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ROVALETTI, ANNA. "A computational outlook on the catalysis exerted by the unique active site of MoCu CO dehydrogenases." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2021. http://hdl.handle.net/10281/305403.
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I processi di produzione e consumo negli ecosistemi del suolo contribuiscono ai cicli biochimici globali di molti gas in tracce (CH4, CO, H2, N2O e NO) che sono rilevanti per la chimica atmosferica e il clima. Tali piccole molecole di gas svolgono ruoli diversi nel metabolismo dei microrganismi posti nel suolo che si basano su metalloenzimi specifici per la loro trasformazione. Tra questi, è stato dimostrato che i metalloenzimi a base di molibdeno sono cruciali in tale contesto. In particolare, è stato riportato che un molibdoenzima specifico è coinvolto nell'ossidazione della CO atmosferica. MoCu CO deidrogenasi (MoCu CODH) è un enzima presente nei carbossidobatteri aerobici, come Oligotropha carbossidovorans, che rappresentano uno dei componenti essenziali nel consumo biogeochimico di monossido di carbonio (CO). Essi infatti contribuiscono al mantenimento della concentrazione subtossica di CO nella bassa atmosfera elaborandone circa 2 × 108 tonnellate all'anno. Questa metalloproteina batterica catalizza l'ossidazione della CO a CO2, mentre può anche scindere H2 in due protoni e due elettroni. Tali reazioni vengono eseguite grazie a un sito attivo unico composto da due metalli, uno ione rame e uno molibdeno, legati tra loro tramite un atomo di zolfo. Nonostante siano stati condotti ampi studi teorici e sperimentali su questo enzima, diversi aspetti relativi alla sua reattività non sono stati ancora chiariti. Nella presente tesi ci siamo concentrati sulla descrizione in silico di MoCu CODH al fine di approfondire la comprensione dei meccanismi di reazione catalizzati dall'enzima. Per fare ciò, nel quadro della teoria del funzionale della densità (DFT), abbiamo applicato modelli di diverse dimensioni per ottenere una descrizione accurata del sistema. Nel contesto della catalisi dell'ossidazione della CO, abbiamo evidenziato che se un intermedio simile al tiocarbonato si forma lungo il percorso catalitico, non rappresenta una specie limitante la velocità nel panorama energetico enzimatico, a differenza di quanto proposto in base ai risultati di precedenti studi teorici. Inoltre, siamo stati in grado di suggerire un meccanismo catalitico alternativo per l'ossidazione della CO che coinvolge il ruolo diretto di una molecola d'acqua, attivata dal sito attivo circostante. Per quanto riguarda l'attività idrogenasica di MoCu CODH, sono stati presentati due meccanismi plausibili per la scissione di H2. Per la prima volta abbiamo suggerito che il sito attivo MoCu CODH possa essere visto come Frustrated Lewis Pairs (FLP) e abbiamo proposto un meccanismo tipo FLP per l'ossidazione del diidrogeno. In alternativa, un evento di protonazione, quale la protonazione del residuo di cisteina coordinata allo ione rame prima del legame di H2 al sito attivo, si è rivelato necessario per presentare un canale reattivo plausibile.Production and consumption processes in soil ecosystems contribute to the global biochemical cycles of many trace gases (CH4, CO, H2, N2O and NO) that are relevant for atmospheric chemistry and climate. Such small gas molecules play different role into the metabolism of microorganisms placed in soil that rely on specific metalloenzymes for their transformation. Among these, molybdenum-based metalloenzymes were evidenced to be crucial in such context. In particular, a specific molybdoenzyme was reported to be involved in atmospheric CO oxidation. MoCu CO dehydrogenases (MoCu CODH) is an enzyme found in aerobic carboxidobacteria, such as Oligotropha carboxidovorans which represent one of the essential components in the biogeochemical carbon monoxide (CO) consumption. In fact, they contribute to maintenance of subtoxic concentration of CO in the lower atmosphere by processing approximately 2×108 tons of it annually. This bacterial metalloprotein catalyses the oxidation of CO to CO2, while it can also split H2 in two protons and two electrons. Such reactions are performed thanks to a unique active site composed of two metals, a copper ion and a molybdenum one, linked together through a sulphur atom. Despite extended theoretical and experimental studies had been carried out concerning this enzyme, several aspects related to its reactivity have not been unravelled.In the present thesis, we focused on the in silico description of MoCu CODH in order to deepen the understanding of the reaction mechanisms catalysed by the enzyme. To do so, in the framework of density functional theory (DFT), we applied models of different sizes to obtain an accurate description of the system. In the context of CO oxidation catalysis, we evidenced that if a previously proposed thiocarbonate like intermediate is formed along the catalytic path, it does not represent a rate limiting species on the enzymatic energy landscape, differently from results of previous theoretical studies. Moreover, we were able to suggest an alternative catalytic mechanism for the oxidation of CO that involves the direct role of a water molecule, activated by the sourrounding active site. As for the MoCu CODH hydrogenase activity, two plausible mechanisms for the splitting of H2 were presented. For the first time we suggested that the MoCu CODH active site may be viewed as a Frustrated Lewis Pair (FLP), and we proposed a FLP-like mechanism for oxidation of the dihydrogen. Alternatively, a protonation event–e.g. Cu-bound cysteine residue protonation – prior to binding of H2 to the active site proved to be necessary to present a plausible reactive channel.
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Kluge, Stefan. "Modellierung sequentieller Metalloenzyme auf Magnesiumbasis." lizenzfrei, 2007. http://www.db-thueringen.de/servlets/DocumentServlet?id=10371.
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Kung, Yan. "Structural studies of metalloenzyme complexes in acetogenic carbon fixation." Thesis, Massachusetts Institute of Technology, 2011. http://hdl.handle.net/1721.1/65474.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Acetogenic bacteria use the Wood-Ljungdahl carbon fixation pathway to produce cellular carbon from CO₂. This process requires several metalloenzymes that employ transition metals such as iron, nickel, and cobalt towards the production of acetyl-CoA, the final product. In one stage of the pathway, the cobalt-containing B₁₂ cofactor harbored by the corrinoid iron-sulfur protein (CFeSP) transfers a methyl group from methyltetrahydrofolate (CH₃-H₄folate), which is bound by a methyltransferase enzyme (MeTr), to a nickel-containing metallocluster called the A-cluster of the downstream enzyme, acetyl-CoA synthase (ACS). Such B12-dependent methyl transfer reactions require the construction of large, multimodular enzyme complexes whose threedimensional assemblies are, at present, largely uncharacterized. X-ray crystallography was used to solve the structure of a CFeSP/MeTr complex, the first crystal structure of a B12-dependent methyltransferase to depict all protein domains required for B12 binding, activation, protection, and catalysis. This structure, along with in crystallo activity data, illustrates how conformational movements, which can occur within protein crystals, enable the B12 cofactor to alternate between a sequestered conformation for cofactor protection and an active conformation for catalysis. Small-angle X-ray scattering (SAXS) experiments were also conducted to explore the quaternary composition of the complex in solution and revealed that multiple CFeSP/MeTr complexes can be formed. In another reaction of the Wood-Ljungdahl carbon fixation pathway, a nickel and iron containing metallocluster called the C-cluster of carbon monoxide dehydrogenase (CODH) reduces a second molecule of CO₂ to CO, an intermediate that is channeled to the ACS A-cluster. Although the structure of the C-cluster was first described a decade ago, its catalytic mechanism remained unresolved. To provide mechanistic insight into the chemistry employed at the C-cluster, crystal structures were determined with substrate and inhibitor molecules bound to the C-cluster of the CODH/ACS complex. These structures capture states of the C-cluster at key steps in the reaction and contribute to a consensus model for C-cluster chemistry. With structural descriptions for both CFeSP/MeTr and CODH/ACS complexes, this work has illuminated the molecular details for metalloenzyme complex assembly and catalysis in the acetogenic Wood-Ljungdahl carbon fixation pathway.
by Yan Kung.
Ph.D.
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Murray, Jill Isobel. "A metalloenzyme model for the biotransformation of nitroglycerin to nitric oxide." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2002. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/MQ63344.pdf.
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Schweitzer, Dirk. "Biomimetic models of the active site of the metalloenzyme nitrile hydratase /." Thesis, Connect to this title online; UW restricted, 2001. http://hdl.handle.net/1773/8692.
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Neupane, Kosh Prasad. "Nickel superoxide dismutase insight into the metalloenzyme gained from functional metallopeptide models /." abstract and full text PDF (UNR users only), 2009. http://0-gateway.proquest.com.innopac.library.unr.edu/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3355593.
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Keppetipola, Niroshika. "Characterization of DNA and RNA end modifying enzymes and a triphosphate tunnel metalloenzyme /." Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1619359881&sid=5&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Saysell, Colin G. "Reactivity of the copper containing enzyme galactose oxidase." Thesis, University of Newcastle Upon Tyne, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307890.
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Benini, Stefano. "Structure and function relationships of urease and cytochrome c-553 from Bacillus pasteurii." Thesis, University of York, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325599.
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Huang, Qiongying. "In Vitro Study of Two Virulence Factors of Listeria monocytogenes: Cytolysin LLO and Metalloenzyme PC-PLC." Thesis, Boston College, 2014. http://hdl.handle.net/2345/bc-ir:103619.
