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Obrecht, Lorenz. "Artificial metalloenzymes in catalysis." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/7248.
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This thesis describes the synthesis, characterisation and application of artificial metalloenzymes as catalysts. The focus was on two mutants of SCP-2L (SCP-2L A100C and SCP-2L V83C) both of which possess a hydrophobic tunnel in which apolar substrates can accumulate. The crystal structure of SCP-2L A100C was determined and discussed with a special emphasis on its hydrophobic tunnel. The SCP-2L mutants were covalently modified at their unique cysteine with two different N-ligands (phenanthroline or dipicolylamine based) or three different phosphine ligands (all based on triphenylphosphine) in order to increase their binding capabilities towards metals. The metal binding capabilities of these artificial proteins towards different transition metals was determined. Phenanthroline modified SCP-2L was found to be a promising scaffold for Pd(II)-, Cu(II)-, Ni(II)- and Co(II)-enzymes while dipicolylamine-modified SCP-2L was found to be a promising scaffold for Pd(II)-enzymes. The rhodium binding capacity of two additional phosphine modified protein scaffolds was also investigated. Promising scaffolds for Rh(I)- and Ir(I)-enzymes were identified. Rh-enzymes of the phosphine modified proteins were tested in the aqueous-organic biphasic hydroformylation of linear long chain 1-alkenes and compared to the Rh/TPPTS reference system. Some Rh-enzymes were found to be several orders of magnitude more active than the model system while yielding comparable selectivities. The reason for this remarkable reactivity increase could not be fully elucidated but several potential modes of action could be excluded. Cu-, Co-, and Ni-enzymes of N-ligand modified SCP-2L A100C were tested in the asymmetric Diels-Alder reaction between cyclopentadiene and trans-azachalcone. A promising 29% ee for the exo-product was found for the phenanthroline modified protein in the presence of nickel. Further improvement of these catalyst systems by chemical means (e.g. optimisation of ligand structure) and bio-molecular tools (e.g. optimisation of protein environment) can lead to even more active and (enantio)selective catalysts in the future.
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Anderson, Harry Laurence. "Model enzymes based on porphyrins." Thesis, University of Cambridge, 1990. https://www.repository.cam.ac.uk/handle/1810/272953.
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Casey, John P. Jr. "Capsid catalysis : de novo enzymes on viral proteins." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/99052.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references (pages 107-119).
Biocatalysis has grown rapidly in recent decades as a solution to the evolving demands of industrial chemical processes. Mounting environmental pressures and shifting supply chains underscore the need for novel chemical activities, while rapid biotechnological progress has greatly increased the utility of enzymatic methods. Enzymes, though capable of high catalytic efficiency and remarkable reaction selectivity, still suffer from relative instability, high costs of scaling, and functional inflexibility. Herein, M13 bacteriophage libraries are engineered as a biochemical platform for de novo semisynthetic enzymes, functionally modular and widely stable. Carbonic anhydrase-inspired hydrolytic activity via Zn²+ coördination is first demonstrated. The phage clone identified hydrolyzes a range of carboxylic esters, is active from 25°C to 80°C, and displays greater catalytic efficacy in DMSO than in water. Reduction-oxidation activity is subsequently developed via heme and copper cofactors. Heme-phage complexes oxidize multiple peroxidase substrates in a pH-dependent manner. The same phage clone also binds copper(II) and oxidizes a catechol derivative, di-tert-butylcatechol, using atmospheric oxygen as a terminal oxidant. This clone could be purified from control phage via Cu-NTA columns, enabling future library selections for phage that coördinate Cu²+ ions. The M13 semisynthetic enzyme platform complements biocatalysts with characteristics of heterogeneous catalysis, yielding high-surface area, thermostable biochemical structures readily adaptable to reactions in myriad solvents. As the viral structure ensures semisynthetic enzymes remain linked to the genetic sequences responsible for catalysis, future work could tailor the biocatalysts to high-demand synthetic processes by evolving new activities, utilizing high-throughput screening technology and harnessing M13's multifunctionality.
by John P. Casey, Jr.
Ph. D.
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Allen, Joanne Victoria. "Recent advances in asymmetric catalysis." Thesis, Loughborough University, 1995. https://dspace.lboro.ac.uk/2134/27574.
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CHAPTER ONE reviews the literature, discussing aspects of transition metal mediated asymmetric catalysis in the presence of enantiomerically pure ligands. CHAPTER TWO discusses the asymmetric addition of dialkyl-zinc reagents to aromatic aldehydes. The work presented is particularly concerned with the design and construction of enantiomerically pure oxazoline ligands tethered to alcohols These ligands have proved effective in the acceleration of the alkylation reaction and are able to influence good levels of asymmetric induction in the resultant secondary alcohol products CHAPTER THREE examines the electronic (and steric) effects of enantiomerically pure oxazoline ligands for the palladium catalysed allylic substitution reaction. Using ligands possessing two electronically different donor atoms, it is possible to create electronic distortion upon the intermediate allyl complex. In doing so it is possible to direct nucleophilic addition to one carbon centre preferentially to the other, resulting in asymmetric induction. Manipulation of these ligands enables control in the extent of electron distortion inflicted upon the allyl complex and consequently influences the levels of enantioselectivity observed. CHAPTER FOUR investigates the ability of hydrolytic enzymes to kinetically resolve a series of allylic acetates, under varying conditions. Lipases appeared superior to esterases for the substrates employed. In particular cis-3-acetoxy-5-carbomethoxycyclohexene was smoothly resolved m high yield and enantioselectivity. CHAPTER FIVE reports on the potentiality of a dynamic resolution of allylic acetates, using hydrolytic enzymes in the presence of a palladium catalyst. A proposed mechanism is discussed. Initial results are promising, however, the sensitivity of the reaction is realised and optimisation of conditions still needs to be addressed.
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Brown, Christopher John. "Efficient intramolecular general acid catalysis." Thesis, University of Cambridge, 1995. https://www.repository.cam.ac.uk/handle/1810/272266.
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Qi, Xiaolin. "Enzyme-substrate interactions in PC1 #beta#-lactamase catalysis." Thesis, University of Newcastle Upon Tyne, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.315617.
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Ndi, Cornelius Ndi. "Synthesis of Chemical Models of Hydrolase Enzymes for Intramolecular Catalysis." Digital Commons @ East Tennessee State University, 2011. https://dc.etsu.edu/etd/1356.
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Most nuclease enzymes can hydrolyze phosphoester bonds (in DNA and RNA) by using metal ions cofactors that coordinate and activate water molecules in the enzymes' active sites. However, there are some hydrolase enzymes (including nucleases) that can function without the aid of metal ions. 2,6-Di(1H-imidazol-2-yl)phenol, a model compound for hydrolase enzyme, was synthesized by the reaction between ethylenediamine and dimethyl-3-carboxysalicylate, initially resulting in the formation of diimidazoline. The diimidazoline was subsequently aromatized to the diimidazole by dehydrogenation over palladium. The overall reaction yield was low; therefore, other dehydrogenation transformation reactions were tried but all failed to improve the yield. Converting this diimidazolphenol into diimidazolphenyl monophoshpate derivative was attempted but failed to give desired products.
Synthesis of 2,2'-anthracene-1,8-diylbis-1H-imidazole, another model compound for hydrolase enzymes, was attempted using dimethyl-1,8-anthracenedicarboxylate, but synthesis was unsuccessful due to solubility problem.
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Smith, Graham Michael. "Enzyme immobilisation and catalysis in ordered mesoporous silica /." St Andrews, 2008. http://hdl.handle.net/10023/573.
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Wright, Penelope A. "Mechanistic studies on the catalysis and inhibition of serine proteases." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302492.
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Lawrence, Christopher Ralph. "Studies towards the catalysis of cationic cyclisations using monoclonal antibodies." Thesis, University of Cambridge, 1994. https://www.repository.cam.ac.uk/handle/1810/272265.
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Walter, Christopher John. "Stereoselective acceleration of Diels-Alder reactions by synthetic enzymes." Thesis, University of Cambridge, 1994. https://www.repository.cam.ac.uk/handle/1810/272679.
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Smith, Graham Murray. "Enzyme immobilisation and catalysis in ordered mesoporous silica." Thesis, University of St Andrews, 2008. http://hdl.handle.net/10023/573.
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A range of mesoporous materials based on SBA-15 have been prepared and characterised. The materials were templated by neutral block copolymer P123, and typically have a hexagonal (p6mm) pore structure, with high surface areas and narrow pore size distributions. The removal of the surfactant template by calcination and solvent extraction has been investigated. The aqueous stability of this material, and the hydrolysis of the surface was studied. Organic functional groups were incorporated into the silica surface by co-condensation, or by post synthesis grafting. A range of functional groups were incorporated, including amine, carboxy, allyl and thiol groups. The pore size of the materials was controlled by the addition of trimethoxybenzene during synthesis, which significantly increased the pore size and uptake capacity of the materials. The adsorption of CALB by SBA-15 was investigated, with support materials extracted by calcination or solvent extraction. Rapid uptake at high loading was observed, with a maximum loading of 450 mg g-1 measured. The leaching of the enzyme from the support was investigated, and found to be high with unfunctionalised supports. The leaching from functionalised supports incorporating sulfur groups was significantly reduced. The activity of the immobilised CALB was measured by tributyrin hydrolysis in aqueous media, and by enantioselective transesterification of (R)-1-phenylethanol in organic media. The effect of surface functionalisation for reusability and thermal stability in aqueous systems was investigated. Preliminary studies of supported CALB for dynamic kinetic resolution were carried out, with an investigation of acidic zeolites and a mesoporous supported catalyst for 1-phenylethanol racemisation. The encapsulation of immobilised CALB was investigated, and the activity and reusability of these systems studied.
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Lindzen, Eric C. "Sequencing and characterization of a carrot cDNA clone encoding a protein kinase fragment." Thesis, Georgia Institute of Technology, 1991. http://hdl.handle.net/1853/25375.
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Cater, Philip A. "Chemo-enzymatic studies using hydrolases and dehydrogenases." Thesis, University of Warwick, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340552.
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Bassan, Arianna. "Theoretical studies of mononuclear non-heme iron active sites." Doctoral thesis, Stockholm : Fysikum, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-103.
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BREGLIA, RAFFAELLA. "Quantum-mechanical study of the stereoelectronic and catalytic properties of metallo-enzymes involved in reactions of environmental and technological relevance." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/153275.
