Enzyme dynamics has been established to play a crucial role in catalysis, and it has therefore become an important area of research to better understand enzymatic rate enhancements. The light-activated enzyme protochlorophyllide oxidoreductase (POR) is a well-studied model system where dynamics are known to be important for catalysis. The catalytic reaction involves a sequential hydride and proton transfer to reduce the C17-C18 double bond in the protochlorophyllide (Pchlide) substrate with NADPH as a cofactor to yield the chlorophyllide (Chlide) product. Both H-transfer steps are established to undergo quantum tunneling, as derived from the temperature-dependence of the kinetic isotope effects (KIEs). Furthermore, a role for ‘promoting motions/vibrations’ has been presumed from the temperature-dependence KIE data, which will be investigated further in this thesis by the study of the KIE response to pressure. A general overview of the pressure-dependence as a new experimental probe is presented and compared with temperature-dependencies of KIEs, to establish whether pressure is suitable as an alternative technique for studying the role of enzyme dynamics in catalysis. This involves a comparison of pressure data from other enzyme systems to newly collected data for POR. However, no clear trend between temperature and pressure data is observed and hence, it can be concluded that pressure effects can be difficult to interpret. A case by case analysis is required and needs to be combined with computational simulations based on structural evidence (e.g. X-ray crystallographic), which is not yet available for POR.Solvent-viscosity has been successfully used to probe enzyme dynamics in POR and provides information on the extent of any protein networks that are involved along the reaction coordinate. Here I investigate the solvent-viscosity dependence of both H-transfer reactions in POR for a range of homologous POR enzymes to obtain an evolutionary perspective of the protein dynamics required for catalysis. This has been successfully used in the past on a limited number of POR homologues and has led to the formulation of a hypothesis supporting a twin-track evolution of the two catalytic steps in POR. I observed a lack of solvent-viscosity dependence in case of the hydride transfer across all the investigated lineages, while the proton transfer was shown to be more strongly affected by viscosity in prokaryotic enzymes than in their eukaryotic counterparts. This supports the proposed theory, suggesting an early optimisation of the dynamics involved in the light-activated hydride transfer with a strong reliance on localised motion. Conversely, the proton transfer experienced selective pressure to reduce its dependence on complex solvent-slaved motion and that has led to localised dynamics in eukaryotic POR homologues. Additionally, I found that the enzymes from eukaryotic species have a higher rate of both H-transfer steps, suggesting that an optimisation of the active site architecture occurred upon endosymbiosis. Enzyme dynamics clearly have a pivotal role to play in catalysis of this unique light-activated enzyme and detection of these will only be possible by detailed structural information.

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T10:44:31Z (GMT). No. of bitstreams: 1 Fonseca_FrancineSouzaAlvesda_D.pdf: 8642062 bytes, checksum: fa3ebef40c6304d2e6d2b02ba701f86e (MD5) Previous issue date: 2013 / Resumo: Capítulo I. Este trabalho revelou aspectos inéditos da ecologia química de Phloeophana longirostris e Phloea subquadrata, duas espécies de percevejo da família Phloeidae. Esta família, representada por apenas três gêneros e quatro espécies, é caracterizada por apresentar cuidado maternal com ovos e ninfas e camuflagem sobre o tronco das árvores hospedeiras. O objetivo deste trabalho foi localizar, identificar e caracterizar os principais compostos voláteis emitidos por percevejos e verificar a relação destes com as árvores hospedeiras. Os resultados revelaram que os insetos, após estímulo físico, liberam aldeídos e hidrocarbonetos insaturados, oxoaldeídos e ésteres como compostos majoritários. Além disso, foi possível discriminar compostos característicos de cada grupo estudado. Capítulo II. Na segunda parte deste trabalho, foi explorada a atividade enzimática do micro-organismo Bacillus safensis isolado do petróleo brasileiro. Esse micro-organismo apresenta um arsenal enzimático capaz de degradar compostos orgânicos aromáticos. O objetivo deste trabalho foi purificar, isolar e identificar oxirredutases presentes na cepa (CFA06) de Bacillus safensis. Através dessa bactéria Gram-positiva, altamente resistente à radiação ultravioleta, foi possível identificar duas oxirredutases, sendo uma delas inédita. A oxirredutase inédita, aqui denominada de BsP315, apresenta 21 kDa e ferro e molibdênio ligados a essa proteína. Além dessa oxirredutase, uma catalase, aqui denominada de BsCat, também foi identificada / Abstract: Chapter I. This study revealed novel aspects of the chemical ecology of Phloeophana longirostris and Phloea subquadrata from Phloeidae family. This family, has only three genders and four species with characteristic maternal care of eggs and nymphs and camouflage on the trunk of the host tree. The aim of this study was to the identification of major volatile compounds produced by the stink bugs and their relationship with the host trees. The results revealed that after physical stimulation, the insects release aldehydes and unsaturated hydrocarbons, esters and oxoaldehyde as major compounds. These volatile compounds were important in the discrimination of male, female and nymphs of each species. Chapter II. In the second part of this research, we investigated the enzymatic activity of the micro-organism Bacillus safensis isolated from Brazilian oil. This micro-organism has an enzymatic arsenal capable of degrading aromatic organic compounds. The aim of this study was to purify, isolate and identify oxidoreductases present in strain (CFA06) Bacillus safensis. Through this Gram-positive bacterium, highly resistant to ultraviolet radiation, it was possible to identify two oxidoreductases. The novel oxidoreductase, here named BsP315 has 21 kDa and iron and molybdenum bound to its catalytic site, additionally we have isolated a catalase here named BsCat / Doutorado / Quimica Organica / Doutora em Ciências