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Thesis advisor: Mary F. RobertsThesis advisor: Jianmin Gao
The research reported in this thesis focused on three proteinaceous virulence factors of the intracellular bacterial pathogen Listeria monocytogenes: listeriolysin O (LLO), broad-range phospholipase C (PC-PLC), and phosphatidylinositol-specific phospholipase C (PI-PLC). Based on sequence homology of LLO with other cholesterol-dependent cytolysins (CDC), the protein has four domains of which domain 4 is thought to anchor the protein to cholesterol-containing surfaces while domain 3 mediates protein-protein binding on the membrane and contributes α-helices that convert to two β-strands that form the large β-barrel pore. It was previously assumed that the sequential and cooperative behaviors of domain 3 in each LLO monomer required D4 to bind to cholesterol-enriched membranes. By cloning and expressing a separate protein containing domains 1, 2, and 3 (D123) and the isolated domain 4 (D4) of LLO, I could uncouple some of the events in its membrane binding and pore-formation. Flow cytometry, used to investigate protein binding to vesicles and to red blood cells, showed that D123 had no membrane affinity on its own, but became membrane-bound when sub-lytic amounts of LLO were added. D123, not membrane-lytic by itself, became hemolytic when trace amounts of LLO were present to provide a membrane anchor for D123 proteins. FRET and fluorescence correlation spectroscopy were used to show that D123 and LLO formed oligomers at nanomolar concentration and could also associate with one another in the solution. These results suggest that D4 provides an initial membrane attachment but need not be present on all monomers to trigger the cooperative conformational change that leads to membrane insertion and pore formation. The gene for L. monocytogenes PC-PLC was obtained, expressed in E. coli and the product protein purified and characterized. The zinc content of this metalloenzyme was analyzed with ICP-MS. The dissociation constants of the three zinc ions proposed as necessary for PC-PLC activity ranged from 0.05 to 60 μM. Enzymatic activities of PC-PLC were analyzed for various substrates, include long-chain phospholipid in vesicles (LUVs, SUVs) and micelles (Triton X-100), and short-chain lipids (diC4PC, diC6PC, diC7PC) mono-dispersed in solutions. Key results include the following: (1) the L. monocytogenes PC-PLC has an acidic pH optimum (in contrast to other bacterial PC-PLC enzymes) consistent with its role in vacuole lysis upon acidification; (2) the preference of PC-PLC for longer chain monomeric substrates is not because of a higher kcat but a reduced Km suggesting some amount of hydrophobicity is important for substrate binding in the active site; (3) the apparent Kd of PC-PLC for Zn2+ derived from kinetics at pH 6.0 (1.94 ± 0.22 μM) is lower that that from ICP-MC; and (4) PC-PLC enzymatic activity is not enhanced by added LLO that generates pores in vesicles (likewise, PC-PLC does not affect the membrane lytic activity of LLO) indicating no synergism between the two virulence factors. These results should aid in understanding the function of PC-PLC in L. monocytogenes pathogenicity. The L. monocytogenes PI-PLC and a variant with reduced catalytic activity were expressed and are currently used in a collaborative project with the Portnoy laboratory at the University of California at Berkeley
Thesis (PhD) — Boston College, 2014
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Chemistry
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Akkaya, Engin Umut. "Synthesis and study of cyclodextrin-based metalloenzyme mimics and fluorescence probes of molecular recognition /." The Ohio State University, 1989. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487599963592456.
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Leurs, Melanie [Verfasser], Jörg C. [Akademischer Betreuer] Tiller, and Dieter [Gutachter] Vogt. "Artifizielle Metalloenzyme in organischen Medien / Melanie Leurs ; Gutachter: Dieter Vogt ; Betreuer: Jörg C. Tiller." Dortmund : Universitätsbibliothek Dortmund, 2019. http://d-nb.info/1204636478/34.
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Breece, Robert M. "Spectroscopic Characterization of Co(II)-Substituted VanX, a Zn(II)-Dependent Dipeptidase Required for High-Level Vancomycin Resistance." Miami University / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=miami1077576564.
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Di, Méo Thibault. "Ingénierie de l’architecture protéique artificielle αRep : élaboration de catalyseurs biohybrides par couplage covalent de complexes métalliques." Thesis, Université Paris-Saclay (ComUE), 2017. http://www.theses.fr/2017SACLS012/document.
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Le développement d’une nouvelle génération de catalyseurs dits biohybrides est basé sur l’association d’un complexe métallique et d’une protéine. D’un côté, le complexe métallique est responsable de l’activité catalytique ; de l’autre côté, la protéine protège le complexe métallique vis-à-vis de la dégradation en milieu aqueux et fournit également un environnement chiral propice à une catalyse énantiosélective. Ces catalyseurs fonctionnant de manière sélective en milieu aqueux s’inscrivent tout à fait dans les préceptes de la chimie verte.Une nouvelle famille de protéines artificielles, nommée αRep, a été récemment décrite. Toutes les protéines de la bibliothèque αRep présentent le même repliement en solénoïde incurvé, mais diffèrent à la fois en taille (nombre de motifs répétés) et dans la nature de 5 acides aminés par motif répété. Une surface variable est ainsi générée sur la surface concave du solénoïde. Ces protéines sont extrêmement stables et modifiables. La modularité de ces protéines ouvre la voie à un panel varié d’ingénierie des protéines, notamment la conception de catalyseurs artificiels.Au sein de la bibliothèque αRep, le variant αRep-A3 est une protéine homodimérique pour laquelle les surfaces concaves de chaque monomère génèrent une crevasse. Les résidus formant cette crevasse peuvent être modifiés sans affecter la structure tridimensionnelle de la protéine. Le but de cette thèse a été d’évaluer la capacité de la protéine αRep-A3 à procurer une architecture rigide pour l’incorporation de complexes de métaux de transition. Pour cela, différents ligands de métaux de transition (phénanthroline, terpyridine, porphyrine) ont été couplés covalemment à des variants de αRep-A3 à différentes positions. Des résultats encourageants concernant la réaction de Diels-Alder entre azachalcone et cyclopentadiène suggèrent que ce type d’architecture pourrait fournir une base intéressante pour la création de nouvelles classes de métalloenzymes entièrement artificielles. Des pistes pour l’amélioration des catalyseurs basés sur les αRep par des méthodes d’évolution dirigée sont alors avancées sur la base de ces résultatsThe development of a new generation of so-called biohybrid catalysts is based on the association of a metal complex and a protein. On the one hand, the metal complex is responsible for the catalytic activity; On the other hand, the protein protects the metal complex from degradation in aqueous medium and also provides a chiral environment conducive to enantioselective catalysis. These catalysts, which function selectively in an aqueous medium, fit perfectly into the precepts of green chemistry.A new family of artificial proteins, called αRep, has recently been described. All proteins in the αRep library exhibit the same curved solenoid folding, but differ in size (number of repeating units) and in the nature of 5 amino acids per repeat unit. A variable surface is thus generated on the concave surface of the solenoid. These proteins are extremely stable and modifiable. The modularity of these proteins paves the way for a varied panel of protein engineering, including the design of artificial catalysts.Within the αRep library, the variant αRep-A3 is a homodimeric protein for which the concave surfaces of each monomer generate a crevice. The residues forming this crevice can be modified without affecting the three-dimensional structure of the protein. The aim of this thesis has been to evaluate the ability of the αRep-A3 protein to provide a rigid scaffold for the incorporation of transition metal complexes. To this end, various transition metal ligands (phenanthroline, terpyridine, porphyrin) have been covalently coupled to variants of αRep-A3 at different positions. Encouraging results regarding the Diels-Alder reaction between azachalcone and cyclopentadiene suggest that this type of scaffold could provide an interesting basis for the creation of new classes of fully artificial metalloenzymes. From these results, lines of improvement for αRep-based catalysts by means of directed evolution are then advanced
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ARRIGONI, FEDERICA. "Chemistry and renewable energy: DFT investigation on CO2 reduction and H2 oxidation/production catalyzed by transition metal biomimics." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2017. http://hdl.handle.net/10281/153273.
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My PhD research has been devoted to the investigation of diiron dithiolate compounds, that are structurally and functionally related to [FeFe]-hydrogenase and nitrogenase active sites. DFT computations provided relevant insights for a better comprehension of the chemistry of these systems, at the same time posing new questions and hints for future prospects.
The core of the thesis is dedicated to [FeFe]-hydrogenase biomimics, whose structures and reactivity have been theoretically addressed by various standpoints. Several gaps concerning the activity of these systems still need to be filled. Indeed, besides catalyzing proton reduction at moderate rates (usually accompanied to high overpotentials), synthetic analogues of [FeFe]-hydrogenases are not reversible catalysts, i.e. they cannot oxidize dihydrogen catalytically (except very few examples).
Some of the most puzzling issues in the field of hydrogenase biomimic have been computationally addressed, such as the oxidative behavior of biomimic compounds, as well as their role in H2 binding and activation (one of the most problematic yet less explored topics in this research area).
Then, also the effect on the variation of Fe’s coordination sphere on H2 production catalysis has been investigated. In particular, we studied the catalytic behavior of an hexacarbonyl model compound, in which a highly protophilic bridge-head pendant has proved to play a crucial role in the hindering of bridging hydride formation, a well-known obstacle for catalysis.
An alternative strategy to the hindering of bridging hydride formation is increasing its reactivity towards H2 formation. This aspect has been theoretically unraveled, and key information, regarding the stereoelectronic ingredients necessary to achieve this reactivity, has been provided.
DFT has been also used to perform the rational design of a hydrogenase biomimic with the so called “rotated” conformation (usually achieved only transiently in biomimics), which is considered one of the most relevant active site features, necessary to entail a hydrogenase-like reactivity. The proposed system thus represents the first example of biomimic compound which is theoretically predicted to resemble the actual Fe’s coordination geometry of the natural system.