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L’ oggetto della mia tesi dottorato è stato lo studio teorico-computazionale delle proprietà stereo elettroniche e catalitiche di metallo-enzimi coinvolti in reazioni con potenziale rilevanza ambientale e tecnologica. In particolare, la ricerca si è focalizzata sulle monossido di carbonio deidrogenasi (CODHs) e idrogenasi che rispettivamente catalizzano la reazione di riduzione di CO2 a CO e l’interconversione reversibile di protoni ed elettroni in idrogeno molecolare.
Calcoli quanto-meccanici, basati sulla teoria del funzionale della densità elettronica (DFT), sono stati condotti su modelli del sito attivo di questi enzimi. Modelli di diverse dimensioni, dal sito minimale a sistemi molto estesi contenti la prima e la seconda sfera di coordinazione, sono stati sviluppati al fine di delucidare il ruolo fondamentale dell’intorno proteico nel ciclo catalitico. Oltre allo studio dei possibili percorsi di reazione eseguiti attraverso l'esplorazione della superficie di energia potenziale, alcuni potenziali intermedi sono stati ulteriormente investigati modellandone alcune proprietà spettroscopiche, quali spettri IR, EPR e Mossbauer.
In particolare, per comprendere maggiormente le proprietà stereoelettroniche e catalitiche del sito attivo delle Ni-CODH, è stato investigato il legame dei substrati CO e CO2 al C-cluster in diversi stati redox, in presenza ed in assenza di altri leganti. I risultati ottenuti hanno permesso di identificare e caratterizzare alcuni intermedi del ciclo catalitico, facendo maggiore chiarezza su di esso. Lo studio sulle Mo-CODH ha invece riguardato la reattività del sito attivo verso la molecola di H2.
Al fine di fornire informazioni utili per il diretto utilizzo delle idrogenasi come catalizzatori industriali per la produzione di idrogeno, l’ossidazione del sito attivo delle [NiFe]-idrogenasi è stato investigato in presenza ed assenza di O2. Infine, la riattivazione di due diverse forme inattive dell’enzima è stata studiata per razionalizzare la loro diversa cinetica di riattivazione.The topic of my PhD project was the theoretical investigation of the stereoelectronic and catalytic properties of metallo-enzymes involved in reactions of technological and environmental relevance. In particular, the research focused on carbon monoxide dehydrogenases (CODHs) and hydrogenases enzymes, that catalyse the interconversion of CO and CO2, and the reversible interconversion of protons and reducing equivalents into molecular hydrogen, respectively. Quantum mechanics calculations were carried out in the framework of the Density Functional Theory (DFT) on models of the enzyme active sites. Models of different sizes, ranging from the minimal metal clusters to very large systems, including the second coordination sphere, were developed to elucidate the role of the protein environment during the catalysis. Several potential intermediate species along reaction pathways were further investigated by calculating spectroscopic properties. Different issues for CODHs and hydrogenases were addressed, depending on the current state of knowledge and the still open questions concerning these enzymes. In particular, the theoretical study of Ni-CODHs was aimed at elucidating the catalytic and stereoelectronic properties of the active site, known as C-cluster. Binding of the substrates CO2 and CO to different forms of the C-cluster was investigated to explore the enzymatic reactivity, whereas analysis of charges and spin densities on metallic atoms composing the active site was carried out to explore its electronic structure. The obtained results yielded a more detailed version of the Ni-CODH catalytic mechanism. Concerning Mo-CODHs, the reactivity of the active site towards H2 was instead investigated. With the aim of deepening insights into the nature of a H2-bound paramagnetic form of the enzyme experimentally observed during the reaction of Mo-CODHs with H2, EPR parameters have been predicted for this species and compared with the experimental values. Conversely, DFT calculations on hydrogenases were aimed at providing significant insights for their direct utilization in biotechnological hydrogen production processes and the development of O2-tolerant biomimetic catalysts. Oxidation and consequent inactivation of the active site of [NiFe]-hydrogenases were investigated using a very large-size DFT model. Since it was demonstrated that the oxidation occurs even in the absence of O2, the interconversion mechanisms between active and inactive forms of the enzyme were investigated by simulating both aerobic and anaerobic conditions. Finally, a DFT investigation of the reactivation mechanism of the oxidized inactive forms of [NiFe]-hydrogenases was carried out to rationalize their different reactivation kinetics.
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Zeamari, Kamal. "Investigation par spectroscopie RPE des bases moléculaires de la réactivité d'une enzyme à molybdène : la nitrate réductase périplasmique de Rhodobacter sphaeroides." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0546/document.
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La nitrate réductase périplasmique de Rhodobacter sphaeroides possède, un cofacteur à Mo (site actif), un centre [4Fe-4S] et deux hèmes de type c formant une chaîne de transfert électronique intramoléculaire. Ce travail est centré sur deux aspects moléculaires de la catalyse de cette enzyme : la réactivité au niveau du site actif de l’enzyme et les processus de transfert d’électrons intramoléculaires. Ces questions sont abordées en s’appuyant parallèlement sur des approches de mutagénèse dirigée, d’activités enzymatiques, de spectroscopie de résonance paramagnétique électronique (RPE) en onde continue et impulsionnelle et sur des mesures de potentiels redox associés aux cofacteurs de l’enzyme. La première partie de ce travail est consacrée à la caractérisation spectroscopique et physico-chimique d’intermédiaires Mo(V) du site actif afin de déterminer leur structure et leur positionnement possible dans le cycle catalytique. Nous avons ainsi étudiée de manière détaillée deux intermédiaires Mo(V) en présence de nitrate dont nous montrons qu’ils présentent des différences structurales au-delà de la première sphère de coordination du Mo. Dans la seconde partie, nous mettons en évidence le rôle d'un acide aminé très conservé (Lys) dans le transfert d'électrons intramoléculaire. Cet acide aminé chargé positivement est situé dans la seconde sphère de coordination du centre [4Fe-4S] et joue un rôle majeur dans la modulation des propriétés rédox du centre [4Fe-4S], ce qui affecte fortement les propriétés catalytiques de l'enzyme. L’ensemble de nos résultats permettent ainsi d’identifier dans l’environnement du Mo des éléments déterminants dans la réactivité de l’enzymeThe periplasmic nitrate reductase from Rhodobacter sphaeroides contains, in addition to the Mo-cofactor, a [4Fe-4S] center and two c-type hemes defining an intramolecular electron transfer chain. This work focuses on two molecular aspects of the catalysis: the reactivity of the Mo-cofactor, and the intramolecular electron transfer step. These issues are dealt by combining approaches as site-directed mutagenesis, enzymatic activities, continuous-waves (CW) and pulse electron paramagnetic resonance spectroscopy (EPR), UV-Vis spectroscopy and redox titration of metal cofactors of the enzyme. A first part of this work is devoted to the spectroscopic and physicochemical characterization (thermodynamic and kinetic properties) of Mo (V) intermediates of the active site in order to determine their structure and their catalytic relevance. We have undertaken a detailed characterization of two Mo(V) intermediates generated in presence of nitrate, which display some structural differences beyond the first coordination sphere of the Mo(V) ion. In a second part, we highlight the role of a highly conserved amino acid (Lys) in intramolecular electron transfer. This positively-charged amino acid is located in the second coordination sphere of the [4Fe-4S] center and plays a major role in the redox properties tuning of the [4Fe-4S] center thus strongly affecting the catalytic properties of the enzyme. All together, these data provide some structural insights on the enzyme reactivity beyond the first coordination sphere of the Mo-cofactor
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Tang, Xiao-Jing. "Bioelectrochemical applications of reactions catalyzed by immobilized enzymes." Lund : Department of Analytical Chemistry, Lund University, Swedend, 1997. http://books.google.com/books?id=Yv5qAAAAMAAJ.
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Anstrom, David Michael. "Structural, mutagenic, and kinetic studies on the reaction mechanism of malate synthase /." view abstract or download file of text, 2005. http://wwwlib.umi.com/cr/uoregon/fullcit?p3181081.
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Thesis (Ph. D.)--University of Oregon, 2005.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 90-101). Also available for download via the World Wide Web; free to University of Oregon users.
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Hearne, Jennifer L. "Glutathione transferase M1-1 delineation of xenobiotic substrate sites and the relationship between enzyme structure and catalytic function /." Access to citation, abstract and download form provided by ProQuest Information and Learning Company; downloadable PDF file 4.57 Mb., p, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3220799.
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Aitken, D. J. "Approaches to selective synthesis using modified enzyme systems." Thesis, University of Strathclyde, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381516.
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Yorke, Jake. "Engineering cytochrome P450BM3 into a drug metabolising enzyme." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:92dcddfe-b3fc-46e8-9e5e-77910fb03783.
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Directed evolution studies by Whitehouse et al. identified several variants of P450BM3 (CYP102A1) with enhanced substrate oxidation rates across a range of substrates. This thesis describes the use of these ‘generic accelerator’ variants, in combination with selectivity altering mutations to engineer P450BM3¬ for the oxidation of pharmaceuticals. Using engineered variants the non-steroidal anti-inflammatory drug diclofenac was metabolised to the primary human metabolites 4′- and 5-hydroxydiclofenac, with total conversion of 2 mM substrate by 5 μM enzyme. The local-anaesthetic lidocaine and the steroid testosterone were similarly metabolised to human metabolites. This is the first report of a drug compound being totally converted to the human metabolites by a P450BM3 variant, and is also the first report of lidocaine metabolism by a P450¬BM3 variant. The engineered variants are akin to CYP3A4, the primary human drug metabolising enzyme, as they show activity towards a range of compounds including anionic, cationic and neutral drugs. This range of activity is at the expense of NADPH coupling, which remains low with these substrates. In order to more fully understand the origin of the rate enhancing properties of the generic accelerator variants, spectroelectrochemical, stopped-flow and kinetic studies were performed. A custom optically transparent thin layer electrode system was designed and fabricated for use in spectroelectrochemical titrations. A spectroelectrochemical cell and gold mesh electrode were designed and used in spectroelectrochemical investigations of P450BM3 variants, as well as other P450s and their redox partners. These spectroelectrochemical, stopped-flow and kinetic studies, in combination with X-ray crystal structures provided insight into the origin of the rate enhancing properties of these enzymes and supplied the first example of the complete characterization of the thermodynamic and kinetic properties of WT and mutant P450BM3 for the oxidation of a non-natural substrate. The generic accelerator variants are, in the resting state, in a more catalytically ready conformation than the WT enzyme, and reorganization energy barriers appear to be lowered, so that fewer substrate-induced structural changes are required to promote electron transfer and initiate the catalytic cycle.
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Vance, Nicholas Robert. "Targeting dynamic enzymes for drug discovery efforts." Diss., University of Iowa, 2018. https://ir.uiowa.edu/etd/6517.