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22484号 / 農博第2388号 / 新制||農||1075(附属図書館) / 学位論文||R2||N5264(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 小川 順, 教授 加納 健司, 教授 栗原 達夫 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

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NAD(P)H:Quinone oxidoreductase-1 (NQO1) has been implicated in the bioreductive activation of the clinically active anticancer drug Mitomycin C (MMC) and a polymorphic variant of NQO1 which lacks functional enzyme activity (NQO1*2) has been linked with poor survival in patients treated with MMC. The relationship between NQO1 activity and cellular response to MMC is however controversial and the aim of this study was to determine whether the response of bladder cancer patients to MMC can be forecast on the basis of NQO1*2 genotype status. Genomic DNA was extracted from formalin-fixed, paraffin-embedded tissue from 148 patients with low to intermediate grade (G1/G2) superficial (Ta/T1) bladder cancers and NQO1*2 genotype status determined by PCR-RFLP. NQO1*2 genotype status was retrospectively compared with clinical response to intravesical administered MMC with the primary end-point being time to first recurrence. NQO1 phenotype was determined by immunohistochemistry. Of the 148 patients genotyped, 85 (57.4%) were NQO1*1 (wild-type), 59 (39.8%) were NQO1*1/*2 (heterozygotes) and 4 (2.7%) were NQO1*2/*2. No NQO1 protein expression was detected in NQO1*2/*2 tumours. A broad spectrum of NQO1 protein expression existed in tumours genotyped as NQO1*1 and NQO1*1/*2 although tumours with NQO1*1 typically expressed higher NQO1 protein. A poor correlation existed between NQO1*2 genotype status and clinical response to MMC. The results of this retrospective study suggest that tailoring MMC therapy to individual patients with superficial bladder cancer on the basis of NQO1 genotype status is unlikely to be of clinical benefit.

Le domaine de la bioénérgétique traîte de la circulation et de la transformation de l’énergie dans et entre des organismes et leur environnement. Dans ce manuscrit de thèse, la respiration cellulaire et plus particulièrement la première enzyme de la chaîne respiratoire, la NADH:ubiquinone oxidoreductase (Complexe I) ont été étudiées, dans l’objectif de clarifier sa fonction et son implication dans certaines maladies. Dans une première partie, la création d’une sonde impliquant l’enzyme immobilisée de façon biomimétique est décrite. La caractérisation de ce système est effectuée via spectroscopie infrarouge par exaltation de surface (SEIRAS) couplée à de l’électrochimie. Sa réponse à l’ajout de substrats et d’inhibiteurs est ensuite présentée. Dans une seconde partie, l’interaction du Complexe I avec des lipides et des inhibiteurs (Zn2+ et NADH-OH) ainsi que le rôle d’une Tyrosine située au site de fixation du NADH ont été étudiés par spectroscopies IR et UV-Vis différentielles induites par électrochimie. L’exploration des résultats obtenus sous un angle structural a finalement permis de proposer un modèle pour le mécanisme de couplage entre la réduction d’ubiquinone et le pompage de protons par le Complexe I. / The field of bioenergetics deals with the flow and transformation of energy within and between living organisms and their environment. The work presented in this thesis report focuses on cellular respiration and more specifically on the first enzyme of the respiratory chain, NADH:ubiquinone oxidoreductase (Complex I). This was done to clarify details about its function and its implication in disease. First, the creation of a sensor involving the biomimetically immobilized enzyme is presented and probed through a combination of surface enhanced infrared absorption spectroscopy (SEIRAS) and electrochemistry. This sensor is then tested against different substrates and inhibitors. In a second part, the interaction of Complex I with lipids, inhibitors (Zn2+ and NADH-OH) and the role of a Tyrosine residue situated in the NADH binding pocket are investigated through electrochemically induced UV-Vis and FTIR difference spectroscopies. The results gathered through these experiments are then explored under a structural perspective and a coupling mechanism between quinone reduction and proton translocation by Complex I is proposed.