The last period of my PhD research has been instead devoted to the study of compounds featuring a Fe2S2 scaffold, proposed nitrogenase biomimics. The aim of the work was to verify if the substrate promiscuity shown by the enzyme active site e.g. towards CO2 activation, could also be preserved in biomimic compounds. Thus, we made a preliminary study regarding the role of Fe2S2 biomimics in CO2 activation and conversion. Our results provided some useful general information about theirreactivity towards CO2, as well as some clues to favor CO2 binding and activation.La mia ricerca di dottorato è stata focalizzata sull’indagine di composti Fe2S2, strutturalmente e funzionalmente correlati al sito attivo di [FeFe[-idrogenasi e nitrogenasi. I metodi computazionali , in particolare la Teoria del Funzionale della Densità (DFT), hanno permesso di ottenere informazioni utili riguardo al funzionamento di questi sistemi, in luce del loro impiego come catalizzarori per la produzione di bio fuels. Il cuore della tesi è costituito dallo studio delle [FeFe]-idrogenasi e dei loro complessi biomimetici, la cui struttura e reattività è stata investigata in dettaglio secondo differenti punti di vista. L’attività dei sistemi biomimetici (in contrasto a quello naturale) è affetta da parecchie problematiche, che li rende poco attivi e non reversibili. Infatti, essi catalizzano (con bassi turnover e alte sovratensioni) la reazione di produzione di H2 da protoni ed elettroni, ma sono incapaci (a parte poche eccezioni) di ossidarlo. Alcuni degli aspetti problematici relativi a questi sistemi sono stati quindi investigati per mezzo del DFT. Innanzitutto ne è stato indagato il comportamento ossidativo, la cui conoscenza e controllo sono fondamentali per poter performare efficientemente l’ossidazione di H2. Questa tematica, sebbene la più problematica in quest’ ambito di ricerca, è anche la meno investigata. In particolare si è scoperto che la natura del ponte ditiolato contenuto in questi sistemi è fondamentale per modulare le proprietà redox di questi sistemi, mediante la formazione di interazioni deboli formatesi durante il processo ossidativo. Tuttavia una buona conoscenza delle proprietà redox di questi sistemi non è sufficiente a garantire un buon funzionamento nei confronti dell’ossidazione di idrogeno. Per questo sono stati studiati i quattro step fondamentali del ciclo catalitico, sia prendendo in esame il sito attivo enzimatico sia i suoi biomimetici. Da una comparazione accurata dei risultati ottenute sono emerse differenze che evidenziano il ruolo chiave di alcuni fattori stereo-elettronici, mancanti nei sistemi sintetici. Per quanto riguarda la riduzione protonica invece, a dare H2, una delle cause principali della scarsa efficienza è la formazione di un idruro a ponte come intermedio di protonazione (estremamente stabile) nel ciclo catalitico, il quale non si forma invece nel sistema naturale. Il DFT ha permesso di evidenziare che l’aumento di protofilicità della funzione contenuta nel pendant ditiolato impedisce (rallenta) la formazione di questo intermedio stabile. Inoltre è stato sviluppato un protocollo per rendere questo intermedio (notoriamente poco reattivo) più reattivo rispetto alla sua controparte terminale. Una delle features più importanti del sito attivo è la cosiddetta conformazione “ruotata”, in cui uno dei due Fe espone un sito di coordinazione vacante per il substrato. Questa conformazione è stata ottenuta finora solo transientemente in sistemi biomimetici, per mezzo di trucchi sterici. Durante il mio progetto di dottorato abbiamo progettato razionalmente un sistema biomimetico che (in linea di principio) ha un ground state con conformazione ruotata. Infine, l’ultima parte di dottorato è stata dedicata allo studio delle nitrogenasi, le quali hanno mostrato una rilevante promiscuità di substrato, catalizzando anche la riduzione di CO2 a idrocarburi. Abbiamo quindi studiato l’attivazione di CO2 da parte di biomimetici del tipo F2S2 di nitrogenasi, per verificare se questa promiscuità sia mantenuta anche a livello sintetico.
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Xie, Juan. "Synthèse, étude biologique et pharmacologique de nouveaux inhibiteurs des enzymes de dégradation des enképhalines." Paris 5, 1988. http://www.theses.fr/1988PA05P617.
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Debela, Mekdes Haile Mariam. "Crystal structures of the human tissue kallikreins 4, 5, 7, 10, characterisation of their substrate specificity and analysis of their various zinc inhibition mechanisms." München Verl. Dr. Hut, 2007. http://d-nb.info/988422395/04.
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Saam, Jan. "Identification of dynamic oxygen access channels in 12/15-lipoxygenase." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2008. http://dx.doi.org/10.18452/15744.
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Zellen enthalten zahlreiche Enzyme, deren Reaktionen von molekularem Sauerstoff abhängen. Oft sind deren aktive Zentren tief im inneren des Proteins verborgen, was die Frage nach spezifischen Zugangskanälen, die den Sauerstoff gezielt zum Ort der Katalyse leiten, aufwirft. In der vorliegenden Arbeit wird dies am Beispiel der 12/15-Lipoxygenase, als ein typisches Beipiel Sauerstoff verbrauchender Enzyme, untersucht. Die Sauerstoffverteilung innerhalb des Proteins wurde bestimmt und mögliche Routen für den Sauerstoffzugang definiert. Zu diesem Zweck wurden theoretische Untersuchungen eng mit Experimenten verzahnt. Zuerst wurden Molekulardynamik Simulationen des Proteins in Lösung durchgeführt. Aus den Trajektorien konnte die dreidimensionale Verteilung der Freien Enthalpie für Sauerstoff berechnet werden. Die Analyse der günstigsten Pfade in dieser Energielandschaft führte zur Identifikation von vier Sauerstoffkanälen im Protein. Alle Kanäle verbinden die Proteinoberfläche mit einem Gebiet hoher Sauerstoffaffinität am aktiven Zentrum. Diese Region liegt bezüglich des Substrats gegenüber dem Eisenzentrum, wodurch eine strukturelle Erklärung für die Reaktionsspezifität des Enzyms gegeben ist. Der katalytisch bedeutsamste Weg des Sauerstoffs kann durch L367F Austauschmutation blockiert werden, was zu einer stark erhöhten Michaelis-Konstante für Sauerstoff führt. Diese experimentell nachgewiesene Blockade konnte, mit Hilfe entsprechender Molekulardynamik Simulationen, durch eine Umordnung eines Wasserstoffbrücken-Netzwerks von Wassermolekülen innerhalb des Protein im Detail erklärt werden. Die Ergebnisse ermöglichen den Schluss, dass die Hauptroute für Sauerstoff zum aktiven Zentrum des Enzyms einem Kanal folgt, der aus vorübergehend verbundenen Hohlräumen besteht. Hierbei unterliegt das Öffnen und Schließen des Kanals der Dynamik der Proteinseitenketten.Cells contain numerous enzymes utilizing molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. In the present thesis this question is investigated choosing 12/15-lipoxygenase as a typical example for such oxygen dependent enzymes. The oxygen distribution within the protein was determined and potential routes for oxygen access were defined. For this purpose an integrated strategy of structural modeling, molecular dynamics simulations, site directed mutagenesis, and kinetic measurements has been applied. First, molecular dynamics simulations of the protein in solution were performed. From the trajectories, the 3-dimensional free-energy distribution for oxygen could be computed. Analyzing energetically favorable paths in the free-energy map led to identification of four oxygen channels in the protein. All channels connect the protein surface with a zone of high oxygen affinity at the active site. This region is localized opposite to the non-heme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange which leads to a strongly increased Michaelis constant for oxygen. This experimetally proven blocking mechanism can, by virtue of molecular dynamics studies, be explained in detail through a reordering of the hydrogen bonding network of water molecules. As a conclusion, the results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure is governed by sidechain dynamics.
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Nakatani, Naoki. "Theoretical Studies of Photoproteins and Non-Heme Iron Enzymes: Electronic Structures and Reaction Processes." 京都大学 (Kyoto University), 2010. http://hdl.handle.net/2433/120885.
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Krystof, Monika [Verfasser], Jürgen [Verfasser] Klankermayer, and Walter [Akademischer Betreuer] Leitner. "Artifizielle Metalloenzyme und heterogenisierte Übergangsmetall-Komplexe mit Triphos-Liganden : Synthese, Kupplungsstrategien und katalytische Aktivität / Monika Krystof, Jürgen Klankermayer ; Betreuer: Walter Leitner." Aachen : Universitätsbibliothek der RWTH Aachen, 2016. http://d-nb.info/1130402843/34.
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Urich, Tim. "The sulfur oxygenase reductase from Acidianus ambivalens." Phd thesis, [S.l.] : [s.n.], 2005. https://tuprints.ulb.tu-darmstadt.de/615/1/urich_diss.pdf.
