento com COQ-10 demonstrou efeito antioxidante na NIC em ratos diabéticos com melhora significativa da função e hemodinâmica renal. / Chronic hyperglycemia favors the occurrence of nephropathy induced by iodinated contrast (CIN). Diabetes Mellitus (DM) and CIN share oxidative damage mechanisms and induction of protective and cellular adaptation enzymes as coenzyme Q-10 (CoQ-10). The aim of this study was to investigate the role of COQ-10 in renal function and hemodynamics, oxidative profile and renal histology in diabetic rats submitted to the NIC model. Methods: Wistar rats, male, weighing 250-290 g, were randomized into two groups: Citrate: animals that received citrate buffer 0.01M (streptozotocin), 0.4 ml, intravenous (i.v.), once; Tween 80: animals that received Tween 80, 1% (CoQ-10 vehicle), 0.5 ml, intraperitoneal (i.p.), once; DM: animals given streptozotocin (65 mg/kg) i.v., once on the first day of the protocol; CI+DM: DM animals, on the 26º day protocol, tretated with iodinated contrast (CI, 6 ml/kg) i.p., once; DM+CI+COQ-10: DM animals preconditioned with COQ-10 (10 mg/kg), once a day, for 6 days from the 22º day and treated with CI. The protocol for all groups lasted 4 weeks. Physiological parameters evaluated were (food and water intake, corporal weight, blood glucose and right kidney weight), renal function (inulin clearance), renal hemodynamics (renal blood flow and renal vascular resistance), the oxidative profile (peroxides, nitric oxide and reactive substances to thiobarbituric acid in urine, thiols in renal tissue) and renal histological analysis. Results: DM animals showed hyperglycemia, polydipsia, polyuria, polyphagia, weight loss and increased weight kidney / animal relationship with reduced renal function, as well as a reduction on renal blood flow, increased renal vascular resistance and changes in oxidative profile with increased the excretion of metabolites and oxidative consumption of endogenous antioxidant reserve. DM+CI promoted further reduction in renal function, exacerbated hemodynamic changes and increase in oxidative stress parameters. COQ-10 administration preserved renal function, prevented hemodynamic changes and reduced oxidative stress in the DM + CI + COQ-10. Histological changes in DM and DM + CI were discrete and treatment with CoQ-10 prevented the progression of the histologic damage in the animals receiving CI. Conclusion: COQ-10 presented an antioxidant effect on the NIC in diabetic rats, by improving function and renal hemodynamics and reducing oxidative stress.

Mutantes respiratórios de Saccharomyces cerevisiae podem apresentar uma ampla variedade de instabilidade do mtDNA. Nós analisamos diferentes classes de mutantes e observamos uma elevada instabilidade nos mutantes que não possuem a coenzima Q (CoQ) funcional. O objetivo desse trabalho foi avaliar os efeitos das alterações no estado redox da coenzima Q sobre a estabilidade do mtDNA de diferentes linhagens de S. cerevisiae. No mutante <font face=\"Symbol\">Dcoq10, que sintetiza CoQ não funcional, a inativação das NADH desidrogenases individuais Ndi1p e Nde1p, resultou numa menor instabilidade do mtDNA, acompanhada por uma diminuição na taxa de liberação de peróxido de hidrogênio (H2O2). Por outro lado, a super-expressão de Nde1p aumentou a instabilidade do mutante <font face=\"Symbol\">Dcoq10. A inativação das NADH desidrogenases na linhagem <font face=\"Symbol\">Dcoq4, deficiente na síntese da CoQ, não reduziu a instabilidade do mtDNA. Juntos, os resultados indicam que alterações no estado de oxido-redução da coenzima Q influenciam a estabilidade do mtDNA, provavelmente através da produção de espécies reativas de oxigênio. / Saccharomyces cerevisiae respiratory mutants can show a wide range of mtDNA instability. We analyze different classes of mutants and observed a higher instability among mutants lacking a functional coenzyme Q (CoQ). The aim of this study was to evaluate the effects of alterations in the redox state of coenzyme Q on the stability of mtDNA mitochondrial in different strains of Saccharomyces cerevisiae. In <font face=\"Symbol\">Dcoq10 mutant, which synthesizes CoQ nonfunctional, inactivation of individual NADH dehydrogenases Ndi1p Nde1p has shown a decreased mtDNA instability, which was accompanied by a decrement in the rate of hydrogen peroxide (H2O2) release. Moreover, overexpression of Nde1p increased instability <font face=\"Symbol\">Dcoq10 mutant. The inactivation of individual NADH dehydrogenases in <font face=\"Symbol\">Dcoq4 strain which is deficient in the synthesis of CoQ, did not reduce the instability of the mtDNA. All the results indicate that changes in the redox state of coenzyme Q influence the stability of mtDNA, probably by the production of reactive oxygen species.