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Proteins are dynamic molecules capable of performing complex biological functions necessary for life. The impact of protein dynamics in the development of medicines is often understated. Science is only now beginning to unravel the numerous consequences of protein flexibility on structure and function. This thesis will encompass two case studies in developing small molecule inhibitors targeting flexible enzymes, and provide a thorough evaluation of their inhibitory mechanisms of action.
The first case study focuses on caspases, a family of cysteine proteases responsible for executing the final steps of apoptosis. Consequently, they have been the subject of intense research due to the critical role they play in the pathogenesis of various cardiovascular and neurodegenerative diseases. A fragment-based screening campaign against human caspase-7 resulted in the identification of a novel series of allosteric inhibitors, which were characterized by numerous biophysical methods, including an X-ray co-crystal structure of an inhibitory fragment with caspase-7. The fragments described herein appear to have a significant impact on the substrate binding loop dynamics and the orientation of the catalytic Cys-His dyad, which appears to be the origin of their inhibition. This screening effort serves the dual purpose of laying the foundation for future medicinal chemistry efforts targeting caspase proteins, and for probing the allosteric regulation of this interesting class of hydrolases.
The second case study focuses on glutamate racemase, another dynamic enzyme responsible for the stereoinversion of glutamate, providing the essential function of D-glutamate production for the crosslinking of peptidoglycan in all bacteria. Herein, I present a series of covalent inhibitors of an antimicrobial drug target, glutamate racemase. The application of covalent inhibitors has experienced a renaissance within drug discovery programs in the last decade. To leverage the superior potency and drug target residence time of covalent inhibitors, there have been extensive efforts to develop highly specific covalent modifications to reduce off-target liabilities. A combination of enzyme kinetics, mass spectrometry, and surface-plasmon resonance experiments details a highly specific 1,4-conjugate addition of a small molecule inhibitor with the catalytic Cys74 of glutamate racemase. Molecular dynamics simulations and quantum mechanics-molecular mechanics geometry optimizations reveal, with unprecedented detail, the chemistry of the conjugate addition. Two compounds from this series of inhibitors display antimicrobial potency comparable to β-lactam antibiotics, with significant activity against methicillin-resistant S. aureus strains. This study elucidates a detailed chemical rationale for covalent inhibition and provides a platform for the development of antimicrobials with a novel mechanism of action.
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Raich, Armendáriz Lluís Adrià. "Unveiling Protein-Substrate Interactions and Conformations that Influence Catalysis in Carbohydrate-Active Enzymes." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/586173.
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Enzymes have attracted the attention of chemists and biologists since long ago due to their molecular complexity and tremendous efficiency. They are highly specific catalysts that make chemical reactions possible at mild conditions with an astonishing rate enhancement. For this reason there have been many efforts to understand how these macromolecules work, trying to find out crucial factors that influence their activity. Whereas it is difficult to settle on how enzymes work in general, with hot debates over many years, significant progress has been made in elucidating specific catalytic mechanisms by means of experimental and computational approaches.
In this thesis we have focused on the study of glycoside hydrolases and transferases, enzymes that are known as “carbohydrate-active enzymes” (CAZymes) and that are essential for the processing and remodeling of carbohydrates in living organisms. In spite of the enormous advances in the understanding of how these enzymes work, with several factors that are known or presumed to enhance their reaction rates (such as certain sugar conformations, enzyme-substrate interactions or the flexibility of the enzyme fold), there are still many aspects that remain poorly understood due to the lack of atomistic insights. Given this, in the present thesis we have used cutting-edge computational methods, including all-atom molecular dynamics simulations, hybrid quantum mechanics/molecular mechanics approaches and enhanced sampling techniques, to unveil some of the essential enzymatic interactions and conformations that influence catalysis in CAZymes. With these techniques we have provided proofs for general concepts that are usually assumed, as well as insights for the specific enzymes that have been studied. In particular, we have evaluated the reaction free energy contribution of sugar conformations to catalysis in β-xylanases, the influence of crucial hydrogen bond interactions in β-glucosidases, the importance of enzymatic residues that bind water in the active site of inverting β-mannanases, and the structural flexibility of a human enzyme called glycogenin.Desde los primeros descubrimientos en el campo de la enzimología, las enzimas han atraído la atención de numerosos químicos y biólogos debido a su gran complejidad molecular y su elevada eficiencia. Estas macromoléculas son catalizadores altamente específicos que hacen posible reacciones químicas en condiciones suaves y a velocidades asombrosas. Por esta razón se han destinado muchos esfuerzos en tratar de comprender su funcionamiento, intentando descubrir los factores fundamentales que influyen en su actividad. En la presente tesis doctoral hemos ahondado en la comprensión de una clase de enzimas llamadas “glicosil hidrolasas” y “glicosil transferasas”, englobadas bajo la denominación de “enzimas activas en carbohidratos”. Estas enzimas están encargadas de la degradación y de la síntesis de carbohidratos, moléculas que debido a su diversidad y flexibilidad añaden un grado de complejidad adicional en su estudio. Aún a pesar de los grandes avances en la comprensión general de estas enzimas, habiéndose destacado distintos factores involucrados en su rendimiento catalítico (e.g. ciertas conformaciones del sustrato, interacciones enzima-sustrato o la flexibilidad de la estructura enzimática), la falta de información a nivel molecular dificulta la racionalización de muchas de estas evidencias. Debido a ello, en esta tesis hemos hecho uso de técnicas computacionales de vanguardia, incluyendo simulaciones atomísticas de dinámica molecular, enfoques híbridos de mecánica cuántica/mecánica molecular y técnicas de exploración avanzada del espacio de fases, para revelar el origen molecular de ciertas interacciones y conformaciones esenciales para la catálisis de enzimas activas en carbohidratos. Con estas técnicas hemos proporcionando pruebas que refuerzan concepciones generalmente asumidas, así como detalles específicos para cada una de las enzimas que hemos estudiado. En particular, hemos analizado la contribución de las conformaciones del sustrato en la catálisis de β-xilanasas, la contribución de puentes de hidrógeno en la catálisis de β-glucosidasas, la importancia de residuos que enlazan agua en β-mananasas y la flexibilidad estructural de una enzima humana llamada glicogenina.
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Kim, ChulHwan. "Azotobacter vinelandii nitrogenase : role of the MoFe protein [alpha]-subunit histidine-195 residue in catalysis /." This resource online, 1994. http://scholar.lib.vt.edu/theses/available/etd-06062008-164937/.
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Lonsdale, Thomas. "Dihydrogen driven cofactor recycling for use in bio-catalysed asymmetric organic synthesis." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:a0407748-e34f-410a-9c78-a8316b7a3d4d.
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Asymmetric reductions are used to produce chiral molecules, which are important precursors for the pharmaceutical industry. Bio catalytic reductions often display high enantioselectivity without the cost and toxicity associated with metal catalysis. However, unlike metal catalysts which use H2 directly, many useful redox-enzymes require the hydride donor NADH. NADH is expensive; therefore for a bio-catalytic process to be viable it must be recycled, usually by using a sacrificial carbon based substrate, generating super-stoichiometric amounts of waste. Two different methods for H2-driven NADH recycling are explored in this project: using soluble hydrogenases (SH) and, carbon particles modified with a hydrogenase and an NAD+-reductase moiety. The conductive carbon particles allow electrons from H2-oxidation to be channelled from the hydrogenase to the NAD+ reductase for reduction of NAD+. This project focuses on four main areas. The first looks at using the enzyme-modified particles for the production of high value chiral amines. A yield of >98% was achieved using the enzyme-modified particles with an L alanine dehydrogenase for H2 driven conversion of pyruvate to L-alanine. Moreover, a faster rate of reaction was demonstrated with the L-alanine dehydrogenase immobilised on particles versus with the L-alanine dehydrogenase in solution. The second section focuses on elevated temperature NADH recycling: an SH and an NAD+-reductase from a thermophilic organism were characterised. The NAD+-reductase was subsequently used as part of a system for recycling NADH at >35 °C. When demonstrated in combination with an enoate-reductase a 62 % yield was obtained for the reduction of 2 methyl 2 cyclopentenone. In the third strand SHs and enzyme-modified particles were investigated as recycling systems for NADH analogues. In summary, this thesis expands the scope for application of H2-driven biocatalytic reduction reactions.
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Chevalier, Yoan. "Développement de flavo-enzymes artificielles pour la chimie radicalaire et l’activation du dioxygène dans l’eau." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASS061.
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Le sujet vise à développer des systèmes artificiels bio-inspirés capables de catalyser d'importantes réactions organiques dans l'eau, dans des conditions douces et en utilisant des réactifs inoffensifs tels que O2. Pour cela, nous envisageons de mimer les deux activités principales des flavoenzymes, qui sont capables de catalyser soit des réductions, en délivrant un flux monoélectronique à un partenaire biologique, soit des réactions d'oxydations, par activation réductrice du dioxygène, et ceci avec le même cofacteur flavinique, mais localisé dans différents échafaudages protéiques. Ce projet est basé sur des résultats obtenus récemment, démontrant que l'incorporation de cofacteurs flaviniques (FMN) dans un environnement localement hydrophobe (polyéthylèneimine modifié) peut générer une réductase artificielle capable de collecter des paires d'électrons de NADH et de délivrer des électrons célibataires à un partenaire redox tel que qu’une porphyrine de manganèse (III). Dans ce contexte, selon les conditions aérobies ou anaérobies, la flavine réduite de ces systèmes pourrait soit délivrer en solution des électrons célibataires pour initier des réactions radicalaires, soit activer le dioxygène pour effectuer des réactions oxydations (Baeyer-Villiger, sulfoxydation, époxydation …). Nous présentons ici les résultats obtenus pour les réactions de Baeyer-Villiger et de sulfoxydations réalisées dans l'eau en utilisant le premier système catalytique composé d’un cofacteur flavinique naturel (FMN) et d’un polymère fonctionnalisé pour activer directement le dioxygène de l'air dans des conditions douces. Nous démontrons également que le NADH peut soit être remplacé par un réducteur moins coûteux tel que l'ascorbate de sodium soit être recyclé en cours de catalyse grâce à la combinaison de notre système catalytique avec un formiate déshydrogénase naturel (FDH). Finalement, le système a également été testé pour initier des réactions radicalaires en conditions anaérobiesThe present project aims at developing bioinspired artificial systems capable of catalysing important organic reactions in water, under mild conditions and using harmless reactants such as O2. For this purpose, we are mimicking both activities of flavoenzymes, which are capable of catalysing either reduction reactions, by delivering single electrons to a biologic partner, or oxidation reactions, by the reductive activation of O2, This project is based on recent results, demonstrating that the incorporation of flavin cofactors (FMN) into the local microenvironment of a water-soluble polymer (modified polyethyleneimine), can generate an artificial reductase capable of collecting electron pairs from NADH and then delivering single electrons to redox partners such as a manganese (III) porphyrin. In this context, depending on the aerobic or anaerobic conditions, the reduced flavin of such systems could either deliver single electrons in solution to initiate radical chemistry reactions or activate dioxygen to perform catalytic oxidations (Baeyer-Villiger, sulfoxidation, epoxidation…). We Here, we demonstrate present the results obtained for the Baeyer-Villiger and sulfoxidation reactions performed in water using the first catalytic system utilizing based on a natural flavin cofactor to directly activate dioxygen from the air as the unique source of oxidant under mild conditions. In parallel, we present how NADH could can be replaced by a cheaper reductant such as the sodium ascorbate and how we managed to recycle NADH along the reaction thank to the combination of our catalytic system with a natural formate dehydrogenase (FDH). To conclude, the system has also been tested to initiate radical chemistry reactions under anaerobic conditions
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Sen, Arundhuti. "Explorations in enzymology: investigating dynamics in dihydrofolate reductase." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/2768.
