Deux études de population, en Chine centrale et en Lorraine, ont mis en évidence une association des gènes MTR, MTRR et MTHFR avec la maladie de Crohn et la recto-colite hémorragique. De plus entre MTRR et stress oxydant il existe un lien qui dépend de la concentration de superoxyde dismutase et du degré de sévérité de la maladie. Par ailleurs dans la maladie de Crohn il existe une élévation importante de l'homocystéine, une diminution de la vitamine B12 liée à la résection iléale et à une influence du stress oxydant sur les capacités réductrices de la vitamine B12 par MTRR. Parallèlement, des rats exposés au DSS, qui est un agent inducteur de lésions inflammatoires de la muqueuse colique, présentent une potentialisation de la réaction inflammatoire s’il sont en plus carencés en donneurs de groupement méthyle. Paradoxalement, la carence en groupement méthyle chez ces rats n’entraîne pas d’effet ni sur l'apoptose ni sur le stress oxydant. Par contre, elle induit une augmentation de l'expression des acteurs de la voie p38/cPLA2/COX2 conduisant à une augmentation de la synthèse de PGE2 sans qu’il n’y ait d’influence sur COX1 ni 5-LOX. Ces résultats ouvrent des perspectives physiopathologiques très originales dans les maladies inflammatoires digestives. Ils décrivent des interactions causes-effets dans lesquelles la maladie provoque une malabsorption, donc une carence en donneurs de méthyle, le stress oxydant diminue les capacités de réduction de la vitamine B12 au niveau intra-muqueux et la carence en groupements méthyles exacerbe les mécanismes inflammatoires en réponse à l’agression par des agents exogènes comme les bactéries. / Studies in populations from Central China and Lorraine showed a correlation between MTR, MTRR, MTHFR and hemorrhagic colitis and Crohn disease. In addition there was a correlation between MTRR and oxidative stress depending on superoxyde dismutase concentration and disease severity. Besides, Crohn disease was characterized by elevated homocysteine and decreased vitamin B12 linked to ileal resection and the effect of oxidative stress on MTRR capacity to reduce vitamin B12. On the other hand, in rats exposed to DSS, which is a potent inducer of inflammatory lesions in colonic mucosa, the extent of inflammation was aggravated by additional exposure to a diet deficient in methyl group donors. Unexpectedly, methyl group donor deficiency in rats did not yield any effect on apoptosis nor oxidative stress. However methyl group donor deficiency induced elevated expression of actors in the p38/cPLA2/COX2 pathway that leads to PGE2 production without affecting neither COX1 nor 5-LOX. These results uncover new pathophysiological events in inflammatory bowel disease (IBD). They depict intricated relationships in which the disease may cause malabsorption, and therefore a deficiency in methyl donors, oxidative stress decreases the capacity to reduce vitamin B12 in the digestive epithelium and methyl group donors deficiency exacerbates inflammatory mechanisms triggered by exposure to exogenous agents such as bacteria.

Taxadiene synthase catalyzes the formation of taxadiene from GGPP, the universal building block of diterpenes. The cyclization of GGPP to taxadiene is generally thought to proceed through a series of monocyclic and bicyclic carbocation intermediates, all of which are mechanistically plausible but experimentally not isolable, and therefore, unobservable. To gain a better understanding of the mechanism of the cyclization of GGPP to taxadiene, a series of GGPP analogs were chemically synthesized and fed in vitro to taxadiene synthase (overexpressed in truncated form in E. coli). These analogs were designed to interrupt the cyclization cascade, such that the monocyclic and/or bicyclic carbocation intermediates could not react further and therefore would be quenched to give isolable and observable monocyclic or bicyclic hydrocarbon products. Four monocyclic hydrocarbon compounds were obtained from four reactions of different analogs, and the structure of each product was unambiguously solved by 1D and 2D NMR. These results support the intermediacy and the existence of the cembrenyl cation in the cyclization of GGPP to taxadiene, and indicate that modifications at the 10,11 double bond of GGPP are tolerable in the cyclization.

Taxadiene synthase catalyzes the formation of taxadiene from GGPP, the universal building block of diterpenes. The cyclization of GGPP to taxadiene is generally thought to proceed through a series of monocyclic and bicyclic carbocation intermediates, all of which are mechanistically plausible but experimentally not isolable, and therefore, unobservable. To gain a better understanding of the mechanism of the cyclization of GGPP to taxadiene, a series of GGPP analogs were chemically synthesized and fed in vitro to taxadiene synthase (overexpressed in truncated form in E. coli). These analogs were designed to interrupt the cyclization cascade, such that the monocyclic and/or bicyclic carbocation intermediates could not react further and therefore would be quenched to give isolable and observable monocyclic or bicyclic hydrocarbon products. Four monocyclic hydrocarbon compounds were obtained from four reactions of different analogs, and the structure of each product was unambiguously solved by 1D and 2D NMR. These results support the intermediacy and the existence of the cembrenyl cation in the cyclization of GGPP to taxadiene, and indicate that modifications at the 10,11 double bond of GGPP are tolerable in the cyclization.