Periodontal disease (PD) is a polymicrobial infection characterized by inflammation of the gingiva, alveolar bone resorption, and tooth loss (edentulism). Treponema denticola along with Porphyromonas gingivalis and Tannerella forsythia are among the “Red Complex” and are main etiological agents in PD. Treponemes are a member of the Spirochaeta phylum and are obligate anaerobes, that express pyruvate ferredoxin oxidoreductase (PFOR). The enzyme catalyzes the oxidation of pyruvate to acetyl-CoA and reduced ferredoxin. Amixicile is a novel bacteriostatic derivative of nitazoxanide and an inhibitor of PFOR. In light of the fact that Treponemes express PFOR, this study was conducted to investigate the susceptibility of oral Treponemes to AMX. All oral Treponemes tested were susceptible to AMX and the MIC values were determined ranging of 1.5-4.5 μg mL-1 for an initial starting cell concentration of 1.9x106 cells mL-1. Other potentially therapeutic effects for AMX for T. denticola were investigated: motility, hydrogen sulfide production, and serum sensitivity. AMX reduced overall spirochete motility by 50% at sub-MIC concentrations. There was a dose dependent decrease in H2S production in T. denticola at sub-MIC and MIC values. Furthermore, prior exposure of AMX led to increases in serum sensitivity. Taking into account the fact that other periodontal red complex bacteria express PFOR, AMX could serve as a new selective adjunctive treatment for periodontal disease.

CO2 fixation is a thermodynamically and kinetically challenging process, but nature has its own way of transforming CO2 into diverse organic molecules. Of our particular interest is 2-oxoacid:ferredoxin oxidoreductase (OFOR) that catalyzes the anaerobic, reversible inter-conversion of 2-oxoacids and CO2, making use of a small electron-transfer protein, ferredoxin (Fd), as the redox partner. This dissertation characterizes OFORs and Fds from organisms that exhibit different metabolic patterns and investigates how the interplay of OFOR and Fd could impact the fate of CO2 metabolism, asking the question What controls the catalytic bias of OFOR for CO2 evolution versus fixation? The study of OFORs and Fds from Desulfovibrio africanus and Hydrogenobacter thermophilus through an electrocatalytic assay reveals that the reduction potential of Fd is possibly associated with the biological function of OFOR and that CO2 fixation requires a low-potential electron donor. The Fd from H. thermophilus (HtFd1) is used as a model to probe the factors that govern iron-sulfur cluster potential. The dependence of OFOR activity on Fd potential is systematically studied with HtFd1 and its molecular variants through the electrocatalytic assay and a coupled enzyme assay. The results suggest there is a Fd “potential optimum” for OFOR-catalyzed CO2 fixation. The study of a 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) and three Fds from Magnetococcus marinus MC-1 further highlights other factors such as the intramolecular electron-transfer within Fd and the electrostatic and hydrophobic interactions at the protein-protein interface in determining OFOR-Fd interaction. The characterization of an OGOR from M. marinus MC-1 (MmOGOR) also provides kinetic, structural and spectroscopic details for a CO2-fixing OFOR that contains only one iron-sulfur cluster. Overall, this work furthers the scientific understanding of how nature achieves CO2 fixation through supplying reducing equivalents and with enzymes as efficient catalysts, and how intermolecular electron-transfer mediated by protein-protein interaction could regulate enzyme catalysis. / 2019-10-08T00:00:00Z

The thesis focuses on regulation of the chlorophyll biosynthetic pathway and its coordination with synthesis of chlorophyll-binding proteins in the cyanobacterium Synechocystis sp. PCC 6803. One of the aims was to analyze correlation between syntheses of photosystems and chlorophyll in Synechocystis cells using radioactive labeling of proteins and chlorophyll by 35S and 14C, respectively. I also investigated the role of enzymes catalyzing protochlorophyllide reduction step in the chlorophyll biosynthesis by analyzing the synthesis and accumulation of photosynthetic proteins in Synechocystis mutants lacking one of the enzymes. Further, roles of Ycf54 and Psb27 proteins in stability and assembly of oxidative cyclase and CP43, respectively, are also described.

The thesis summarizes data gathered on various components of respiratory chain of Trypanosoma brucei. Namely, NADH:ubiquinone oxidoreductase (complex I), alternative NADH:ubiquinone oxidoreductase (NDH2) and mitochondrial glycerol-3-phosphate dehydrogenase are discussed themselves and in broader context of energy metabolism. Also, a work done using RNA interference library is described.