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The relationship between enzyme dynamics and enzymatic catalysis has become a central topic in modern enzymology, and studies in this area promise to enrich our current understanding of catalysis in biological systems. Escherichia coli dihydrofolate reductase (EcDHFR) has been a frequent subject of study in the context of protein dynamics, due to its small size, biological ubiquity, and the fact that its structural, kinetic and mechanistic characteristics are well established. Intrinsic kinetic isotope effects (KIEs) have proven to be highly sensitive probes of the role of dynamics in EcDHFR catalyzed reaction, as they circumvent the kinetic complexity of the enzyme-catalyzed reactions, and extract information directly pertaining to the chemical step. Previously, studies of their temperature-dependence were used to probe the effect of mutations at residues distant from the active site upon the hydride-transfer reaction catalyzed by EcDHFR. The results of these experiments supported the presence of a network of residues that were dynamically linked to the hydride-transfer step, and were in excellent agreement with computational studies predicting the presence of such a network. This thesis aims to extend upon these results to study the nature and extent of the dynamic network in EcDHFR, both by using an established experimental methods and by developing new biophysical probes of protein dynamics in this system. The major experimental methodology utilized in the following chapters is the determination and analysis of KIEs in a variety of EcDHFR mutants. To facilitate these measurements, new synthetic routes to a range of isotopically labeled nicotinamide cofactors have been developed. Some of the labeled materials have been used to establish a sensitive, triple-isotope technique to competitively measure deuterium isotope effects in enzyme-catalyzed reactions in EcDHFR. Synthesized materials were usd to measure the temperature dependence of intrinsic KIEs in selected dynamically altered mutants of EcDHFR, viz. W133F and F125M DHFR. Crystal structures have been obtained for both these mutants as well as for the previously studied G121V isozyme, and the combination of kinetic and structural information discussed in the context of catalytically important dynamic fluctuations in EcDHFR. Pressure-dependence of deuterium KIEs is also developed as a tool to probe the role of dynamics and tunneling in the EcDHFR reaction, with the ultimate aim of establishing high-pressure KIE measurements as a complementary method to variable temperature measurements. Finally, molecular recognition force spectroscopy (MRFS) measurements of an EcDHFR self-assembled monolayer (SAM) on gold are described. The surprisingly active enzymatic SAM has been shown to be a promising platform for future MRFS experiments to measure the forces involved in EcDHFR dynamics. All together, these studies advanced our ability to study the role of enzyme dynamics and quantum tunneling in enhancing their chemistry.
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Gkaniatsou, Effrosyni. "Elaboration of novel enzymatic immobilization matrices, based on Metal-Organic Frameworks for the catalytic degradation of environmental pollutants." Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLV005.
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Les enzymes sont des biocatalyseurs de plus en plus utilisés pour la transformation de molécules organiques (chimie fine, bioconversions, dépollution, chimie du pétrole) car elles possèdent de très bonnes sélectivité et réactivité, générant rapidement de larges quantités de produit. Cependant, la fragilité des enzymes, notamment en solution, limite souvent leur utilisation. Il est donc crucial de les immobiliser et de les stabiliser dans des supports adaptés. Une grande variété de matrices d’immobilisation (organiques ou inorganiques) a déjà étudiée, mais aucune ne satisfait pleinement aux critères nécessaires pour le développement de bio-réacteurs (accessibilité au site actif de l’enzyme, relargage de l’enzyme, diffusion des réactifs, recyclabilité, stabilité..). En outre, la majorité de ces matrices présente une porosité désordonnée, inadaptée pour une immobilisation homogène. L’utilisation de matériaux hybrides, cristallins et poreux de type Metal-Organic Frameworks (MOFs) a été récemment proposée comme alternative avec des applications en biocatalyse et en biodétection.Le travail de cette thèse a consisté à associer des matériaux de type Metal-Organic Frameworks à une mini-enzyme, la microperoxidase 8 (MP8), afin d’obtenir des matériaux multifonctionnels. Dans une première partie, le MOF mésoporeux, MIL-101(Cr), a été utilisé pour encapsuler la MP8, ce qui a conduit à une amélioration de son activité catalytique dans des conditions qui ne sont pas adéquates pour l’activité enzymatique (conditions acides, forte concentration en H2O2), démontrant ainsi le rôle protecteur du MOF vis-à-vis de l’enzyme. De plus, il a été possible de recycler le biocatalyseur. Cette approche a également permis d’améliorer considérablement la sélectivité de la MP8 pour la dégradation d’un colorant organique toxique négativement chargé, le méthyl orange, grâce à son adsorption sélective par interaction électrostatique avec les particules de MIL-101(Cr). La seconde partie a été consacrée à l’utilisation de matériaux MIL-101(Cr) fonctionnalisés. Tout d’abord, l’influence de la fonctionnalisation du ligand (avec un groupement –NH2 ou –SO3H) sur l’encapsulation de la MP8 ainsi que sur son activité catalytique pour des réactions de sulfoxydation a été étudiée. Il a été montré que l’activité catalytique et la réactivité de la MP8 sont affectées par le microenvironnement spécifique des pores du MOF, notamment pour des réactions de sulfoxydation mettant en jeu des dérivés thioanisole. Ensuite, un MOF à métal mixte (MIL-101(Cr/Fe)) choisi pour ses propriétés catalytiques stables, a été synthétisé et caractérisé. Enfin, la dernière partie de cette thèse a été consacrée à la synthèse in-situ d’un MOF (le microporeux MIL-53(Al)-FA) en présence de biomolécules (BSA) dans des conditions compatibles avec la préservation de la structure protéique (en solution aqueuse à température ambiante). Les matériaux hybrides obtenus ont été caractérisés en couplant de nombreuses techniques. Cette méthode d’encapsulation a conduit à des taux d’immobilisation extrêmement élevés. Une étude préliminaire a été initiée avec l’enzyme, Horseradish Peroxidase , qui conserve son activité catalytique après immobilisationThe use of enzymes in biocatalytic processes has been a challenging goal over the years. While enzymes present exceptional catalytic properties, their fragility hinders their industrial application. Their stabilization and protection are therefore of paramount importance. This can be effectively addressed through their immobilization within host solid matrices. Traditional materials (silica, clays, polymers, biopolymers, porous carbons…) have been widely studied as supports. Their pure organic or inorganic nature often requires a compromise between affinity with enzymes and robustness of the matrix. Besides, most of them have non-ordered porosity, with non-homogenous pore size distributions, unsuitable for homogeneous immobilization. Metal-Organic Frameworks (MOFs) have been recently introduced as alternative supports, thanks to their hybrid nature and their crystalline and highly porous structures.The aim of this PhD was to combine Metal-Organic Frameworks (highly porous and chemically stable polycarboxylate MOFs) and a mini-enzyme, microperoxidase 8 (MP8) to obtain multifunctional biocatalysts. In a first part, the mesoporous MIL-101(Cr) was used as a host matrix to encapsulate MP8. The encapsulation led to an increased catalytic activity under conditions (acidic conditions, high concentration of H2O2) detrimental to the catalytic activity of MP8, thereby demonstrating the protecting effect of MIL-101(Cr) matrix. The biocatalyst was also efficiently recycled. The selectivity of MP8 for the degradation of the harmful negatively charged organic dye methyl orange was also enhanced, thanks to the charged-based selective adsorption of the dye in MIL-101(Cr) porosity. A second part of the work was devoted to the use of functionalized MIL-101(Cr) analogs. First, functionalized ligands (bearing –NH2 and –SO3H groups) were used, and their influence on MP8 encapsulation was evaluated. The catalytic activity toward sulfoxidation reactions was also studied. The successful encapsulation of MP8 was strongly dependent on charge matching between the enzyme and the MOFs particles, while its catalytic activity was affected by the specific microenvironment of the pores. The MOF frameworks also modified the reactivity of MP8 toward different thioanisole derivatives. Then, a mixed metal MOF (MIL-101(Cr/Fe)), selected for its stable catalytic properties, was synthesized and characterized. Finally, the last part was devoted to the in-situ synthesis of MOFs (microporous MIL-53(Al)-FA) in presence of biomolecules (BSA) under compatible conditions with the preservation of the protein’s quaternary structure (aqueous media and room temperature). The resulting hybrid materials were thoroughly characterized and presented high loadings of BSA. A preliminarily study was performed with the enzyme, Horseradish Peroxidase, which retained its catalytic activity after immobilization
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Wirth, Petra. "Enzymes en solvants organiques." Grenoble 2 : ANRT, 1988. http://catalogue.bnf.fr/ark:/12148/cb37619244x.
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Rayder, Thomas M. "Modulation of Catalyst@MOF Host-Guest Composites in Pursuit of Synthetic Artificial Enzymes:." Thesis, Boston College, 2020. http://hdl.handle.net/2345/bc-ir:108930.