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The microbial oxidation of reduced inorganic sulfur compounds and elemental sulfur to sulfate is one of the major reactions in the global sulfur cycle. Despite its importance, only limited information is available about molecular details of the enzymes involved. The present work was aimed to contribute to the understanding of the underlying molecular mechanisms by investigating the function and structure of the sulfur oxygenase reductase (SOR) from the thermoacidophilic crenarchaeote Acidianus ambivalens. The expression of the sor gene in Escherichia coli resulted in active, soluble SOR and in inclusion bodies from which active SOR could be refolded as long as ferrous ions were present in the refolding solution. The wild type and recombinant SOR preparations possessed indistinguishable properties when analyzed for activity and by gel permeation chromatography, CD spectroscopy and electron microscopy. The analysis of the quaternary structure showed a multi-subunit shell-like assembly with a central hollow core. The subunits formed homodimers as the building blocks of the holoenzyme, as shown by denaturation experiments. Conformational stability studies showed that the apparent unfolding free energy in water was ~5 kcal mol-1, at pH 7. Iron was found in the wild type enzyme at a stoichiometry of one iron per subunit. EPR spectroscopy of the colorless SOR resulted in a single isotropic signal at g = 4.3 characteristic of high-spin ferric iron. The signal disappeared upon reduction with dithionite or incubation with sulfur at elevated temperature. The iron center had a reduction potential of E0´ = -268 mV at pH 6.5. Protein database inspection identified five SOR protein homologues which allowed the prediction of amino acids putatively involved in catalysis. The recombinant SOR was crystallized by the sitting drop vapor diffusion method. The crystal structure was determined at 1.7 Å resolution. The homo-icosatetrameric holoenzyme was a highly symmetrical hollow protein particle with 432 point group symmetry and a molecular mass of 871 kDa. The subunits were αβ-proteins and comprised a central β-barrel surrounded by α-helices. Each monomer contained one mononuclear non-heme iron site with the ligands H85, H89, E113 and two water molecules in an octahedral arrangement. The protein ligands formed a 2-His-1-carboxylate facial triad for iron binding. The cysteines C30, C100 and C103 were in the vicinity of the iron site and located along the same cavity within the interior of the subunit, therefore defining the enzyme´s active site. C30 was persulfurated. The 24 active sites were spacially separated from each other, making an electronic interaction during catalysis unlikely. They were accessible solely via the inner compartment. Access of substrate to the inner compartment is most probably provided by six hydrophobic channels along the four-fold symmetry axes of the particle. Furthermore, the structure suggested that a linear polysufide species and not the cyclic α-S8 is the substrate of the SOR. Crystal structures of the SOR in complex with the inhibitors p-hydroxy-mercury-benzoic acid and iodoacetamide identified the cysteines as the inhibitor binding sites. The iron-binding residues H85, H89 and E113 and the three cysteines C30, C100 and C103 were altered by site-directed mutagenesis and the mutant proteins were analyzed for activity and iron content. Mutations of the iron ligands and C30 resulted in inactive enzyme, whereas mutations of C100 and C103 resulted in a reduced activity. All mutations affected the oxygenase and reductase partial reactions to a similar degree. These analyses allowed the first detailed insight into the mode of action of this self-compartmentalizing metalloenzyme. C30 is most probably the sulfur binding residue which aligns the substrate for the initial oxygenation catalyzed by the Fe site. The role of C100 and C103 is not clear, but they might act in the subsequent sulfur disproportionation reaction. The comparison of the SOR with SoxAX, the only other sulfur compound oxidizing enzyme from prokaryotes for which a high resolution structure is available, showed no structural similarity. SoxAX and other sulfur oxidizing enzymes contain different cofactors, demonstrating the diversity of mechanistic approaches utilized for sulfur compound oxidation. In contrast, a basic functional principle seems to be the central role of cysteine residues, acting as covalent binding sites for the substrate.
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David, Rolf. "Chemins de protonation et réactivité des métalloenzymes : application à la superoxide réductase." Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV087/document.
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L’obtention, dans des conditions douces, sélectives et de chimie durable de molécules ciblées est aujourd’hui un enjeu majeur. Les métalloenzymesartificielles représentent une voie d’investigation importante, car en jouant, par exemple, sur la seconde sphère de coordination,il est possible de modifier fortement la réactivité de ces systèmes bio-inspirés. Le développement de cette chimie suppose une connaissanceapprofondie des différentes étapes du mécanisme de la réaction envisagée. Pour cela, la chimie théorique est essentielle à la rationalisation dela réactivité chimique mais elle souffre encore de nombreuses insuffisances pour les systèmes que nous nous proposons d’étudier.Dans ce travail, nous avons choisi d’étudier la superoxyde réductase, enzyme détoxifiante du radical superoxyde. Si de nombreuses expérimentalessont disponibles détaillant certains intermédiaires, le mécanisme précis est peu documenté. Le but a été de mettre en place uneméthodologie complète allant du développement de paramètres MM spécifiques à l’étude de la réactivité par métadynamiques QM/MM.Le développement de paramètres MM pour le site actif à fer a permis son étude en dynamique MM donnant des informations sur la conformationsdu squelette peptidique ainsi que l’interaction avec les molécules de solvant. De part la nature du fer, une description QM du site actifà été nécessaire via l’utilisation de DFT hybride. Les métadynamiques QM/MM ont permis quant à elles d’explorer les chemins réactionnelset de caractériser les espèces ainsi formées et les énergies d’activations.Cette méthodologie a permis la compréhension en premier lieu de la réactivité native de la forme sauvage et elle a aussi permis d’explorer lesréactivités nouvelles des mutations de la SOR permettant ainsi de définir le rôle crucial de la seconde sphère de coordinationObtaining targeted molecules under gentle, selective and sustainable conditions is still a major challenge. Artificial metalloenzymes are animportant line of enquiry, because by playing, for example, with the second sphere of coordination, it is possible to strongly modify thereactivity of these bio-inspired systems. The development of this chemistry presupposes a thorough knowledge of the different stages of themechanism of the reaction under study. For this reason, theoretical chemistry is essential to rationalize chemical reactivity, but it still suffersfrom many shortcomings for the systems we propose to study.In this work, we study the superoxide reductase, a detoxifying enzyme of the superoxide radical. While many experiments are available detailingsome intermediates, the precise mechanism is not well documented. The aim was to implement a complete methodology ranging from thedevelopment of specific MM parameters to the study of reactivity by QM/MM metadynamics.The development of MM parameters for the iron active site allowed its study by MM dynamics giving informations on the conformation ofthe peptide backbone as well as on the interaction with solvent molecules. Due to the nature of the iron, a QM description of the active sitewas required using hybrid DFT. QM/MM metadynamics have allowed us to explore reaction pathways and to characterize the compoundsformed to obtain the needed activation energies. This methodology made it possible to understand the native reactivity of the wild form ofthe SOR, but also to explore the new reactivity of the mutations of the SOR and thus to define the crucial role of the second coordination sphere
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Inoue, Takahiro. "Genetic engineering studies of Ni-carbon monoxide dehydrogenase from a thermophilic carboxydotrophic bacterium." Kyoto University, 2014. http://hdl.handle.net/2433/188777.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第18339号
農博第2064号
新制||農||1023(附属図書館)
学位論文||H26||N4846(農学部図書室)
31197
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 左子 芳彦, 教授 澤山 茂樹, 教授 菅原 達也
学位規則第4条第1項該当
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Gu, Jiesi. "New applications of immobilised metal ion affinity chromatography for pharmaceutics processing and drug discovery." Thesis, The University of Sydney, 2016. http://hdl.handle.net/2123/16722.
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The development of more efficient extraction methods is the key to improving access to natural products with therapeutic uses. Immobilised metal affinity chromatography (IMAC) has conventionally been used to purify proteins from biological mixtures. This thesis aimed to expand the applications of IMAC beyond protein purification by developing two new innovations of IMAC. First, multi-dimensional IMAC (MD-IMAC) was developed for the simultaneous purification of multiple non-protein-based bacterial secondary metabolites from a single culture. The MD-IMAC system, comprised of two in-series IMAC resins charged with different metal ions, was designed to exploit differences between the coordination chemistries of different compounds. A system was optimised for two clinical metabolites, desferrioxamine B (DFOB) and bleomycin (BLM). Retention of compounds was dependent on the metal ion used to charge the resin, the immobilised chelating ligand denticity, and column order. The technique was used successfully to resolve native DFOB and BLM from a culture of Streptomyces verticillus. Second, the utility of IMAC as a method of screening natural sources for metalloenzyme inhibitors was examined. Three microbial sources (Penicillium citreonigrum, Streptomyces incarnatus, Salinispora tropica) were processed on Ni(II)-, Cu(II), and Zn(II)-IMAC, followed by assay screens for inhibition against histone deacetylases (HDACs), 5-lipoxygenase and tyrosinase. Proof-of concept was established, with a putative HDAC inhibitor ([M + H+]+, m/z = 827.2800) identified from S. incarnatus. More material and further analyses are required to structurally characterise the bioactive species. This study is the first to demonstrate these applications of IMAC. As IMAC is an aqueous-compatible, green technology, MD-IMAC could be used to expedite pharmaceutics processing and reduce reliance on organic solvents. IMAC could also provide a new way of screening natural sources for metalloenzyme inhibitors.
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Pabon, Sanclemente Miguel Alejandro. "A Comparative Study of the Structural Features and Kinetic Properties of the MoFe and VFe Proteins from Azotobacter Vinelandii." DigitalCommons@USU, 2009. https://digitalcommons.usu.edu/etd/233.
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Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown. By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, could account for the differences in catalytic properties depicted by these enzymes. The quaternary structure of the dinitrogenases also differs. Such component in the Mo-nitrogenase is an α2β2 tetramer while for the V- an Fe-only nitrogenase is an α2β2δ2 hexamer. The latter enzymes are unable to reduce N2 in the absence of a functional δ subunit, yet they reduce H+ and the non-physiological substrate C2H2. Therefore, the δ subunit is essential for V- and Fe-only dependent nitrogen fixation by a mechanism that still remains unknown. In attempt to understand why the δ subunit is essential for V-dependent N2 reduction from a structural stand point, this work presents the strategy followed to clone the vnfG gene and purify its expression product, the δ subunit. The purified protein was subjected to crystallization trials and used to stabilize a histidine-tagged VFe protein that would otherwise purify with low Fe2+ content and poor H+ and C2H2 reduction activities. The VFe preparation was used to conduct substrate reduction assays to assess: i) The electron allocation patterns to each of the reduction products of the substrates C2H2, N2, N2H4, and N3−; and ii) Inhibition patterns among substrate and inhibitor of the nitrogenase reaction. This work also reports on the effect N2H4 and N3− has on the electron flux to the products of the C2H2 reduction. The work presented herein provides information with which to compare and contrast biological nitrogen fixation as catalyzed by the Mo- and V-nitrogenases from Azotobacter vinelandii.
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Blanchard, Vincent. "Synthèse de composés phosphorés chélatants à visée phytosanitaire." Thesis, Montpellier, Ecole nationale supérieure de chimie, 2015. http://www.theses.fr/2015ENCM0020/document.