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has long been the scourge of humanity, claiming millions of lives. It is the most devastating infectious disease of the world in terms of mortality as well as morbidity (WHO, 2009). The lack of a uniformly effective vaccine against TB, the development of resistance in the Mycobacterium tuberculosis against the present antitubercular drugs and its synergy with AIDS has made the situation very alarming. This therefore necessitates a search for new antitubercular drugs as well as the identification of new and unexplored drug targets (Broun et aI., 1992). Coenzyme A is an essential cofactor for all organisms and is synthesized in organisms from pantothenate by a universally conserved pathway (Spry et al., 2008; Sassetti and Rubin, 2003). The first enzyme of the pathway, pantothenate kinase catalyzes the most important step of the biosynthetic process, being the first committed step of CoA biosynthesis and the one at which all the regulation takes place (Gerdes et aI., 2002) This thesis describes the successful cloning of PanK from Mycobacterium tuberculosis, its expression in E. coli, single step affinity purification, and complete biochemical and biophysical characterization. In this work, pantothenol, a widely believed inhibitor of pantothenate kinase, has been shown to act as a substrate for the mycobacterial pantothenate kinase. Further it was shown that the product, 4'phosphopantothenol, thus formed, inhibited the next step of the CoA biosynthesis pathway in vitro. The study was extended to find outthe fate of pantothenol inside the cell and it was demonstrated that the CoA biosynthetic enzymes metabolized the latter into the pantothenol derivative of CoA which then gets incorporated into acyl carrier protein. Lastly, it was decisively shown that pantothenate kinase is not only regulated by feedback inhibition by CoA but, also regulated through feed forward stimulation by Fructose 1, 6 biphosphate (FBP), a glycolytic intermediate. The binding site of FBP was determined by docking and mutational studies of MtPanK. Chapter 1 presents a brief survey of the literature related to Coenzyme A biosynthesis pathway and describes the objective of the thesis. It also presents a history of TB and briefly reviews literature describing TB as well as the life cycle, biology, survival strategy, mode of infection and the metabolic pathways operational in the TB parasite, Mycobacterium tuberculosis. The chapter details the enzymes involved in CoA biosynthesis pathway from various organims. Chapter 2 In this chapter, cloning of the ORF (Rv1092c), annotated as pantothenate kinase in the Tuberculist database (http://genolist.pasteur.frfTubercuList), its expression in E. coli and purification using affinity chromatography has been described. Protein identity was confirmed by MALDI-TOF and by its ability to complement the pantothenate kinase temperature sensitive mutant, DV70. This chapter also illustrates the oligomeric status of MtPanK in solution and describes the biochemical characterization of MtPanK by means of two different methods, spectrophotometrically by a coupled assay and calorimetrically by using Isothermal Titration Calorimetry. Feedback inhibition of MtPanK by CoA is also discussed in this chapter. Chapter 3 describes the biophysical characterization of MtPanK. It discusses the enthalpy (~H) and free energy change (~G) accompanying the binding of a non-hydrolysable analogue of ATP; CoA; acetyl CoA and malonyl-CoA to MtPanK. The chapter details the energetics observed upon ATP binding to pantothenate-saturated MtPanK further elucidating the order of the reaction. This chapter also describes the various strategies which were designed and tested to remove CoA from the enzyme as the latter is always purified from the cell in conjunction with CoA. Validation of these strategies for complete CoA removal (by studying the n value from ITC studies) is further described. Chapter 4 discusses the interaction of the well-studied inhibitor of pantothenate kinases from other sources (e.g. the malarial parasite), pantothenol, with the mycobacterial enzyme. In order to investigate the interaction of this compound with MtPanK, its effect on the kinetic reaction carried out by the enzyme was studied by several methods. Surprisingly, a new band corresponding to 4'phosphopantothenol appeared when the reaction mix of MtPanK with pantothenol and ATP was separated on TLC. The identity of the new spot was confirmed by mass spectrometry analyses of the MtPanK reaction mixture.. These findings established the fact that pantothenol is a substrate of pantothenate kinase. To delve deeper into the mechanism of interaction of this compound with the enzymes of the coenzyme A biosynthesis pathway, the ability of pantothenol to serve as a substrate for the next step of the pathway, MtCoaBC was studied. Using various approaches it was established that pantothenol is actually a substrate for the MtPanK and the inhibition observed earlier (Saliba et aI., 2005) is actually due to the inability of CoaBC to utilize 4' -phosphopantothenol as substrate. Chapter 5 takes the story from Chapter 4 further detailing the effects of pantothenol on cultures of E. coli and M. smegmatis. I observed that pantothenol does not inhibit the culture of E. coli or M. smegmatis. So, further studies were carried out to know the fate of pantothenol once it is converted into 4'phosphopantothenoi. Since, the next enzyme of the pathway does not utilize 4'phosphopantothenol, I checked the further downstream enzyme of the pathway, CoaD, and found that it converts 4'-phosphopantothenol to thepantothenol derivative of dephospho-CoA. The next enzyme of the pathway, CoaE, took up this pantothenol derivative of dephospho-CoA as a substrate and converted it to the pantothenol