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Thesis advisor: Jeffery A. ByersThesis advisor: Chia-Kuang (Frank) Tsung
Biological systems have evolved over time to favor structures beneficial for the efficient transformation of simple feedstocks to sophisticated products. In particular, enzymes have evolved such that cooperative and geometrically controlled interactions between active sites and substrates enhance catalytic activity and selectivity. Separation of these active sites from other incompatible catalytic components allows for chemical transformation in a stepwise fashion, circumventing the inherent limitations to performing reactions in a single step. This dissertation describes the use of porous crystalline materials called metal-organic frameworks (MOFs) as hosts to mimic the component separation and precise active site control observed in nature. The first phase of these efforts explores the use of dissociative “aperture-opening” linker exchange pathways in a MOF to encapsulate transition metal complexes for carbon dioxide hydrogenation to formate. This strategy is then used to separate two incompatible complexes and perform the cascade conversion of carbon dioxide to methanol, resulting in unique and previously unobserved network autocatalytic behavior. Finally, the modularity of the MOF host is leveraged to install beneficial functionality in close proximity to the encapsulated transition metal complex, leading to activity exceeding that of any reported homogeneous system for carbon dioxide reduction. The insights gained through these studies can inform the development of composites for other reactions, allowing for access to new and unique reaction manifolds
Thesis (PhD) — Boston College, 2020
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Setser, Jeremy Wayne. "Conformational dynamics control catalysis in disparate systems : structural insights from DNA repair and antibiotic biosynthetic enzymes." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/91114.
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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2014.Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Chemical reactions allow biological systems to function. The majority of these biochemical reactions occur due to the work of protein catalysts known as enzymes. These biocatalysts are often thought of as pre-formed, static 'locks' that bind, and subsequently transform, their substrate molecule 'keys'. However, scientists are increasingly finding that dynamic movements of enzymes are a crucial aspect of catalysis. One such example of a system that relies on conformational flexibility is the human DNA repair protein alkyladenine DNA glycosylase (AAG). To efficiently repair DNA, AAG must search the million-fold excess of unmodified DNA bases to find a handful of DNA lesions. Such a search can be facilitated by the ability of glycosylases, like AAG, to interact with DNA using two affinities: a lower-affinity interaction in a searching process, and a higher-affinity interaction for catalytic repair. We have captured crystallographic snapshots of AAG bound to DNA in both high- and lower-affinity states. These depictions reveal several significant and unexpected protein structural rearrangements, providing molecular insight into the DNA-searching process adopted by AAG. By combining these new insights with existing biochemical and structural data, we are able to relate AAG to the big picture question of how DNA binding proteins find their binding sites in the vast expanse of the genome. In another study, a member of a biosynthetic pathway for antibiotic natural products, called kutznerides, was shown to be dependent on conformational changes. The enzyme in question, KtzI, uses a bound flavin cofactor, reducing equivalents from NADPH, and molecular oxygen to install a hydroxyl group on the side-chain nitrogen of the amino acid L-ornithine, which is subsequently incorporated into the kutzneride scaffold. KtzI was structurally characterized after being subjected to various chemical and environmental factors, capturing the enzyme in several states along its catalytic trajectory. These states suggest that a novel conformational change of both the protein backbone and the flavin moiety must take place in order to complete the enzymatic cycle of KtzI. This drastic rearrangement was also shown to be chemically interchangeable in the protein crystal, suggesting that these dynamic motions are catalytically relevant.
by Jeremy Wayne Setser.
Ph. D.
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Crawford, Luke. "Mechanistic insights into enzymatic and homogeneous transition metal catalysis from quantum-chemical calculations." Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/7818.
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Catalysis is a key area of chemistry. Through catalysis it is possible to achieve better synthetic routes, exploit molecules normally considered to be inactive and also attain novel chemical transformations. The development of new catalysts is crucial to furthering chemistry as a field. Computational chemistry, arising from applying the equations of quantum and classical mechanics to solving chemical problems, offers an essential route to investigating the underlying atomistic detail of catalysis. In this thesis calculations have been applied towards studying a number of different catalytic processes. The processing of renewable chemical sources via homogeneous reactions, specifically cardanol from cashew nuts, is discussed. All routes examined for monoreduction of a diene model by [Ru(H)(iPrOH)(Cl)(C₆H₆)] and [Ru(H)(iPrOH)(C₆H₆)]⁺ are energetically costly and would allow for total reduction of the diene if they were operating. While this accounts for the need of high temperatures, further work is required to elucidate the true mechanism of this small but surprisingly complex system. Gold-mediated protodecarboxylation was examined in tandem with experiment to find the subtle steric and electronic effects that dictate CO₂ extrusion from gold N-heterocyclic carbene activated benzene-derived carboxylic acids. The origin of a switch in the rate limiting step from decarboxylation to protodeauration with less activated substrates was also clearly demonstrated. Studies of gold systems are closed with examinations of 1,2-difluorobenzene C–H activation and CO₂ insertion by [Au(IPr)(OH)]. Calculations highlight that the proposed mechanism for oxazole-derived substrates cannot be extended to 1,2-difluorobenzene and instead a digold complex offers more congruent predicted kinetics. The lens of quantum chemistry was turned upon palladium-mediated methoxycarbonylation reactions. An extensive study was undertaken to attempt to understand the bidentate diphosphine ligand dependency on forming either methylpropanoate (MePro) or copolymers. Mechanisms currently suggested in literature are shown to be incongruous with the formation of MePro by Pd(OAc)₂ and bulky diphosphines. A possible alternative route is proposed in this thesis. Four mechanisms for methoxycarbonylation with Pd(2-PyPPh₂)ₙ are detailed. The most accessible route is found to be congruent with experimental reports of selectivity, acid dependency and slight steric modifications. A modification of 2-PyPPh₂ to 2-(4-NMe₂-6-Me)PyPPh₂ is shown to improve both selectivity and turnover, the latter by four orders of magnitude (highest transition state from 22.9 kcal/mol to 16.7 kcal/mol ∆G), and this new second generation in silico designed ligand is studied for its applicability to wider substrate scope and different solvents. The final chapter of this thesis is a mixed quantum mechanics and molecular mechanics (QM/MM) examination of an enzymatic reaction, discussing the need for certain conditions and the role of particular amino acid residues in an S[sub]N2 hydrolysis reaction.
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Subramaniam, Srisunder. "Studies of conformational changes and dynamics accompanying substrate recognition, allostery and catalysis in bacteriophage lambda integrase." The Ohio State University, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=osu1111655332.
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Sen, Mustafa Yasin. "Green Polymer Chemistry: Functionalization of Polymers Using Enzymatic Catalysis." University of Akron / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=akron1258422775.
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Kim, ChulHwan. "Azotobacter vinelandii nitrogenase: role of the MoFe protein α-subunit histidine-195 residue in catalysis." Diss., Virginia Tech, 1994. http://hdl.handle.net/10919/38311.
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Site-directed mutagenesis and gene replacement procedures were used to isolate mutant strains of Azotobacter vinelandii that produce altered MoFe proteins where the α-subunit residue-195 position, normally occupied by a histidine residue, was individually substituted by a variety of other amino acids. Structural studies have revealed that this histidine residue is associated with the FeMo-cofactor binding domain and probably provides an NH→S hydrogen bond to a central bridging sulfide located within FeMo-cofactor. The present study investigates the role of the α-histidine-195 residue in nitrogenase catalysis by examining the altered MoFe proteins.
Comparisons of the catalytic and spectroscopic properties of altered MoFe proteins produced by the Azotobacter vinelandii mutant strains suggest that the α-histidine-195 residue has a structural role which serves to keep the FeMo-cofactor attached to the MoFe protein and to correctly position the FeMo-cofactor within the polypeptide matrix such that N₂ binding is accommodated. Substitution of the α-His-195 residue by a glutamine residue results in an altered MoFe protein that binds but does not reduce N₂, the physiological substrate. Stopped-flow spectroscopic analyses indicate that the α-195gln MoFe protein is unable to reduce N₂ even though the altered MoFe protein can reach the redox state necessary for N₂ reduction. Although, N₂ is not a substrate for the altered MoFe protein, it is an inhibitor of both acetylene and proton reduction, both of which are otherwise effectively reduced by the altered MoFe protein. This result provides evidence that N₂ inhibits proton and acetylene reduction by simple occupancy of the active site. The α-195gln MoFe protein catalyzes HD formation in the presence of N₂ and D₂. Moreover, N₂ binding at the active site of the altered MoFe protein is inhibited by the addition of D₂. These observations indicate that binding of nitrogen to the enzyme is necessary but its reduction is not required for the formation of HD. N₂ uncouples MgATP from proton reduction catalyzed by the α-195gln MoFe protein, but does so without lowering the overall rate of MgA TP hydrolysis. Thus, the quasi-unidirectional flow of electrons from the Fe protein to the MoFe protein that occurs during nitrogenase turnover is controlled, in part, by the substrate serving as an effective electron sink. N₂-induced uncoupling of ATP hydrolysis from substrate reduction by the α-195gln MoFe protein is reversed by the addition of H₂ (D₂) in the assay atmosphere. This observation can successfully be explained if it-is assumed that the altered MoFe protein has a much greater binding affinity for H₂ (D₂) than for N₂. Substitution of the α-histidie-195 residue by glutamine also imparts hypersensitivity of acetylene reduction and N2 binding to inhibition by CO, indicating that the imidazole group of the α-histidine- 195 residue might protect an Fe contained within FeMo-cofactor from attack by CO.Ph. D.
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Goldman, Peter John. "The roles of redox active cofactors in catalysis : structural studies of iron sulfur cluster and flavin dependent enzymes." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/82313.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.Cataloged from PDF version of thesis.
Includes bibliographical references.
Cofactors are highly prevalent in biological systems and have evolved to take on many functions in enzyme catalysis. Two cofactors, flavin adenine dinucleotide (FAD) and [4Fe-4S] clusters, were originally determined to aid in electron transfer and redox chemistry. However, additional activities for these cofactors continue to be discovered. The study of FAD in the context of rebeccamycin and staurosporine biosynthesis has yielded another role for this cofactor in the enzyme StaC. A homolog of this enzyme, RebC, uses its FAD cofactor in the oxidation of 7-carboxy-K252c. StaC also uses 7-carboxy-K252 as a substrate, but its reaction does not result in a redox transformation. Biochemical and X-ray crystallographic methods were employed to determine that, indeed, the role of FAD in the StaC system is not to catalyze redox chemistry. Instead, FAD sterically drives an initial decarboxylation event. Subtle differences in the active sites of RebC and StaC promote this redox neutral decarboxylation, by activating water for a final protonation step. In another system, the characterization of the S-adenosyl-L-methionine (AdoMet) radical superfamily showed the versatility of these cofactors. In this superfamily, which includes over 40,000 unique sequences, [4Fe-4S] clusters are responsible for the initiation of radical chemistry. A recently described subclass of this superfamily, the dehydrogenases, require additional [4Fe-4S] cluster for activity. This requirement led to the hypothesis that these enzymes are catalyzing redox chemistry by directly ligating substrates to auxiliary (Aux) clusters. X-ray structures of 2-deoxy-scyllo-inosamine dehydrogenase (BtrN), required for the biosynthesis of 2-deoxystreptamine, and an anaerobic sulfatase maturating enzyme, anSMEcpe, which installs a required formylglycine posttranslational modification, refute this hypothesis. In these structures, substrate binding is distal from each enzymes' Aux clusters. However, the Aux cluster binding architecture shared between BtrN, anSMEcpe, and another AdoMet radical enzyme, MoaA, involved in molybdenum cofactor biosynthesis, suggests that the structural features will be a staple in the AdoMet radical superfamily, common to - 30% of the AdoMet radical reactions.
by Peter John Goldman.