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La voie de synthèse des acides aminés ramifiés est présente chez les plantes, les bactéries et les champignons mais est absente chez les mammifères. C’est pourquoi il est intéressant de cibler cette voie métabolique par des inhibiteurs spécifiques des enzymes qui la compose afin de développer des herbicides non sélectifs. Une famille d’herbicide déjà commercialisée agit sur la première enzyme de cette chaine enzymatique. Néanmoins des cas de mauvaises herbes résistantes à ces composés sont apparus et leur incidence continue d’augmenter. Il apparait donc important de cibler une autre enzyme de la voie de synthèse de ces acides aminés ramifiés afin de contourner ce problème de résistance.L’enzyme cétoacide réductoisomérase (KARI) intervient également dans la voie métabolique citée précédemment. Deux inhibiteurs IpOHA et Hoe 704 ont été développés dans les années 1980, mais malheureusement n’ont pas montré d’activité lors de traitements en plein champ. Cependant ils restent tous les deux, les références en tant qu’inhibiteurs in vitro.L’analyse de la diffraction par rayons X de KARI cristallisée avec un des inhibiteurs ou son substrat naturel montre que les groupements fonctionnels portés par ces derniers viennent complexer deux cations métalliques au sein du site actif. De plus la comparaison des structures des inhibiteurs ou des états de transition du substrat permet de dégager une structure générale pour le développement de nouvelles molécules potentiellement actives. Ainsi trois sous-structures doivent être présentes avec une double pince complexante, un groupement lipophile et un groupement accepteur de liaison hydrogène.Dans ce contexte, différents groupements comme des oxydes de diorganylphosphines, des dihydropyrimidinediones, des dérivés d’acides carboxyliques ou hydroxamiques peuvent être modifiés et utilisés comme motifs complexants. Les fonctions et groupements fonctionnels cités ont déjà prouvé leur efficacité lors du développement d’autres inhibiteurs de métalloenzymesThe branched-chain amino acids metabolic pathway is present in many living beings such as plants, bacteria and fungi but not in mammals. This is why it has been interesting to target this enzymatic pathway with specific inhibitors in order to develop non-selective herbicides. The main non-selective herbicides commercialised inhibit the first enzyme of this metabolic route. However more and more cases of resistant weed appeared and spread. Thus it raises the interest and importance of designing new compounds targeting another enzyme from the biological pathway in order to circumvent the resistance issue.The Ketolacid Reductoisomerase (KARI) protein also intervenes in the aforementioned enzymatic pathway. Although two inhibitors, IpOHA and Hoe 704, were synthesised during the 80s they have not proven active enough in field treatment. Nonetheless both inhibitors remain as references for in vivo biological activity.The X-ray diffraction representations of KARI including each inhibitor or its natural substrate show that the functional groups borne by the latter chelate two metal cations within the active site. Moreover the comparison between inhibitor structures and the substrate transition states reveals a general pattern in order to design and develop new potential biologically active compounds. For that purpose three major substructure units have to be considered: a double chelating pincer, a lipophilic group and a hydrogen bond accepting moiety.In this context different functional groups such as diorganylphosphine oxides, dihydropyrimidinediones, carboxylic acid or hydroxamic acid derivatives could be modified and used as chelating motifs. The functional groups listed have already proven their efficacy as part of other metalloenzyme inhibitors
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McMillen, Lyle, and l. mcmillen@sct gu edu au. "Isolation and Characterisation of the 5'-Nucleotidase from Escherichia coli." Griffith University. School of Biomolecular and Biomedical Science, 2001. http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20030226.153545.
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Escherichia coli 5'-nucleotidase is a periplasmically localised enzyme capable of hydrolysing a broad range of substrates, including all 5'-ribo- and 5'-deoxyribonucleotides, uridine diphosphate sugars, and a number of synthetic substrates such as bis (r-nitrophenyl) phosphate. The enzyme has been shown to contain at least one zinc ion following purification, and to have two metal binding sites in the catalytic cleft. 5'-Nucleotidase activity is significantly stimulated by the addition of particular divalent metal ions, most notably cobalt which results in a 30-50 fold increase in activity. Significant sequence homology between the E. coli 5'-nucleotidase and members of the Ser/Thr protein phosphatase family in the catalytic site has lead to 5'-nucleotidase being included in this protein family. This thesis describes the development of a rapid purification methodology for milligram quantities of 5'-nucleotidase, and the investigation of a number of physical and biochemical properties of the enzyme with the aim of comparing these properties to those of certain catalytic site mutants. The molecular weight of the mature protein was estimated as 58219 daltons, with a specific activity for 5'-AMP, in the presence of 4 mM Co2+ and 13 mM Ca2+ at pH 6.0, of 730 mmol/min/mg. The presence of up to two zinc ions associated with the purified enzyme was observed using ICP-ES analysis, suggesting both metal ion binding sites are occupied by zinc in vivo, and some degree of displacement of zinc by cobalt could be observed. Mass spectrometry data, gathered at 60 and 70 mS orifice potential, suggested the presence of a small proportion of material with a mass 118 to 130 daltons greater than the main 5'-nucleotidase mass estimation. This study suggests that this mass difference, only evident at the lower orificepotential, is due to the presence of two zinc ions closely associated with 5'-nucleotidase. To account for the observed high level of activation of 5'-nucleotidase activity by particular divalent metal ions, this thesis describes a proposed model in which these divalent ions may displace the zinc ion at one of the metal ion binding sites. This displacement only occurs at one of the two metal ion binding sites, with the other metal binding site retaining the zinc ion already present. Studies with purified enzyme, each with a single amino acid substitution, lend support to this hypothesis and suggest the identity of the metal ion binding site at which displacement occurs. Seven key catalytic site residues (Asp-41, His-43, Asp-84, His-117, Glu-118, His-217 and His-252) were selected on the basis of sequence conservation within the Ser/Thr protein phosphatases and 5'-nucleotidases. X-ray crystallographic data published by others during this study implicated five of the selected residues (Asp-41, His-43, Asp-84, His-217 and His-252) directly in metal ion binding, including two residues from each metal ion binding site and one directly involved in both sites (Asp-84). The remaining two residues (His-117 and Glu-118) are highly conserved but were not thought to play direct roles in metal ion binding. The seven selected residues were modified by site-directed mutagenesis, and the effect of the amino acid substitutions upon the kinetic properties of 5'-nucleotidase activity was determined. Residues hypothesised to be involved in metal ion displacement, and subsequent activation of 5'-nucleotidase activity, were identified by reductions in metal ion affinity and increased levels of activation by cobalt compared to the wild type 5'-nucleotidase. This study suggests that the metal binding site, M2, that includes residues Asp-84, His-217 and His-252, is involved in metal ion displacement, while the other metal binding site, M1, is not. This, in turn, suggests the metal binding sites are functionally non-equivalent and kinetically distinct. No residues were identified in this study as playing significant roles in substrate binding, as there was no significant reduction observed in affinity for 5'-AMP observed in any of the catalytic site mutants.
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Commeinhes, Frédéric. "Structure tridimentionnelle de la carboxypeptidase A par diffraction des rayons X." Paris 5, 1994. http://www.theses.fr/1994PA05P019.
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Pauff, James Michael. "Structure-Function Studies of Xanthine Oxidoreductase." The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1227480976.
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Manesis, Anastasia C. "Bioorganometallic Chemistry within Nickel-Substituted Azurin: From Protein Design to Reactivity." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1542735809462775.
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McMillen, Lyle. "Isolation and Characterisation of the 5'-Nucleotidase from Escherichia coli." Thesis, Griffith University, 2001. http://hdl.handle.net/10072/366487.
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Escherichia coli 5'-nucleotidase is a periplasmically localised enzyme capable of hydrolysing a broad range of substrates, including all 5'-ribo- and 5'-deoxyribonucleotides, uridine diphosphate sugars, and a number of synthetic substrates such as bis (r-nitrophenyl) phosphate. The enzyme has been shown to contain at least one zinc ion following purification, and to have two metal binding sites in the catalytic cleft. 5'-Nucleotidase activity is significantly stimulated by the addition of particular divalent metal ions, most notably cobalt which results in a 30-50 fold increase in activity. Significant sequence homology between the E. coli 5'-nucleotidase and members of the Ser/Thr protein phosphatase family in the catalytic site has lead to 5'-nucleotidase being included in this protein family. This thesis describes the development of a rapid purification methodology for milligram quantities of 5'-nucleotidase, and the investigation of a number of physical and biochemical properties of the enzyme with the aim of comparing these properties to those of certain catalytic site mutants. The molecular weight of the mature protein was estimated as 58219 daltons, with a specific activity for 5'-AMP, in the presence of 4 mM Co2+ and 13 mM Ca2+ at pH 6.0, of 730 mmol/min/mg. The presence of up to two zinc ions associated with the purified enzyme was observed using ICP-ES analysis, suggesting both metal ion binding sites are occupied by zinc in vivo, and some degree of displacement of zinc by cobalt could be observed. Mass spectrometry data, gathered at 60 and 70 mS orifice potential, suggested the presence of a small proportion of material with a mass 118 to 130 daltons greater than the main 5'-nucleotidase mass estimation. This study suggests that this mass difference, only evident at the lower orificepotential, is due to the presence of two zinc ions closely associated with 5'-nucleotidase. To account for the observed high level of activation of 5'-nucleotidase activity by particular divalent metal ions, this thesis describes a proposed model in which these divalent ions may displace the zinc ion at one of the metal ion binding sites. This displacement only occurs at one of the two metal ion binding sites, with the other metal binding site retaining the zinc ion already present. Studies with purified enzyme, each with a single amino acid substitution, lend support to this hypothesis and suggest the identity of the metal ion binding site at which displacement occurs. Seven key catalytic site residues (Asp-41, His-43, Asp-84, His-117, Glu-118, His-217 and His-252) were selected on the basis of sequence conservation within the Ser/Thr protein phosphatases and 5'-nucleotidases. X-ray crystallographic data published by others during this study implicated five of the selected residues (Asp-41, His-43, Asp-84, His-217 and His-252) directly in metal ion binding, including two residues from each metal ion binding site and one directly involved in both sites (Asp-84). The remaining two residues (His-117 and Glu-118) are highly conserved but were not thought to play direct roles in metal ion binding. The seven selected residues were modified by site-directed mutagenesis, and the effect of the amino acid substitutions upon the kinetic properties of 5'-nucleotidase activity was determined. Residues hypothesised to be involved in metal ion displacement, and subsequent activation of 5'-nucleotidase activity, were identified by reductions in metal ion affinity and increased levels of activation by cobalt compared to the wild type 5'-nucleotidase. This study suggests that the metal binding site, M2, that includes residues Asp-84, His-217 and His-252, is involved in metal ion displacement, while the other metal binding site, M1, is not. This, in turn, suggests the metal binding sites are functionally non-equivalent and kinetically distinct. No residues were identified in this study as playing significant roles in substrate binding, as there was no significant reduction observed in affinity for 5'-AMP observed in any of the catalytic site mutants.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Science, Environment, Engineering and Technology
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Rondot, Laurianne. "Design de nouvelles métalloenzymes artificielles." Thesis, Université Grenoble Alpes (ComUE), 2016. http://www.theses.fr/2016GREAV056/document.
