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Biochemical Characterization of 2-Nitropropane Dioxygenase from Hansenula MRAKIIMijatovic, Slavica 22 April 2008 (has links)
2-Nitropropane dioxygenase from Hansenula mrakii is a flavin-dependent enzyme that catalyzes the oxidation of anionic nitroalkanes into the corresponding carbonyl compounds and nitrite, with oxygen as the electron acceptor. Although nitroalkanes are anticipated to be toxic and carcinogenic, they are used widely in chemical industry for a quick and effective way of synthesizing common reagents. Consequently, the biochemical and biophysical analysis of 2-nitropropane dioxyganase has a potential for bioremediation purposes. In this study, recombinant enzyme is purified to high levels, allowing for detailed characterization. The biochemical analysis of 2-nitropropane dioxygenase presented in this study has established that enzyme utilizes alkyl nitronates as substrates by forming an anionic flavosemiquinone in catalysis. The enzyme is inhibited by halide ions, does not contain iron and has a positive charge located close to the N(1)-C(2)=O locus of the isoalloxazine moiety of the FMN cofactor.
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On the Mechanistic Roles of the Protein Positive Charge Close to the N(1)Flavin Locus in Choline OxidaseGhanem, Mahmoud 12 June 2006 (has links)
Choline oxidase catalyzes the oxidation of choline to glycine betaine. This reaction is of considerable medical and biotechnological applications, because the accumulation of glycine betaine in the cytoplasm of many plants and human pathogens enables them to counteract hyperosmotic environments. In this respect, the study of choline oxidase has potential for the development of a therapeutic agent that can specifically inhibit the formation of glycine betaine, and therefore render pathogens more susceptible to conventional treatment. The study of choline oxidase has also potential for the improvement of the stress resistance of plant by introducing an efficient biosynthetic pathway for glycine betaine in genetically engineered economically relevant crop plant. In this study, codA gene encoding for choline oxidase was cloned. The cloned gene was then used to express and purify the wild-type enzyme as well as to prepare selected mutant forms of choline oxidase. In all cases, the resulting enzymes were purified to high levels, allowing for detailed characterizations. The biophysical and biochemical analyses of choline oxidase variants in which the positively charged residue close to the flavin N(1) locus (His466) was removed (H466A) or reversed (H466D) suggest that in choline oxidase, His466 modulates the electrophilicity of the bound flavin and the polarity of the active site, and contributes to the flavinylation process of the covalently bound FAD as well as to the stabilization of the negative charges in the active site. Biochemical, structural, and mechanistic relevant properties of selected flavoproteins with special attention to flavoprotein oxidases, as well as the biotechnological and medical relevance of choline oxidase, are presented in Chapter I. Chapter II summarizes all the experimental techniques used in this study. Chapter III-VII illustrate my studies on choline oxidase, including cloning, expression, purification and preliminary characterizations (Chapter III), spectroscopic and steady state kinetics (Chapter IV), the catalytic roles of His466 and the effects of reversing the protein positive charge close to the flavin N(1) locus (Chapter V and VI), and the roles of His310 with a special attention to its involvement in a proton-transfer network (Chapter VII). Chapter VIII presents a general discussion of the data presented.
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Roles of Serine 101, Histidine 310 and Valine 464 in the Reaction Catalyzed by Choline Oxidase from Arthrobacter GlobiformisFinnegan, Steffan 05 March 2010 (has links)
The enzymatic oxidation of choline to glycine betaine is of interest because organisms accumulate glycine betaine intracellularly in response to stress conditions, as such it is of potential interest for the genetic engineering of crops that do not naturally possess efficient pathways for the synthesis of glycine betaine, and for the potential development of drugs that target the glycine betaine biosynthetic pathway in human pathogens. To date, one of the best characterized enzymes belonging to this pathway is the flavin-dependent choline oxidase from Arthrobacter globiformis. In this enzyme, choline oxidation proceeds through two reductive half-reactions and two oxidative half-reactions. In each of the reductive half-reactions the FAD cofactor is reduced to the anionic hydroquinone form (2 e- reduced) which is followed by an oxidative half-reaction where the reduced FAD cofactor is reoxidized by molecular oxygen with formation and release of hydrogen peroxide. In this dissertation the roles of selected residues, namely histidine at position 310, valine at position 464 and serine at position 101, that do not directly participate in catalysis in the reaction catalyzed by choline oxidase have been elucidated. The effects on the overall reaction kinetics of these residues in the protein matrix were investigated by a combination of steady state kinetics, rapid kinetics, pH, mutagenesis, substrate deuterium and solvent isotope effects, viscosity effects as well as X-ray crystallography. A comparison of the kinetic data obtained for the variant enzymes to previous data obtained for wild-type choline oxidase are consistent with the valine residue at position 464 being important for the oxidative half-reaction as well as the positioning of the catalytic groups in the active site of the enzyme. The kinetic data obtained for the serine at position 101 shows that serine 101 is important for both the reductive and oxidative half-reactions. Finally, the kinetic data for histidine at position 310 suggest that this residue is essential for both the reductive and oxidative half-reactions.