derivative of CoA which was then transferred to apo-ACP by holo-ACP synthase. The holo-ACP thus synthesized enters into the dedicated pathway of fatty acid synthesis. Extensive investigations have been carried out on the regulation of pantothenate kinases, by the product of the pathway, Coenzyme A and its thioesters, xx establishing the latter as the feedback regulators of these enzymes. In order to determine if the cell employs mechanisms to sense available carbon sources and consequently modulate its coenzyme A levels by regulating activity of the enzymes involvedin CoA biosynthesis, glycolytic intermediates were tested against MtPanK for their possible role in the regulation of MtPanK activity. Chapter 6 details my identification of a novel regulator of MtPanK activity, fructose-I, 6-bisphosphate (FBP), a glycolytic intermediate, which enhances the MtPanK catalyzed phosphorylation of pantothenate by three fold. Further, the possible mechanisms through which FBP mediates MtPanK activation are also discussed. This chapter also describes the experiments carried out to identify the binding site of FBP on MtPariK.Interestingly, docking of FBP on MtPanK revealed that FBP binds close to the ATP binding site on the enzyme with one of its phosphates overlapping with the 3'~phosphate of CoA thereby validating its competitive binding relative to CoA on MtPanK. Based on these observations I propose that the binding of FBP to MtPanK lowers the activation energy of pantothenate phosphorylation by PanK. Chapter 7 presents a summary of the findings of this work. Coenzyme A biosynthesis pathway harbors immense potential in the development of drug against many communicable diseases, thanks to its essentiality for the pathogens and the differences between the pathogen and host CoA biosynthetic enzymes. The work done in this thesis extensively characterizes the first committed enzyme of the CoA biosynthetic pathway, pantothenate kinase, from Mycobacterium tuberculosis (MtPanK). The thesis also deals with the fate of a known inhibitor of PanK and proves it as a substrate for MtPanK. Finally this thesis describes a new link between glycolysis and CoA biosynthesis. Biotin, like coenzyme A, is another essential cofactor required by several enzymes in critical metabolic pathways. De novo synthesis of this critical metabolite has been reported only in plants and microorganisms. Therefore targeting the synthesis of biotin in the tubercular pathogen is another effective means of handicapping the tubercle pathogen. During the course of my studies, I also investigated the mycobacterial biotin biosynthesis pathway, studying the first enzyme of the pathway, 7-keto-8-aminopelargonic acid (KAPA) synthase (bioF) in extensive detail. Appendix 1 elucidates the kinetic properties of 7-keto-8aminopelargonic acid synthase (bioF) from Mycobacterium tuberculosis and proves beyond doubt that D-alanine which has previously been reported to act as a competitive inhibitor for the B. sphaericus enzyme (Ploux et al., 1999), is actually a substrate for the mycobacterial bioF.

Malonyl-thioesters are reactive at the thioester carbonyl and the carboxylate moieties, as seen in acyl transfer or hydrolysis and decarboxylation. Enzymes use these reactive centers to perform different enzyme chemistry throughout metabolism. This enzyme chemistry coupled with the inherent reactivity of malonyl-thioesters makes structure-function studies difficult. When malonyl-thioesters are used for structure-function studies, it usually results in a hydrolyzed or decarboxylated product. There are examples, however, where this is overcome, many of which are discussed throughout this thesis. To overcome the inherent reactivity of malonyl-thioesters and enzymes, analogs have been synthesized to perform structure-function studies. Initial studies focused on altering the thioester carbonyl to limit hydrolysis and decarboxylation; however, these studies revealed the importance of retaining the thioester carbonyl to be positioned in the oxyanion hole. My thesis work focused on the synthesis, characterization, and use in structure-function studies of malonyl-thioester analogs that either preserve the thioester carbonyl or alter it to an ester or amide, and alter the carboxylate to a sulfonate or nitro group. After synthesizing the methylmalonyl-CoA analogs, we performed structure-function studies with methylmalonyl-CoA decarboxylase. This case study revealed the potential of these analogs to both inhibit decarboxylase activity and their use in structure-function studies to gain mechanistic insights. This successful study prompted us to continue these structure-function studies in enzymes with different chemistries such as an epimerase or bi-functional acyltransferase/decarboxylase. The widespread use of these methylmalonyl-CoA analogs also motivated us to add more malonyl-thioester analogs to our toolbox. I have preliminary data that these malonyl-thioester analogs inhibit β-keto-acyl-synthase III, an enzyme involved in fatty acid production in <i>E. coli</i>. This inhibition gives us confidence that these analogs will be useful in structure-function studies that will reveal answers to long standing mechanism and protein-protein interaction questions in the polyketide and fatty acid synthase field.