Thesis (Ph. D.)--University of Iowa, 2007. / Thesis supervisor: Amnon Kohen. Includes bibliographical references (leaves 108-114).

München, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

München, Techn. Univ., Diss., 2001. / Computerdatei im Fernzugriff.

München, Techn. Universiẗat, Diss., 2001.

A limitation to investigations of homodimeric protein dissociation is that the signals produced from methods such as fluorescence and circular dichroism represent both dissociation and protein unfolding that may be occurring simultaneously within a sample. Although size exclusion chromatography examines the state of a protein’s quaternary structure, complicated overlapping peaks representative of oligomer and monomer can result. To address these limitations the mixed dimer system has been adopted to investigate the dissociation of a homodimeric protein. A mixed dimer is a species in which each subunit of a homodimeric protein is associated with a different affinity tag. The two tags used are the His6-tag and the Glu7-tag. Such a mixed dimer will bind to a metal chelating column such as Ni-NTA so long as the dimer is intact. Denaturant- or temperature induced dimer dissociation can be detected by the amount of Glu7-tagged subunit present in the unbound fraction after the protein is passed over an Ni-NTA resin. SDS PAGE and densitometry assess the amount of Glu7-tagged subunit present in those unbound fractions. The experimental conditions necessary to implement this method were developed, and then applied to mammalian inducible nitric oxide synthase (iNOS) and Staphylococcus aureus NOS (SaNOS). With respect to both urea and temperature, the stability of SaNOS is higher than that of iNOS in spite of the bacterial enzyme having a much smaller dimer contact surface. We have also used the mixed dimer method to estimate an equilibrium dissociation constant (KD) for iNOS dissociation of no greater than 2.3μM. This value is compared to the results obtained for iNOS by analytical ultracentrifugation, which can characterize protein complexes and their stoichiometry. / Thesis (Master, Chemistry) -- Queen's University, 2007-10-26 15:28:14.182

Human mitochondrial ATP synthase is an enzyme containing 18 unlike subunits located in the inner mitochondrial membrane (IMM), where the catalytic F1 domain extends into the mitochondrial matrix and the FO domain, which contains the c8-ring rotor, the a-subunit and the supernumerary subunits, is anchored in the IMM. All the subunits, apart from the a- and A6L-subunits, are encoded in the nucleus and require transport into the mitochondria before being assembled. The a- and A6L-subunits are encoded on the mitochondrial genome. The respiratory complexes generate the proton motive force (PMF), which ATP synthase uses to generate ATP from ADP and Pi. Rotation of the α- and β-subunits with the central stalk γ-, δ- and ε-subunits is prevented by coupling the F1 domain to the FO domain via the peripheral stalk (the OSCP-, F6-, d- and b-subunits). ATP hydrolysis is prevented by the natural inhibitor of the enzyme, IF1, binding to the F1 domain. In addition to the aand, b-subunits, the FO domain contains the c8-ring and six supernumerary subunits not involved in the catalytic activity of ATP synthase. The roles of five of these subunits in the assembly of ATP synthase, the e-, f-, g-, DAPIT- and 6.8 kDa proteolipid-subunits, were investigated by suppressing or disrupting their expression individually. The e-subunit is the first of the supernumerary subunits to assemble, then the g-subunit followed by the f-, 6.8 kDa proteolipid- and DAPIT-subunits. All five supernumerary subunits investigated were required to facilitate the dimerisation and oligomerisation of ATP synthase. The e-, f- and g-subunits were found to be important for maintaining mitochondrial respiratory capacity.

Indiana University-Purdue University Indianapolis (IUPUI) / Acetolactate synthase (ALS) and acetohydroxyacid synthase (AHAS) are two thiamin diphosphate (ThDP)-dependent enzymes that catalyze the formation of acetolactate from two molecules of pyruvate. In addition to acetolactate, AHAS can catalyze the formation of acetohydroxybutyrate from pyruvate and α-ketobutyrate. When formed by AHAS, these compounds are important precursors to the essential amino acids valine and isoleucine. Conversely, ALS forms acetolactate as a precursor to 2,3-butanediol, a product formed in an alternative pathway to mixed acid fermentation. While these enzymes catalyze the same reaction, they have been found to be quite different. Such differences include: biological function, pH optimum, cofactor requirements, reaction kinetics and quaternary structure. Importantly, AHAS has been identified as the target of the widely-used sulfonylurea and imidazolinone herbicides, which has led to many structural and kinetic studies on AHAS enzymes from plants, bacteria, and fungi. ALS, on the other hand, has only been identified in bacteria, and has largely not seen such extensive characterization. Finally, although some bacteria contain both enzymes, they have never been studied in detail from the same organism. Here, the ALS and AHAS enzymes from Klebsiella pneumoniae were studied using steady-state kinetic analyses, X-ray crystallography, site-directed and site-saturation mutagenesis, and cell growth complementation assays to i) compare the kinetic parameters of each enzyme, ii) compare the active sites to probe their differences in substrate profile and iii) test the ability of ALS to function in place of AHAS in vivo.