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The crystal structures of xenobiotic reductase A and B from pseudomonas putida II-B and pseudomonas fluorescens I-C: structural insight into regiospecific reactions with nitrocompoundsManning, Linda 28 November 2005 (has links)
Nitrochemicals are currently widely used as solvents, drugs, biocides, fuels and explosives and are consequently widely distributed in the environment. The reductive nitrite elimination from explosive compounds is catalyzed by two FMN-dependent, xenobiotic reductases (XenA or XenB). These genes for these regiospecific enzymes were cloned from Pseudomonas putida and P. fluorescens I-C respectively and isolated from the soil of a contaminated World War II munitions manufacturing plant. These enzymes enable the microbes to fulfill their nitrogen requirements from nitroglycerin by catalyzing the regiospecific, NADPH dependent, reductive denitration of nitroglycerin with differing selectivities. The two enzymes also transform a number of additional nitrocompounds in vitro, e.g. TNT and metronidazole, a leading drug in the treatment of Helicobacter pylori, a causative agent of human ulcers. Single crystals were obtained for XenA and XenB and complete X-ray diffraction datasets have been collected and analyzed to better understand these characteristics. The 1.6 Å resolution structure of XenA reveals a dimer of β/α)₈-TIM barrels, but the 2.3 Å resolution structure for XenB is a monomer. The (β/α)₈-TIM barrel protein fold is the most common fold in the PDB. However, the XenA structure exhibits a unique, C-terminal domain-swapped topology. Thus a portion of each active site is comprised of residues from the neighboring monomer. To probe the reaction cycle, crystal structures of ligand complexes and the reduced enzyme have been refined. For example, our structure of the XenA-metronidazole complex shows that ligands bind parallel to the FMN si-face. Our 1.5 Å resolution structure for reduced XenA reveals an FMN isoalloxazine ring with an angle of ~165° along the N5-N10 axis. We have also generated models of the reduced enzyme-nitroglycerin complexes by molecular dynamics. The results with both XenA and XenB reveal differences in enzyme-ligand hydrogen bonding. These differences correlate remarkably well with the regiospecific differences observed for nitrite elimination from nitroglycerin and reduction of TNT by the two enzymes.
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Prokaryotické proteíny antioxidačnej obrany v hydrogenozómoch Trichomonas vaginalis / Prokaryotic proteins of antioxidant defense in Trichomonas vaginalis hydrogenosomesSmutná, Tamara January 2016 (has links)
Parasitic protists with modified mitochondria represent important and exciting group of organisms, not only from the view of eukaryotic cell evolution but also because these parasites are causative agents of serious and widespread diseases. The study and understanding of their biology is thus necessary for the development of new antiparasitic drugs. These organisms reside in host body cavities with low concentrations of oxygen and while they lack typical mitochondria, they possess mitochondrion-related organelles which still integrate many physiologically important processes. Trichomonas vaginalis is an anaerobic flagellate inhabiting mucosal surface of vagina. Instead of canonical mitochondria, T. vaginalis possesses organelles termed hydrogenosomes. These organelles harbor pathways of ATP-generating metabolism via substrate-level phosphorylation, dependent on enzymes prone to oxidative damage, such as pyruvate:ferredoxin oxidoreductase and Fe-Fe hydrogenase. Because the environment of trichomonads is not fully anaerobic, the parasite had to develop complex strategies to cope with both oxygen and reactive oxygen species (ROS) generated by host immune system cells. Recent data from T. vaginalis proteomic and genomic analyses revealed the presence of bacterial-type proteins potentially participating...