<p>  </p> <p>Biosynthesis of fatty acids and specialized metabolites, such as polyketides, is dependent on the C-C bond forming enzymatic activity of carboxylases and <u>k</u>eto<u>s</u>ynthases (KS). Carboxylases and KS perform complex carbon-carbon bond forming reactions via a ping-pong mechanism; the catalytic interactions of which are still unclear. The KS reaction involves the Claisen condensation of an acylated enzyme with a malonyl-thioester, driven forward by the energy of the malonyl-thioester decarboxylation. Similarly, the carboxylase proceeds via a carboxyl-biotin-enzyme intermediate, and a subsequent C-C bond forming reaction. Engineering the substrate specificity of these enzyme involved in producing polyketides is sought after for the purpose of producing novel, derivative polyketides. These derivative polyketides may have serve as effective new antibiotics, of which discovery has waned. Unfortunately, incomplete understanding of protein-protein interactions, conformational changes, and substrate orientation in catalysis leads to not well informed engineering attempts. A challenge in deducing the catalytic details of enzymes acting on malonyl-thioesters in general is the hyper-reactivity of their β-ketoacid and thioester substrates, which are prone to hydrolysis and decarboxylation. Many structures of malonyl-CoA bound enzymes feature hydrolysis of the thioester, preventing determination of enzyme:substrate interactions in structure-function studies. To work around this problem of innate reactivity, groups have synthesized a variety of acyl-thioester analogs for probing the details of enzyme catalysis with mixed success. The success of these enzyme:analog mechanistic studies appears to hinge upon the similarity of the analog to the natural substrate. Here, we demonstrate the synthesis of near-natural, acyl-thioester analogs, featuring single atom substitutions. Using a novel UV-vis assay, we have determined <em>K</em>i values of our analogs with paradigmatic KSs <em>E. coli</em> FabH. These <em>K</em>i values are marginally higher than the substrate <em>K</em>m values, suggesting the KSs bind the analogs as they would natural substrates. Using this information, we have conducted preliminary X-ray crystallography experiments to determine the carboxylase:analog and KS:analog catalytic interactions, which will allow for new insight into debated C-C bond forming catalytic details. The information presented in this thesis and additional studies on protein-protein interactions can be leveraged into informed engineering studies of PKS enzymes.</p>

It is well-known that the large diversity of protein functions and structures derives from the broad spectrum of physicochemical properties of the 20 canonical amino acids that constitute modern proteins. According to the generally accepted coevolution theory of the genetic code, evolution of protein structures and functions was continuously associated with enrichment of the genetic code, with aromatic amino acids being considered the latest addition to the genetic code to increase structural stability of proteins and diversification of their catalytic functions. The main objective of this master thesis was to test whether enzymatic catalysis could precede the appearance of aromatic amino acids in the standard genetic code. For that purpose, the effect of amino acid alphabet reduction on structure and function of dephosphocoenzyme A kinase (DPCK) was studied. Dephosphocoenzyme A kinase catalyses the final step in the biosynthesis of coenzyme A, a very conserved cofactor. Two aromatic amino acid-lacking mutants of DPCK from a thermophilic bacterium, Aquifex aeolicus, were designed by substituting aromatic amino acid residues by (i) leucines and (ii) various non-aromatic amino acids to best preserve the structural stability of the protein. Wild type protein and the two mutants were cloned and...

The mechanism by which nicotinamide adenine dinucleotide (NAD) stimulates the activity of adenylate cyclase (AC) in canine plasma membrane has been studied. Using [3 2P]-NAD, the activation by NAD was correlated with the radiolabeling of the stimulatory guanosine triphosphate (GTP) binding protein Gsa. Further characterization demonstrated that the modification occurred only in the presence of G-protein activators and that arginine residue(s) were modified by ADP-ribose by the action of a mono-ADP-ribosyltransferase. Inhibitors of the transferase blocked both the modification of Gsa and the activation of AC. Collectively, these studies suggest that ADP-ribosylation of Gsa by an endogenous mono-ADP-ribosyltransferase may regulate cardiac AC.