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Dans l’industrie chimique, de nombreux composés organiques sont issus d’étapes d’oxydation, pouvant être énantiosélectives et mettant en jeux des conditions dangereuses et polluantes, comme par exemple l’emploi du tétraoxide d’osmium comme oxydant. Dans un souci de respect de l’environnement, il est alors nécessaire de repenser les procédés de synthèse vers un développement de la chimie verte et durable.Dans cet objectif, mes travaux de thèse consistent à développer des nouveaux catalyseurs d’oxydation de molécules organiques en rassemblant les mondes de la catalyse inorganique et de la biocatalyse par la conception de Métalloenzymes artificielles. Ces hybrides catalytiques développés en ancrant un complexe inorganique au sein d’une protéine hôte permettent ainsi des catalyses d’oxydation de façon douce et propre. Premièrement, nous avons développé et caractérisé une métalloenzyme artificielle à centre ruthénium (II) scorpionnate ancrée dans la protéine bactérienne NikA. Nous nous sommes ensuite intéressés à sa réactivité en oxydation asymétrique d’alcène en milieu aqueux en présence de diacétate d’iodobenzène. Ceci a permis de mettre en évidence une activité singulière de l’hybride par la formation de de produit d’oxydation de type chlorhydrine. Enfin, l’énantiosélèctivité de cette activité catalytique a été étudiée en fonction d’un panel de substrat et de l’influence de la cavité protéique.Dans un second temps mes travaux de thèses ont consisté à concevoir et caractérisé une nouvelle oxygénase artificielle FeLn (III)-NikA. La seconde étape fut ensuite de vérifier la capacité de cet hybride catalytique à activer l’oxygène moléculaire en présence de réducteur, Puis d’étudier son aptitude à le transférer à un substrat exogène en condition de catalyse d’oxydation d’alcène aromatique. En parallèle, nous avons développé un système de réduction photocatalytique associé à cette nouvelle métalloenzyme artificielle sous apport de source lumineuse bleue et en présence de photosensibilisateur (chlorure de ruthénium (II) ) couplé à un donneur d’électron sacrificiel (triéthanolamine)Dans l’industrie chimique, de nombreux composés organiques sont issus d’étapes d’oxydation, pouvant être énantiosélectives et mettant en jeux des conditions dangereuses et polluantes, comme par exemple l’emploi du tétraoxide d’osmium comme oxydant. Dans un souci de respect de l’environnement, il est alors nécessaire de repenser les procédés de synthèse vers un développement de la chimie verte et durable.Dans cet objectif, mes travaux de thèse consistent à développer des nouveaux catalyseurs d’oxydation de molécules organiques en rassemblant les mondes de la catalyse inorganique et de la biocatalyse par la conception de Métalloenzymes artificielles. Ces hybrides catalytiques développés en ancrant un complexe inorganique au sein d’une protéine hôte permettent ainsi des catalyses d’oxydation de façon douce et propre. Premièrement, nous avons développé et caractérisé une métalloenzyme artificielle à centre ruthénium (II) scorpionnate ancrée dans la protéine bactérienne NikA. Nous nous sommes ensuite intéressés à sa réactivité en oxydation asymétrique d’alcène en milieu aqueux en présence de diacétate d’iodobenzène. Ceci a permis de mettre en évidence une activité singulière de l’hybride par la formation de de produit d’oxydation de type chlorhydrine. Enfin, l’énantiosélèctivité de cette activité catalytique a été étudiée en fonction d’un panel de substrat et de l’influence de la cavité protéique.Dans un second temps mes travaux de thèses ont consisté à concevoir et caractérisé une nouvelle oxygénase artificielle FeLn (III)-NikA. La seconde étape fut ensuite de vérifier la capacité de cet hybride catalytique à activer l’oxygène moléculaire en présence de réducteur, Puis d’étudier son aptitude à le transférer à un substrat exogène en condition de catalyse d’oxydation d’alcène aromatique. En parallèle, nous avons développé un système de réduction photocatalytique associé à cette nouvelle métalloenzyme artificielle sous apport de source lumineuse bleue et en présence de photosensibilisateur (chlorure de ruthénium (II) ) couplé à un donneur d’électron sacrificiel (triéthanolamine)
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Lator, Alexis. "Métalloenzymes artificielles et nouvelles réactivitées des complexes de fer." Thesis, Normandie, 2018. http://www.theses.fr/2018NORMC256.
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Les nouveaux aspects économiques et écologiques de la chimie moderne (à savoir la gestion des déchets, l’économie d’atome) font de la réduction de liaisons polarisées C=X (X = O, N) catalysée par des complexes organométalliques, une réaction importante en chimie organique. De plus, ces complexes permettent le contrôle de la stéréo-, régio- et chimiosélectivité de ces réactions. Les complexes de fer tricarbonyle à ligand cyclopentadiènone ont montré ces dernières années une excellente réactivité pour les hydrogénations de liaisons C=O et C=N. Grâce à de précédents travaux réalisés au laboratoire, nous avons envisagé l’approche de ces réductions polarisées via l’utilisation de métalloenzymes artificielles incorporant un complexe de fer cyclopentadiènone. Parallèlement à cette étude, une optimisation des performances catalytiques de complexes de fer a été réalisée et a permis de développer l’alkylation de cétones par les alcools, la méthylation d’amines par le méthanol et la réduction de liaisons C=C pour les cétones ,-insaturées. Enfin, grâce à un nouveau complexe aminocyclopentadiènyle de fer, les réactions d’amination réductrice étudiées précédemment au laboratoire ont pu être amélioréesEnvironmental and economic concerns within modern chemistry (waste management, atom economy), lead metal-catalyzed reduction of polarized C=X bonds (X= O, N) with organometallic complexes an important reaction in organic chemistry. Additionally, stereo-, regio- and chemoselective reductions can be involved in organometallic catalysis. In the past decade, cyclopentadienone iron tricarbonyl complexes were reported as efficient for the hydrogenation of C=O and C=N bonds. According to previous investigations in the group, we developed a new approach within artificial metalloenzyme involving iron complexes for the hydrogenation of polarized bonds. Simultaneously, we described new reactivities of the complex prepared in the group for ketone alkylation and amine methylation through auto-hydrogen borrowing process, and chemoselctive C=C bonds of enones. We developed as well a new aminocyclopentadienyl iron complex for the enhancement of reductive amination previously described
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Myers, Andrew Ross. "Cloning, Expression, and Sequence Analysis of Camelysin, a Zinc Metalloprotease from Bacillus anthracis and B. cereus." [Tampa, Fla.] : University of South Florida, 2005. http://purl.fcla.edu/fcla/etd/SFE0001218.
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Mei, Xiaonan. "HOW A SILENT MUTATION SUPPRESSES THE ACTIVITY AND IRON INCORPORATION IN SUPEROXIDE DISMUTASE." UKnowledge, 2012. http://uknowledge.uky.edu/chemistry_etds/9.
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A mutation (CTG to TTG) of FeSOD gene was found in Escherichia coli. Since they both encode leucine, it is a silent mutation. Site-‐directed mutagenesis was applied to correct the mutation, and the mutant FeSOD (before gene correction) and wild type FeSOD (after gene correction) were purified. The FeSODs from the two genes were Characterized using different assays and spectroscopic methods including EPR and CD. The requirement for the rare codon TTG may result in slowed translation and heavy demand on a scarce tRNA. Cultures expressing wild type FeSOD are better able to grow for long times after addition of IPTG and more mature to incorporate Fe atoms to the active sites than are cultures expressing the mutant gene. Moreover, the wild type FeSOD has more activity than the mutant. To our knowledge, this is the first time that a silent mutation has been demonstrated to affect metal incorporation into a metalloenzyme.
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Nedonchelle, Elsa. "Les anticorps catalytiques : des outils pour la production et l'étude des anticorps catalytiques semi-synthétiques et auto-immuns." Compiègne, 2000. http://www.theses.fr/2000COMP1320.