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Strategien zur Charakterisierung von ausgesuchten Streptomyces lividans Genen und deren FunktionenOverbeck, Jens 16 October 2007 (has links)
Das lineare Chromosom von S.lividans zeichnet sich durch eine hohe Variabilität insbesondere der chromosomalen Endbereiche aus. Hier finden sich unter anderen auch verschiedene Gene, die bisher einzigartig sind. Nach Klonierung der Gene in E.coli wurden die entsprechenden Genprodukte als His-Tag Fusionsproteine überproduziert, aufgereinigt und zur Herstellung von Antikörpern verwendet. Der untersuchte Abschnitt, als Ganzes und in Unterabschnitten, wurde auf einem Hoch Kopien Vektor in S.lividans transformiert. In extra hierfür konstruierten Vektorsystemen erfolgte die Produktion von His-Tag Proteinen in S.lividans. Nach Fusion von potentiellen Promoterbereichen mit dem promoterlosen EGFP-Gen, gelang deren Identifizierung in enhanced green fluorescent protein (EGFP) produzierenden S.lividans Transformanten. Mit Hilfe eines Vektors, der ein Temperatur sensitives Replikon besitzt, wurden Gene durch die Integration eines Hygromycin-Resistenzgenes ersetzt, bzw. als Fusionsgen mit dem EGFP-Gen erstellt. Ein Flavoprotein wurde zur Homogenität gereinigt. Es wurde nachgewiesen, dass in S.lividans pro Monomer ein FAD-Molekül interagiert. Physiologische Studien zeigen, dass die Synthese des chromosomal determinierten Proteins in S.lividans nur erfolgt, wenn dieser Stamm ein Plasmid- oder chromosomal- kodiertes Thiostrepton Resistenzprotein (23S rRNA Methylase) enthält. Es muss geschlussfolgert werden, dass die Methylierung der 23S rRNA die Translation verschiedener mRNAs beeinflusst. Die Synthese dieses Proteins ist des Weiteren abhängig von hohen Konzentrationen an NaCl und KCl im Medium, wie auch die zweier Aldo-Keto Reduktasen. Disruptionsmutanten eines dieser zwei Aldo-Keto Reduktase-Gene zeigen jeweils eine erhöhte und verfrühte Produktion eines rot gefärbten Mycel-assoziierten Antibiotikums (Undecylprodigiosin), während die eines weiteren (Actinorhodin) unbeeinflusst blieb.
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Charge-density Features of Protein Molecules Revealed with Ultra-high Resolution X-ray Crystallography / 超高分解能X線解析法によるタンパク質電荷分布の解明Takaba, Kiyofumi 26 March 2018 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20938号 / 理博第4390号 / 新制||理||1631(附属図書館) / 京都大学大学院理学研究科化学専攻 / (主査)教授 三木 邦夫, 教授 杉山 弘, 教授 秋山 芳展 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM
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Biochemical and Crystallographic Investigations of Flavin Dependent Tryptophan-6 Halogenase BorHLingkon, Kazi January 2020 (has links)
No description available.
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Insight into the structure of tetrameric flavoprotein WrbA involved in oxidative-stress response / Insight into the structure of tetrameric flavoprotein WrbA involved in oxidative-stress responseWOLFOVÁ, Julie January 2012 (has links)
This Ph.D. thesis addresses the structural characterization of the unique family of tetrameric flavoproteins WrbA, the role of which in the life of cells is still largely unknown but its enzymatic activity and expression properties implicate it in the cell protection against oxidative stress. Proteins of the WrbA family were proved to carry out two-electron reductions of quinones and in this way to prevent generation of the free radicals, similarly to other flavoproteins known as quinone oxidoreductases. Crystal structures of the liganded and unliganded forms of the prototypical WrbA from Escherichia coli were determined. Comparative analyses of these structures with the related flavoproteins were intended to identify and explain the defining structural features of the WrbA family and to clarify its structural and functional relationships to the other flavoproteins.
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Development and Commercialization of Functional, Non-Invasive Retinal Imaging Device Utilizing Quantification of Flavoprotein Fluorescence for the Diagnosis and Monitoring of Retinal DiseaseHeise, Erich A. 27 May 2016 (has links)
No description available.
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