Ph.D.
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Arhab, Yani. "Caractérisation structurale et fonctionnelle des phospholipases D." Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1225/document.
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Les phospholipases D (PLD, EC 3.1.4.4) sont des enzymes ubiquitaires retrouvées aussi bien chez les procaryotes (bactéries) que chez les eucaryotes (plantes, animaux et champignons). Les PLD catalysent l'hydrolyse des glycérophospholipides au niveau distal de la liaison phosphodiester pour former de l'acide phosphatidique, un important messager cellulaire impliqué dans de nombreuses voies telles que la prolifération cellulaire, la formation et le trafic vésiculaire, mais aussi la transcription et la survie cellulaire. Les PLD appartiennent à une superfamille de protéines (superfamille des PLD) qui ont en commun un site catalytique HXKX4D, X étant un acide aminé quelconque, contenant les résidus H (Histidyl), K (Lysyl) et D (Aspartyl). Ce site est nommé séquence consensus "HKD" et est dupliqué dans la plupart des membres de la superfamille des PLD. L'étude des PLD de plante est le moyen le plus sûr d'étudier cette famille d'enzyme car ce sont les seules PLD eucaryotiques purifiées à homogénéité et en grande quantité à ce jour. Ces travaux proposent une caractérisation fonctionnelle des résidus conservés au sein des PLD végétales menant à une caractérisation structurale avec la cristallisation de cette protéine. Dans un second temps l'activité de l'enzyme est modulée avec l'étude du domaine minimum, de la maturation post-traductionnelle de l'enzyme et le recherche d'un nouvel inhibiteur. Enfin, nous proposons le clonage d'une nouvelle PLD et la mise au point d'un système de détection in vivo de l'activité PLDPhospholipases D (PLD, EC 3.1.4.4) are ubiquitary enzymes found in prokaryotes (bacteria) as well as in eukaryotes (plant, animals and fungi). PLD catalyzes the hydrolysis of the distal phosphoester bound of phospholipids thus forming phosphatidic acid, an important cell signaling messenger implicated in numerous pathways such as cell proliferation, vesicular formation and trafficking but also transcription and cell survival. PLDs belong to a superfamily of protein which share a common catalytic site called “HKD†for HXKX4D, X is a random amino acid, containing H (Histidyl), K (lysyl) and D (aspartyl) residues. This consensus sequence is duplicated in most of the PLD superfamily members. The study of plant PLD is the best way to understand this family of proteins as they are the sole eukaryotic PLDs to be purified to homogeneity so far. This work provides a functional characterization of the most conserved residues in plant PLDs leading to a structural characterization with the crystallization of this enzyme. A second part of this work proposes the modulation of the enzyme hydrolysis activity by studying the minimal domain necessary for the activity and post-translational maturation undergone by plant PLDs. Also, we look for a new specific inhibitory molecule. Finally, we propose the cloning of a new plant PLD and the development of a new way to detect in vivo PLD activity
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Daude, David. "Molecular evolutionary perspectives of amylosucrases : from natural substrate promiscuity to tailored catalysis." Thesis, Toulouse, INSA, 2013. http://www.theses.fr/2013ISAT0036/document.
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La promiscuité des enzymes joue un rôle primordial pour l’évolution des protéines et la divergence des fonctions catalytiques. Comprendre les déterminants moléculaires qui régissent cette promiscuité enzymatique représente un enjeu scientifique majeur pour identifier les trajectoires évolutives pouvant entrainer l’émergence de nouvelles fonctions. L’objectif de cette thèse a consisté d’une part à sonder la promiscuité catalytique de l’amylosaccharase de Neisseria polysaccharea, une transglucosidase d’intérêt biotechnologique majeur, dans le but d’identifier des substrats alternatifs et d’autre part à étendre ses capacités naturelles par des techniques d’évolution moléculaire. Une trentaine de molécules ont été testées et ont permis de mettre en évidence la forte spécificité de l’enzyme pour son substrat donneur ainsi que sa large promiscuité vers les substrats accepteurs. Le rôle des résidus impliqués dans la reconnaissance des substrats a par la suite été considéré au travers d’une stratégie rationnelle basée sur des prévisions de stabilité thermodynamique. Deux histidines (H187 et H392), ont ainsi été ciblées par mutagénèse à saturation. La stabilité de ces mutants a été étudiée ainsi que ainsi que leur activité envers différents substrats. Des enzymes à la stabilité améliorée ou montrant des changements de spécificité de produits ont ainsi été identifiées. Afin d’étudier plus amplement la promiscuité de cette amylosaccharase, une deuxième stratégie d’ingénierie a été menée pour mimer in vitro les mécanismes moléculaires de la dérive génétique neutre. Ce phénomène dit de “Neutral-Drift” a préalablement été décrit comme un facteur impliqué dans les changements de promiscuité catalytique. Quatre cycles de mutagénèse ont ainsi été réalisés et 440 clones ont été sélectionnés pour avoir conservé leur fonction originelle (i.e. leur activité sur saccharose). Des variants aux propriétés catalytiques améliorées envers des substrats alternatifs ont été caractérisés et des groupes de positions corrélées ont été identifiés. L’effet des mutations neutres sur la thermostabilité a également été étudié. Enfin, de façon remarquable, une nouvelle activité envers un substrat non reconnu par l’enzyme native, le méthyl-α-L-rhamnopyranoside, a été détectée. Ce variant possédant quatre substitutions a été caractérisé et la résolution de sa structure tridimensionnelle par cristallographie aux rayons-X permettra d’approfondir les relations unissant séquence, structure et activité de l’enzymeInvestigation of substrate promiscuity is of prime interest to understand the way enzymes evolve. Understanding the molecular determinants involved in substrate promiscuity is challenging to determine the evolutionary trajectories that lead to the emergence of catalytic functions and to take further advantage of their evolvability to develop original biocatalysts. The objective of this thesis aimed to investigate the substrate promiscuity of the amylosucrase from Neisseria polysaccharea, a transglucosidase of prime biotechnological interest, to identify alternative donor and acceptor molecules and further extend its catalytic properties through enzyme engineering. About thirty molecules were assayed and the strong specificity for the natural donor sucrose was emphasized, as well as the broad acceptor promiscuity. The rational engineering of active site residues responsible for substrate recognition was undertaken through thermodynamic stability predictions. Two residues, namely H187 and H392, were rationally targeted for site-directed mutagenesis. The stability of these variants was investigated as well as their activity toward both natural and promiscuous substrates. Variants with enhanced stability or altered product distribution were identified. These results highlighted that mutations responsible for stability changes may also lead to substrate promiscuity or product specificity changes. To further investigate the promiscuity of amylosucrase, we considered another engineering strategy to mimic in vitro the neutral enzyme evolution. Neutral genetic drift was previously shown to be related to promiscuity changes. On this basis, four repeated round of mutagenesis were performed and 440 clones were selected because they maintained the protein original function (i.e. the activity on sucrose). Variants with enhanced properties towards promiscuous donors and acceptors were characterized and clusters of correlated amino acid substitutions were identified. The impact of neutral mutations on thermodynamic stability was also discussed. Remarkably, a totally new activity towards methyl-α-L-rhamnopyranoside, an acceptor not recognized by the parental wild-type enzyme, was detected. The variant harboring four amino acid substitutions was characterized and the determination of its three-dimensional structure by X-ray crystallography will be useful to further investigate the structure-sequence-activity relationships of this enzyme
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Tull, Dedreia L. "An investigation of the mechanism of the Cellulomonas fimi exoglucanase." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/30402.
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The exoglucanase from Cellulomonas fimi catalyses the hydrolysis of cellobiose units from the non-reducing terminus of cello-oligosaccharides with overall retention of anomeric configuration. Its mechanism of action is therefore thought to involve a double displacement reaction, involving as the first step, formation of a glycosyl-enzyme intermediate (glycosylation) and as a second step, the hydrolysis of this intermediate (deglycosylation). This mechanism is investigated here through the study of the kinetics of hydrolysis of aryl β-glucosides and aryl β-cellobiosides and by employing the mechanism-based irreversible inactivators, 2', 4'-dinitrophenyl 2-deoxy-2-fluoro-β-D-glucoside (2F-DNPG) and 2", 4"-dinitrophenyl 2-deoxy-2-fluoro-β-D-cellobioside (2F-DNPC).
The study with the aryl β-glucosides revealed that this enzyme is indeed active on glucosides, a feature that had previously been undetected. A linear relationship was found to exist between the logarithm of Vmax for hydrolysis and the phenol pKa as well as between the logarithm of Vmax/Krn and me phenol pKa, showing that glycosylation is both the rate determining step and the first irreversible step for all substrates. The reaction constant calculated, ρ = 2.21, indicates a considerable amount of charge build up at the transition state of glycosylation.
The linear free energy relationship study of the aryl β-cellobiosides revealed no significant dependence of the logarithm of Vmax on the pKa of the phenol, indicating that deglycosylation is rate determining. However, the slight downward trend in this Hammett plot at higher pKa values may suggest that the rate determining step is changing from deglycosylation to glycosylation. However, the logarithm of Vmax/Km does correlate with the pKa of the phenol, thus showing that the first irreversible step is glycosylation. The reaction constant (ρ = 0.60) which reflects the development of charge at the glycosylation transition state for the cellobiosides is less than that calculated for the glucosides, thus suggesting a glycosylation transition state with either a greater degree of acid catalysis or less C-O bond cleavage than that for the glucosides. The inactivators, 2F-DNPC and 2F-DNPG, are believed to inactivate the exoglucanase by binding to the enzyme and forming covalent glycosyl-enzyme intermediates. The inactivated-enzyme was stable in buffer but reactivated in the presence of a suitable glycosyl-acceptor such as cellobiose, presumably via a transglycosylation reaction. These results indicate that covalent 2F-glycosyl-exoglucanase intermediates are stable and are catalytically competent to turn over to product, thus supplying further evidence for the Koshland mechanism. The exoglucanase is inactivated more rapidly by 2F-DNPC than by 2F-DNPG. However, both inactivated forms of the enzyme reactivated at comparable rates in the presence of cellobiose, showing that the second glucosyl unit present on the cellobiosides increases the rate of glycosylation relative to that found for the glucosides but not the rate of deglycosylation.