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Les anticorps catalytiques associent les propriétés de reconnaissance des anticorps aux propriétés de catalyse des enzymes. Différentes approches ont été envisagées pour leur faire mimer les enzymes : analogues d'états de transition, copie du site actif par le réseau idiotypique, ingénierie des protéines. De nombreuses enzymes s'associent avec des métaux pour assurer leurs fonctions. Le zinc est un élément intéressant dans ce cas car retrouvé dans plus de 300 enzymes. Ces sites de fixation étant bien caractérisés, ils ont pu semir de modèle en biotechnologie dans l'ingénierie des protéines. L'approche que nous proposons est basée sur la biosynthèse de nove du site de fixation du zinc catalique. Pour cela, nous nous appuyons sur les règles de reconnaissance de l'IDA-Zn (Il) par les protéines (IMAC). L'immunisation d'un animal contre l'IDA-Zn(ll) devrait produire des anticorps présentant au niveau de leur paratope une structure de ligands capable de fixer un ion métallique dans une conformation catalytique. Pour développer ce type d'anticorps, une nouvelle méthode ELISA a d'abord été développée. L'utilisation d'un présentateur dIhaptène non protéique (le PEG bifonctionalisé) nous permet d'éviter toute réaction croisée avec la protéine de trans ort utilisée pour l'immunisation. Les anticorps anti-IDA-Zn(ll) ont ensuite été réalisés selon deux tech¬niques. D'abord la méthode des hybridomes nous a permis d'isoler à partir de 1152 clones 14 clones présentant de bonnes affinités. Ensuite, le répertoire immunologique de la souris immunisée contre l'IDA-Zn(ll) a été exprimé en banque de phages (banque de scFv). L'avantage d'une telle banque réside dans l'expression de tout le répertoire immunologique de la souris immunisée, ce qui offre la possibilité de "screener" des spécificités plus vastes vis-à-vis d'autres métaux, chélates métalliques ou d'autres antigènes. Enfin, la production de ces anticorps anti-chélate métallique à grande échelle nous a fait apparaître la nécessité de développer une méthode de purification douce, respectueuse de la structure tridimensionnelle des anticorps. Une méthode, faisant appel au ligand pseudobiospécifique l'histidine, a été développée au laboratoire. Pour valider son utilisation aux anticorps catalytiques, nous avons décidé de l'appliquer à la purification des anticorps catalytiques naturels présents dans le sérum des malades afteints de maladies auto-immunes. Les résultats sont comparés avec les méthodes classiques protéine A et protéine G, en termes de pureté des fractions et d'activité catalytique.
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TIBERTI, MATTEO. "Computational investigation of structure-function relationship in metalloenzymes." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2014. http://hdl.handle.net/10281/53873.
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Important processes such as carbon dioxide capture, hydrogen evolution and oxidation as well as bioremediation are of paramount importance for the development of an environmental-friendly economy. In nature, the reactions underlying these processes are naturally performed by metalloenzymes.
The understanding of how they work will ease the development of important technological and biotechnological applications, such as bioinspired inorganic catalysts or more effective enzymes. In the present work, we have used computational methods to study structure-function relationships in metalloenzymes which catalyze important reactions involved in environment-friendly processes. In particular, we selected three cases of study; the formate dehydrogenase (FDH) from Escherichia coli, which catalyzes the conversion between carbon dioxide and formate, the [NiFe]-hydrogenase from Allochromatium vinosum, which performs hydrogen oxidation, and the phosphotriesterase (PTE) from Agrobacterium radiobacter that is able to hydrolyse organophosphates. Since the study of structure-function relationship in enzymes generally requires both a
view on the reaction mechanism and on the functional dynamics, we had to employ different levels of theory. At first, we focused on the study of metallo-enzymes for which the catalytic mechanisms still have to be clarified in details, as FDH and a [NiFe]-hydrogenase. In particular, we studied the oxidation of formate to carbon dioxide at the Mo-binding cofactor in the active site of FDH, using Density Functional Theory (DFT).
We employed for the first time a cluster model for the molybdopterin cofactor which is meant to best reproduce the stereoelectronic properties of the whole moiety. By testing several reaction pathways for the ratedetermining step of FDH catalytic cycle, we were able to propose a novel reaction mechanism which includes a β-hydride elimination step with a metal hydride intermediate. We then employed similar methods to study hydrogen oxidation in [NiFe]-hydrogenase. We have investigated the dihydrogen
coordination mode and subsequent oxidation, using model clusters of different sizes. It turned out that the spin state and the distorted seesaw coordination geometry of the Ni ion are two crucial factors that tune the energetics and regiochemistry of H2 binding. We were able to propose a reaction pathway involving the oxidative addition of H2 followed by proton transfer to the sulfur atom of one of the terminally coordinated cysteines as the lowest energy one. We then focused on another metalloenzyme, in which experimental evidences support a role of conformational dynamics for the catalysis, PTE. In this enzyme, it has been shown that the substrate hydrolysis can occur on fast time-scales, whereas one of the rate limiting steps seems to be the transition between so-called "open" and "closed" conformations with respect to the metal-binding site.
Indeed, in PTE mutations of residues, which are not directly involved in the active site, are known to affect the population of the closed and open states, as well as to alter the kcat. We thus studied this enzyme by atomistic Molecular Dynamics (MD) simulations to investigate the long-range structural communication routes between mutation sites and active site residues. We did so by considering both wild-type PTE (arPTE-WT) and a multiple mutant (arPTE-8M) in which mutations alter the equilibrium between the “closed” and “open” populations. By employing both freely available and in-house produced MD trajectories analysis tools, we were able to identify the major communication routes in the enzymes as well as to point out which mutation sites are more likely to be involved in the transmission of structural information. Comparing the communication
pathways in the two variants allowed us to investigate how the presence of mutations influence the communication pathways in the protein and to relate them to the effects induce by the mutations on protein function.
In conclusion, we have studied function-structure relationships in three different metalloenzymes, FDH, [NiFe] hydrogenase and PTE, by using high level DFT.
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Mulako, Inonge. "Characterization of the Xerophyta humilis desiccation induced-1 (Xhdsi-1voc) gene : a member of the Vicinal Oxygen Chelate (VOC) metalloenzyme superfamily upregulated in X. humilis (BAK) DUR and SCHINZ during desiccation." Doctoral thesis, University of Cape Town, 2008. http://hdl.handle.net/11427/4303.
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Includes abstract.Includes bibliographical references (leaves 172-191).
Has accompanying material on CD.
The resurrection plant, Xerophyta humilis is used as a model system to identify and characterise genes which play an important role in conferring desiccation tolerance in plants. In this study, the expression of a novel gene named desiccation induced-1 (dsi-1VOC) during desiccation in X. humilis and desiccationsensitive plants is characterised.
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Arndt, Joseph W. "Characterization and structural determination of metalloenzymes DNA polymerase beta, carboxypeptidase, and acetyl coenzyme-A decarbonylase/synthase /." Columbus, OH : Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1061312369.
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Thesis (Ph. D.)--Ohio State University, 2003.Title from first page of PDF file. Document formatted into pages; contains xxii, 172 p. : ill., some col. Includes abstract and vita. Advisor: Michael K. Chan, Dept. of Chemistry. Includes bibliographical references (p. 165-172).
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Chan, Alice. "Structure et Mécanisme de la Quinolinate Synthase : enzyme à centre [4Fe-4S]2+ et cible d'agents antibactériens." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV036.
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Le Nicotinamide Adénine Dinucléotide (NAD) est un cofacteur clé du métabolisme cellulaire. Synthétisé à partir d'acide quinolinique (QA) chez tous les organismes vivants, la biosynthèse du QA diffère entre les eucaryotes et les procaryotes. Chez les eucaryotes, il est produit à partir de L-tryptophane alors que chez les procaryotes et les plantes, il est synthétisé par l'action concertée de deux enzymes: la L-aspartate oxydase (NadB) qui permet la formation d'iminoaspartate (IA) à partir de L-aspartate et la quinolinate synthase (NadA) qui permet la condensation de deux molécules, la dihydroxyacétone-phosphate (DHAP) et l'iminoaspartate, pour former l'acide quinolinique. En plus de cette voie dite « de novo », la plupart des organismes possèdent une voie de secours qui produit le NAD à partir de niacine provenant de l'alimentation ou de la dégradation du NAD. Chez certains pathogènes tels que Mycobacterium leprae et Helicobacter pylori, cette voie de secours n'existe pas. Ceci fait de NadA une cible particulièrement attractive pour la conception d'antibactériens et ceci d'autant plus qu'elle est absente chez l'homme.NadA est la seule enzyme de la voie de biosynthèse de novo du NAD dont le mécanisme moléculaire et la structure tridimensionnelle sous forme active (avec son centre [4Fe-4S]2+) sont inconnus. Grâce à l'utilisation d'analogues de substrats ou d'intermédiaires réactionnels, nous avons pu non seulement avancer dans l'élucidation du mécanisme moléculaire de NadA et notamment dans la compréhension du rôle du centre [4Fe-4S]2+ dans la catalyse mais en plus, nous avons été en mesure de proposer un 1er inhibiteur in vitro et in vivo de NadA : l'acide 4,5 Dithiohydroxyphtalique (DTHPA). Le DTHPA nous a fourni de bonnes bases pour la conception d'inhibiteurs puissants et spécifiques de NadA grâce à une étude Structure-Activité. Par ailleurs, nous avons résolu la 1ère structure aux rayons X de NadA sous forme holoprotéine dont les données structurales nous ont grandement aidé dans la compréhension du mécanisme de NadAThe Nicotinamide Adenine Dinucleotide (NAD) is a key cofactor essential for cellular metabolism. Synthesized from quinolinic acid (QA) in all living organisms, NAD biosynthesis is different between eucaryotes and procaryotes. Indeed, most of eukaryotes produce QA from L-tryptophan, whereas most of prokaryotes and plants synthesize QA by the concerted action of 2 enzymes: L-aspartate oxydase (NadB), an FAD enzyme, which catalyzes L-Aspartate oxidation to form iminoaspartate (IA) while quinolinate synthetase (NadA) allows condensation between IA and Dihydroxyacetone Phosphate (DHAP) to produce QA. Besides this « de novo » pathway, most eukaryotes and some bacteriae have a salvage pathway which allows NAD synthesis from nutrients and metabolites of NAD degradation in order to maintain a correct pool of NAD in the cell. However, some pathogens like Mycobacterium leprae, Helicobacter pylori do not possess this pathway. As a consequence, NadA represents a very attractive target for designing specific antibacterial agents since it does not exist in Human.NadA is the only metalloenzyme of NAD de novo biosynthesis whose molecular mechanism and tridimensional structure with its [4Fe-4S]2+ cluster are unknown. Using substrate and intermediate analogues, we have been able to understand better NadA mechanism, especially [4Fe-4S]2+ cluster role in catalysis. Moreover, we proposed the first in vitro and in vivo inhibitor of NadA : the 4,5 Dithiohydroxyphtalic Acid (DTHPA) which gave us basis to design powerful and specific NadA inhibitors thanks to a structure-activity relationship study. Besides, we resolved the first X-rays structure of NadA under its holoprotein form. Datas we extracted from it helped us greatly to understand NadA mechanism
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Dutoit, Raphaël. "The Paradigm of Self-compartmentalized M42 Aminopeptidases: Insight into Their Oligomerization, Substrate Specificities, and Physiological Function." Doctoral thesis, Universite Libre de Bruxelles, 2020. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/314337.