The stable covalent nature of the 2F-glycosyl-enzyme intermediates provided an excellent opportunity to identify the enzymic nucleophile. This was accomplished by radiolabelling the exoglucanase with a tritiated analogue of 2F-DNPG cleaving the protein into peptides and purifying the radiolabelled peptides. Sequencing of this peptide resulted in the identification of the active site nucleophile as glutamic acid residue 274. This residue was found to be highly conserved in this family of β-glycanases, further indicating its importance in catalysis.Science, Faculty of
Chemistry, Department of
Graduate
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Heath, Rachel Sarah. "Studies of a 'blue' copper oxidase electrocatalyst." Thesis, University of Oxford, 2008. http://ora.ox.ac.uk/objects/uuid:e8359408-d3d4-4fe3-910a-cc69265a1546.
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This thesis concerns the electrochemical investigation of high-potential laccases. These multicopper oxidases are efficient electrocatalysts for the dioxygen reduction reaction. A method for stabilising laccase on a graphite electrode was established. The method involved modification of the graphite surface by diazonium coupling of a 2-anthracene molecule. A laccase ‘film’ adsorbed on this modified surface remained stable for over two months and, typically, the current density for dioxygen reduction was doubled compared to a laccase ‘film’ on an unmodified surface. Protein film voltammetry was used to investigate thermodynamic and kinetic aspects of the electrochemical behaviour of laccase. The effect of inhibitors on the magnitude of reduction current and the position of the wave (related to the overpotential for the reaction) was also studied. Fluoride, chloride and azide showed different modes of inhibition and inhibition constants ranged from micromolar for azide to millimolar for chloride. In cyclic voltammetry experiments it was only in the presence of high concentrations of the inhibitors fluoride, chloride and azide that a non-turnover signal, corresponding to a one electron transfer process, was revealed. The evidence suggested that the non-turnover signal arose from interfacial electron transfer between the electrode and the type 1 or ‘blue’ copper. Evaluation of the peak areas allowed determination of the catalytic rate constant, kcat, as 300 s–1, and the electroactive surface coverage as four pmol cm–2. The rate of interfacial electron transfer was rapid enough to not limit catalysis at high overpotentials. A spectroelectrochemical cell was designed to investigate the behaviour of the type 1 copper in the presence of inhibitors and at different pH values. The inhibitors fluoride, chloride and azide had little effect on the reduction potential of the type 1 copper, but at higher pH values the reduction potential of the type 1 copper was decreased.
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Duchiron, Stéphane. "Etude de la polymérisation enzymatique de monomères hétérocyclocarbonyliques." Thesis, Strasbourg, 2016. http://www.theses.fr/2016STRAE039/document.
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Le domaine des polymères biosourcés connait une croissance rapide mais se pose toujours le problème d’une synthèse plus respectueuse de l’environnement. En cela, la catalyse enzymatique est une voie prometteuse. Ce travail vise donc à étudier et comprendre la polymérisation enzymatique par ouverture de cycle (eROP) afin d’en dépasser les limitations qui sont principalement : une cinétique lente, une faible masse molaire des polymères obtenus ou une variété limitée des fonctions chimiques polymérisables. La première partie de notre étude, portant sur les lactones, a mis en évidence la possibilité d’activer la réaction via une amine tertiaire. La seconde, qui traite des thiolactones, a mis en exergue des mécanismes spécifiques de copolymérisation avec plusieurs étapes de croissance de chaînes. Enfin, nous avons synthétisé des polyesters porteurs d’acides aminés, ouvrant ainsi la voie à des polymères « fonctionnalisables » et à de nouvelles architectures macromoléculairesThe field of biobased polymers is experiencing a rapid growth but the development of more environmentally friendly synthesis methods remains a problem. With this in mind, enzymatic catalysis is a promising tool but it has some limitations such as slow polymerization kinetics, the low molecular weight of the produced polymers and a limited range of polymerizable monomers. This work aims at a better understanding of the enzymatic ring opening polymerization reactions (eROP) with a view to overcoming these limitations. To achieve this, three main topics have been investigated. The first, focusing on lactones, has demonstrated the possibility of activating the polymerization reaction with a tertiary amine. The second part, dealing with thiolactones as sulfur-based monomers, highlighted specific copolymerization mechanisms comprising of several distinct steps of polymer chain growth. Finally, polyesters bearing amino acids were synthesized, thus paving the way for functionalizable polymers and new macromolecular architectures
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Netto, Caterina Gruenwaldt Cunha Marques. "Desenvolvimento de catalisadores e sistemas enzimáticos para a redução do CO2." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46136/tde-08052013-084051/.
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O gás carbônico é a fonte primária de carbono ideal para se obter novos compostos, devido principalmente à sua abundância, atoxidade e ao fato de ser renovável. Desta forma, novos métodos de introduzir o CO2 em rotas sintéticas se fazem necessários. Dentre os possíveis métodos, nesta tese foram estudadas duas rotas diferentes de utilização do gás carbônico: uma, empregando complexos bimetálicos como catalisadores da inserção do CO2 em alcenos e alcanos e outra utilizando enzimas imobilizadas em partículas magnéticas, atuantes no ciclo de redução do CO2 ao metanol. Os complexos bimetálicos foram construídos a partir do ligante de ponte, bispirrolidyl- fenil (BPP) incorporando grupos difenilmetanol e difenilfosfino em suas arquiteturas moleculares. Sua inspiração partiu de sistemas biomiméticos para a redução do CO2, e foi direcionada para a carboxilação de hidrocarbonetos. Nas reações de acoplamento carbono-carbono, observou-se que com o iodeto de metila, os complexos foram capazes de transformar o gás carbônico e produzir acetato de metila. Já nas reações com 1-deceno, isobutano e iso-octano, apenas três complexos se mostraram eficientes: BPP(ONi,ONi), BPP(OZn,OPPh2Pd) e BPP(OPPh2Pd,OPPh2Pd). Na rota enzimática, fez-se uso de enzimas imobilizadas do tipo desidrogenase e três tipos de nanopartículas magnéticas (MagNP) como suporte (MagNP-APTS, MagNP@SiO2-APTS e MagNP-APTS/Glioxil-Agarose, APTS = aminopropiltrimetoxissilano). Observou-se que para a imobilização da álcool desidrogenase e da formaldeído desidrogenase, o melhor suporte foi a MagNP@SiO2- APTS, enquanto para a formato desidrogenase, o melhor suporte de imobilização foi a MagNP-APTS. Para a glutamato desidrogenase, um sistema com imobilização via múltiplos pontos, como na MagNP-APTS/Glioxil-Agarose, conduziu a um melhor desempenho, . Os melhores sistemas enzima-suporte foram utilizados em uma reação multi-enzimática com CO2, NADH e glutamato para a obtenção de formaldeído e metanol. Os dois métodos de redução do gás carbônico se mostraram capazes de realizar o objetivo da tese, que é a transformação do CO2 em produtos de maior valor agregadoCO2 is a primary world carbon source readily available for the production of new compounds, under sustainable conditions due to its great abundance and non-toxic, renewable characteristics. Hence, there is a compulsive interest to develop new methodologies capable of introducing carbon dioxide in the chemical synthetic routes. Among the many possible alternatives, two different strategies were pursued this thesis: one using bimetallic complexes as catalysts, and the other one using enzymes supported on superparamagnetic nanoparticles. The bimetallic complexes were based on the bridging bis-pyrrolidyl-phenol (BPP) architecture encompassing diphenylmethanol and diphenylphosphino groups. They were inspired in biomimetic systems for the chemical reduction of CO2 and employed in the carboxylation of hydrocarbons. In such carbon-carbon coupling reactions, the bimetallic complexes were able to catalyse the reaction between CO2 and methyl iodide in order to obtain methyl acetate. However, in the reaction with 1- decene, isobutene and iso-octane, only three of them were efficient: BPP(ONi,ONi), BPP(OZn,OPPh2Pd) and BPP(OPPh2Pd,OPPh2Pd). In the studies focusing on immobilized enzymes, dehydrogenase-like enzymes and three different kind of magnetic particles (MagNP) were employed (MagNP-APTS, MagNP@SiO2-APTS and MagNP-APTS/Glioxyl-Agarose, APTS = aminopropyltrimethoxisylane). The best immobilization support for alcohol dehydrogenase and formaldehyde dehydrogenase was MagNP@SiO2-APTS, while for formate dehydrogenase the best immobilization support was MagNP-APTS. For glutamate dehydrogenase a multi-point immobilization was required, turning MagNPAPTS/ Glioxyl-Agarose the best catalytic support. These enzyme-support systems were used in a multi-enzymatic reaction using CO2, NADH and glutamate in order to obtain methanol and formaldehyde. Both CO2 redution methods were successful explored, and the results fulfilled the major objective of this thesis, which is the conversion of CO2 into higher value products.
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Galmés, Ordinas Miquel Àngel. "Molecular insights into the promiscuity of serine hydrolases. Towards a computationally guided protocol for the redesign of enzymes." Doctoral thesis, Universitat Jaume I, 2022. http://dx.doi.org/10.6035/14122.2022.725777.
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Two serine hydrolases, Candida antarctica Lipase B (CALB) and para-nitrobenzyl (Bs2) esterase from Bacillus subtilis, were used as a model to study enzyme promiscuity through QM/MM methods and experimental enzymes kinetics. Both, the catalytic and the substrate promiscuity were studied. The hydrolysis of amides and the epoxidation of alkenes catalyzed by CALB were explored. Moreover, a computational scheme for the redesign of the Bs2 was also proposed. The electrostatic environment around the active site was analyzed and a map of structural determinants in the vicinity of the active site pocket was done using 3D Convolutional Neural Networks. The proposed computationally guided protocol for the mutagenesis of enzymes based on the detailed analysis of the electrostatic environment of two structurally aligned trajectories using rotation quaternions was applied. A new mutant variant of the Bs2 was suggested as an improved catalytic variant by combining the best electrostatic features of both enzymes.Programa de Doctorat en Química Teòrica i Modelització Computacional
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Andrejić, Milica. "Development of Hybrid QM/QM Local Correlation Methods for the Study of Metal Sites in Biomolecular Catalysis." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://hdl.handle.net/11858/00-1735-0000-0022-6011-C.
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Blomberg, Mattias. "Redox Reactions of NO and O2 in Iron Enzymes : A Density Functional Theory Study." Doctoral thesis, Stockholms universitet, Fysikum, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-863.
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In the present thesis the density functional B3LYP has been used to study reactions of NO and O2 in redox active enzymes. Reduction of nitric oxide (NO) to nitrous oxide (N2O) is an important part in the bacterial energy conservation (denitrification). The reduction of NO in three different bimetallic active sites leads to the formation of hyponitrous acid anhydride (N2O22-). The stability of this intermediate is crucial for the reaction rate. In the two diiron systems, respiratory and scavenging types of NOR, it is possible to cleave the N-O bond, forming N2O, without any extra protons or electrons. In a heme-copper oxidase, on the other hand, both a proton and an electron are needed to form N2O. In addition to being an intermediate in the denitrification, NO is a toxic agent. Myoglobin in the oxy-form reacts with NO forming nitrate (NO3 -) at a high rate, which should make this enzyme an efficient NO scavenger. Peroxynitrite (ONOO-) is formed as a short-lived intermediate and isomerizes to nitrate through a radical reaction. In the mechanism for pumping protons in cytochrome oxidase, thermodynamics, rather than structural changes, might guide protons to the heme propionate for further translocation. The dioxygenation of arachidonic acid in prostaglandin endoperoxide H synthase forms the bicyclic prostaglandin G2, through a cascade of radical reactions. The mechanism proposed by Hamberg and Samuelsson is energetically feasible.
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Idris, Zulkifli. "Electrocatalytic cycling of nicotinamide cofactors by Ralstonia eutropha soluble hydrogenase." Thesis, University of Oxford, 2012. http://ora.ox.ac.uk/objects/uuid:3d458a13-ce61-4ae4-bc93-5a7db3bb371d.
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Nicotinamide cofactors in their reduced and oxidised forms are important redox agents in biology. Of about 3000 dehydrogenases available to date, many require these cofactors for their activity. Dehydrogenases are of interest to chemists as they offer asymmetric catalysis to yield chiral products. The requirement of dehydrogenases for nicotinamide cofactors necessitates research into finding the best way of recycling the oxidised or reduced forms of these cofactors. Electrocatalytic NAD(P)H oxidation and NAD(P)⁺ reduction on standard electrodes is problematic due to unwanted side reactions and high overpotential requirements, but in Nature efficient enzyme catalysts are available to facilitate these reactions. The focus of this Thesis, the Soluble Hydrogenase of R. eutropha (SH) is a multimeric bidirectional hydrogenase that couples H2 oxidation to the reduction of NAD⁺ to NADH. Protein Film Electrochemistry (PFE) has been employed to study NAD⁺-reducing catalytic moieties of the SH for the first time. It is shown that SH subunits on an electrode are able to catalyse NADH oxidation and NAD⁺ reduction efficiently with minimal overpotential, which is significant because in vivo, NAD(H) cycling is coupled to 2H⁺/H₂ cycling and these reactions are closely spaced in potential. Substrate affinities and inhibition constants for the SH, determined using PFE are discussed in the context of the SH function and the related catalytic domains of respiratory Complex I. A range of molecules that are known to inhibit the related Complex I have been investigated for their ability to inhibit the SH moieties: the similarity between inhibition constants is consistent with structural and functional similarity between the SH and Complex I. The ability of the SH moieties to sustain NAD(H) catalysis in the presence of O₂ is also demonstrated and is consistent with the requirement for the SH to function under aerobic conditions and to reactivate the inactivated hydrogenase moiety by supplying low potential electrons from NADH. Engineered variants of the SH, designed to enhance the affinity towards NADP⁺, were investigated for the first time, using PFE. Electrochemical characterisation of the variants is presented and results are discussed alongside findings on the wild type SH. The variants are shown to exhibit NADP⁺ reduction, and to have higher affinity towards NADP⁺ than the wild type SH. The first efficient NADP⁺ reduction and NADPH oxidation is observed for one of the variants on a graphite electrode and the best variant showed a KM of 1.7 mM for NADP⁺. This Thesis also provides evidence for the ability of moieties of the SH to be used in cofactor regeneration systems. Two novel systems are demonstrated. The first involves H₂ driven NADH recycling based on the NAD⁺-reducing moiety of the SH immobilised on graphite particles together with a hydrogenase or platinum, with electrons from H₂ passed from the hydrogenase through the graphite to the NAD⁺-reducing moiety. The second involves an electrode modified with the NAD⁺-reducing moiety of the SH, and is demonstrated as an electrochemical NADH recycling system coupled with NADH-dependent pyruvate reduction to lactate by lactate dehydrogenase. The ability of variants of the SH to catalyse NADP⁺ reduction suggests that it may also be possible to use these systems for recycling NADPH for catalysis of important biotransformation reactions by NADPH-dependent dehydrogenases.
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Coines, Lopez-Nieto Juan. "Mechanistic insights into substrate-assisted catalysis in glycosidases by means of QM/MM molecular dynamics." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/670537.
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Carbohydrates are essential molecules in biotechnology and for the proper functioning of living organisms. They are involved in energy storage, being structural components or participating in crucial biological processes such as cellular signaling or the development of diseases. For this reason, carbohydrates have gained great attention among the scientific community. “Carbohydrate-active enzymes” are the catalytic machinery that degrade, synthesize, and modify carbohydrates, even that saccharides show a vast variety of configurations, conformations and stereochemical properties. This thesis is focused on the study of glycosidases, enzymes in charge of hydrolyzing glycosidic bonds. In particular, glycosidases that use the substrate-assisted mechanism, in which the N-acetyl group of the substrate actively participate in catalysis. This reaction mechanism still hinders several mysteries such as the nature of the reaction intermediate, the role of the catalytic residues or the conformational itinerary. The latter corresponds to the conformations that a carbohydrate ring adopts during the enzymatic reaction. Deciphering glycosidase catalytic itineraries and their reaction mechanism boosts the design of specific inhibitors and provides further knowledge of these enzymes. To study glycosidase reaction mechanisms, we used computational techniques based on molecular dynamics, describing the systems with molecular mechanics, quantum mechanics or hybrid methods that combine the benefits from both. We investigated chitinase B from family GH18 that degrades chitin and β-N-acetylglucosaminidases from families GH84 and GH85, which cleave O- and N-glycans, respectively. Furthermore, we elucidated the conformational intrinsic properties of carbohydrates and carbohydrate-based inhibitors related to the substrate-assisted mechanism. In summary, we provide an overview of this reaction mechanism.Els carbohidrats són molècules primordials tant en l’àmbit biotecnològic com en el bon funcionament de tot organisme. Es troben implicats en el l’emmagatzematge d’energia, són elements estructurals o participen en processos biològics crucials pels éssers vius com ara la senyalització cel·lular o el desenvolupament de situacions patològiques. Per aquest motiu, els carbohidrats han atret l’atenció de la comunitat científica. Els “enzims actius en carbohidrats” són la maquinària catalítica que degrada, sintetitza i modifica els carbohidrats, tot i la gran varietat de configuracions, conformacions i estereoquímiques que aquests presenten. Aquesta present tesi doctoral s’ha dedicat a l’estudi de glicosidases, enzims encarregats d’hidrolitzar enllaços glicosídics. En concret, glicosidases que empren un mecanisme d’assistència de substrat, en el qual el substituent acetil del carbohidrat participa activament en la reacció enzimàtica. Aquest mecanisme de reacció encara amaga incògnites com la naturalesa del intermedi de reacció, la disposició dels residus catalítics o l’itinerari conformacional. Aquesta última característica correspon a les conformacions que adopta un anell de sucre en el centre actiu de l’enzim al llarg de la reacció enzimàtica. Desxifrar aquests itineraris, així com el mecanisme de reacció, permet assistir en el disseny d’inhibidors específics per glicosidases i empènyer més enllà el coneixement sobre aquests enzims. Per realitzar aquesta tasca, s’han usat mètodes computacionals basats en dinàmica molecular, tot descrivint els sistemes estudiats amb mecànica molecular, mecànica quàntica o mètodes híbrids que combinen els avantatges de les anteriors. S’ha investigat la quitinasa B de la família GH18 que degrada el polisacàrid quitina, així com β-N-acetilglicosaminidases de les famílies GH84 i GH85, que tallen O- i N-glicans, respectivament. Així mateix, s’han estudiat les propietats conformacionals de carbohidrats i inhibidors de glicosidases basats en aquests últims per tal de comprendre la inhibició en diverses glicosidases que segueixen el mecanisme d’assistència de substrat. D’aquesta manera, s’ha obtingut una visió general d’aquest mecanisme.
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Wulff, Philip. "Principles of hydrogen catalysis in the presence of oxygen by a [NiFe] hydrogenase from E. coli." Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:9e434467-d50b-484a-a17e-ef3091636269.
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[NiFe] hydrogenases are metalloenzymes that act as highly efficient molecular electrocatalysts for the interconversion of protons and molecular hydrogen. Unlike any other known molecular electrocatalyst, the members of a subgroup of respiratory membrane-bound [NiFe] hydrogenases are able to maintain H2 catalysis in the sustained presence of O2. This O2-tolerance depends on the ability to respond to oxidative inactivation by O2 by exclusively forming rapidly reactivated active site states, thus implying a catalytic cycle in which O2 acts as a competing substrate to H2. Using isotope ratio mass spectrometry it is proven that the O2-tolerant Escherichia coli Hydrogenase 1 responds to O2 attack by acting as a four-electron oxidoreductase, catalysing the reaction 2 H2 + O2 → 2 H2O, equivalent to hydrogen combustion. Special features of the enzyme’s electron relay system enable delivery of the required electrons. A small fraction of the H2O produced arises from side reactions proceeding via reactive oxygen species, an unavoidable consequence of the presence of low-potential relay centres that release electrons from H2 oxidation. While the ability to fully reduce O2 to harmless H2O at the active site to generate the rapidly reactivated state Ni-B, determines if a hydrogenase is O2-tolerant, the ratio of oxidative inactivation to reductive reactivation rates determines how tolerant the enzyme is. It is shown by protein film electrochemistry that the (αβ)2 dimeric assembly of Hyd-1 plays an important role in O2-tolerance by aiding reactivation of one catalytic unit through electron transfer from the other. The teamwork between two redundant partners implicates a new role for dimerisation and represents a new example of cooperativity in biology. Finally, the non-natural amino acid p-azido-L-phenylalanine was synthesised and incorporated into Hyd-1, testing the possibility of introducing labels at specific sites.
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Blomberg, Mattias. "Redox Reactions of NO and O2 in Iron Enzymes : A Density Functional Theory Study." Doctoral thesis, Stockholm : Department of Physics, Stockholm University, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-863.
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