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M42 aminopeptidases are dinuclear enzymes widely found in prokaryotes but completely absent from eukaryotes. They have been proposed to hydrolyze peptides downstream the proteasome or other related proteolytic complexes. Their description relies mainly on the pioneering work on four M42 aminopeptidases from Pyrococcus horikoshii. Their quaternary structure consists of twelve subunits adopting a tetrahedral-shaped structure. Such a spatial organization allows the compartmentalization of the active sites which are only accessible to unfolded peptides. The dodecamer assembly results from the self-association of dimers under the control of the metal ion cofactors. Both oligomers have been shown to co-exist in vivo and heterododecamers with broadened substrate specificity may even occur. Yet, the molecular determinants behind the dodecamer assembly remain unknown due the lack of a high-resolution structure of a stable dimer. In addition, the bacterial M42 aminopeptidases are still ill-described due to the paucity of structural studies. This work focuses mainly on the characterization of TmPep1050, an M42 aminopeptidase from Thermotoga maritima. As expected, TmPep1050 adopts the genuine tetrahedral-shaped structure with twelve subunits. It also displays a leucyl-aminopeptidase activity requiring Co2+ as a cofactor. In addition to its catalytic function, Co2+ has a role in the enzyme thermostability and oligomerization. The absence of Co2+ provokes the disassembly of active TmPep1050 dodecamers into inactive dimers. The process, however, is reversible since Co2+ triggers the self-association of dimers into dodecamers, as shown by native MS. The main achievement of this work is the determination of the first high-resolution structure of a dimer, allowing to better understand the dimer-dodecamer transition. Several structural motifs involved in oligomerization are displaced or highly flexible in the TmPep1050 dimer structure. Furthermore, a loop bringing two catalytic relevant residues is displaced outside the catalytic site. These residues are the catalytic base and a ligand involved in the Co2+ binding at the M1 site. The metal ion binding sites have been further investigated to define how they influence the oligomerization of TmPep1050. A mutational study shows that the M1 site strictly controls the dodecamer formation while the M2 site contributes only partly to it. A strictly conserved aspartate residue of the M2 site second shell also plays an important structural role in maintaining the active site integrity. Indeed, its substitution prevents the formation of dodecamer probably due to the lack of stabilization of the active site loop. The characterization of TmPep1050 supports that bacterial M42 aminopeptidases probably share the quaternary structures and dodecamer assembly with their archaeal counterparts. The dimer structure highlights several structural modifications occurring in the dimer-dodecamer transition. Yet, based on current knowledge, no general rules can be drawn for the role of the M1 and M2 sites in oligomerization. Besides, the physiological function of the M42 aminopeptidases is under-examined albeit the proposed link to the proteasome. In this work, this has been investigated using the Escherichia coli M42 aminopeptidases as a model. Yet, no phenotype has been associated to the deletion of their coding genes. Preliminary results have shown that the three enzymes (i) display a redundant substrate specificity, (ii) could be localized partly to the membrane, and (iii) form heterocomplexes. Further experiments are still required to crack the function of these M42 aminopeptidases.Option Biologie moléculaire du Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Carfi, Andrea. "Étude structurale des métallo-(beta)-lactamases de Bacillus cereus et Bacteroides fragilis : proposition d'un mécanisme enzymatique." Grenoble 1, 1997. http://www.theses.fr/1997GRE10080.
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Le probleme de la resistance des bacteries aux antibiotiques resulte directement de leur faculte d'adaptation. Des le debut du developpement de la penicilline, dans les annees 1940, on a vu l'apparition de bacteries resistantes a cette famille d'antibiotiques. Le mecanisme de resistance le plus courant, resulte de la production par la bacterie des b-lactamases. Ces enzymes hydrolysent le noyau b-lactame de l'antibiotique, ce qui a pour effet de l'inactiver et de rendre la souche resistante. Actuellement, quatre classe de b-lactamases (de a jusqu'a d) ont ete identifiee biochimiquement. Les classes a, c et d comprennent des enzymes a serine active. La classe b regroupe les metallo-proteines qui necessitent toujours des ions zn(ii) comme cofacteur. Cette these a abouti a la determination et a l'affinement a haute resolution des structures cristallines des metallo-b-lactamases de bacillus cereus et bacteroides fragilis. Cette etude a permis, entre autre, de mettre en evidence le repliement tout a fait nouveau de cette famille d'enzymes, de comprendre la capacite de ces enzymes a hydrolyser un large spectre de substrats et enfin de proposer un mecanisme catalytique base sur les structures obtenues et sur les donnees cinetiques.
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Mendonça, Jordana Dutra de. "O estudo da enzima deidroquinato sintase de Mycobacterium tuberculosis H37Rv como alvo para o desenvolvimento de fármacos antituberculose." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2010. http://hdl.handle.net/10183/25547.
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Apesar da incidência per capita da tuberculose (TB) ter se mantido estável em 2005, o número de novos casos que surgem a cada ano continua a aumentar no mundo todo. De acordo com a Organização Mundial de Saúde, foram estimados 9,4 milhões de novos casos de TB em 2008, dos quais 1,4 milhões eram HIV - positivos, e com 1,8 milhões de mortes - o equivalente a 4.500 mortes por dia. Fatores como migração, privação sócio-econômica, co-infecção TB-HIV e o aparecimento de cepas resistentes contribuíram para o aumento do número de casos de TB no mundo, principalmente nos países onde a TB já foi considerada erradicada, e criaram a necessidade do desenvolvimento de novas terapêuticas. Alvos moleculares específicos, que são essenciais para o patógeno, e ausentes no hospedeiro, como as enzimas da via do ácido chiquímico são alvos atraentes para o desenvolvimento de novas drogas antituberculose. Essa via leva à síntese de compostos aromáticos, como aminoácidos aromáticos, e é encontrada em plantas, fungos, bactérias e parasitas do phylum Apicomplexa, mas está ausente em humanos. No ano de 2000, foi comprovada a essencialidade dessa via para a viabilidade do bacilo, tornando todas essas enzimas alvos validados para estudo. A segunda enzima da via, deidroquinato sintase (DHQS), catalisa a conversão de 3-deoxi-D-arabino heptulosonato-7-fosfato em 3-deidroquinato, o primeiro composto cíclico. Neste trabalho, são descritos o requerimento de metais divalentes na reação e a determinação do mecanismo cinético da DHQS. Os parâmetros cinéticos verdadeiros foram determinados e, juntamente com os experimentos de ligação, o mecanismo rápido-equilíbrio aleatório foi proposto. O tratamento com EDTA aboliu completamente a atividade de DHQS, sendo que a adição de Co+2 e Zn+2 levam a recuperação total e parcial da atividade enzimática, respectivamente. O excesso de Zn+2 inibe a atividade DHQS, e os dados de ITC indicaram a presença de dois sítios seqüenciais de ligação, o que é consistente com a existência de um sítio secundário inibitório. O protocolo de cristalização foi estabelecido e experimentos em andamento proporcionarão a elucidação da estrutura tridimensional da DHQS, que irá beneficiar tanto o desenho de novos inibidores como uma análise detalhada dos rearranjos do domínio da proteína. Em conjunto, estes resultados representam um passo essencial para o desenho racional de inibidores específicos que podem fornecer uma alternativa promissora para um novo, eficaz, e mais curto de tratamento para TB.Although the estimated per capita tuberculosis (TB) incidence was stable in 2005, the number of new cases arising each year is still increasing globally. According with World Health Organization, there were estimated 9.4 million new TB cases in 2008, from which 1.4 million were HIV-positive, with 1.8 million deaths total – equal to 4500 deaths a day. Migration, socio-economic deprivation, HIV co-infection and the emergence of extensively-resistance strains, have all contributed to the increasing number of TB cases worldwide, mainly in countries where it was once considered eradicated, and have created an urgent need for the development of new therapeutics against TB. Specific molecular targets, that are essential to the pathogen, and absent in the host, like the enzymes of the shikimate pathway, are attractive targets to development of new antitubercular drugs. This pathway leads to the biosynthesis of aromatic compounds, including aromatic amino acids and it is found in plant, fungi, bacteria and Apicomplexa parasites, but is absent in humans. In 2000, this pathway was proved to be essential to the viability of the pathogen, which validates all its enzymes as potential targets. The second enzyme of this pathway, dehydroquinate synthase (DHQS), catalyzes the conversion of 3-deoxy-D-arabinoheptulosonate 7-phosphate in 3-dehydroquinate, the first cyclic compound. In this work, we described the metal requirement and kinetic mechanism determination of the dehydroquinate synthase. The determination of the true kinetic parameters was performed, and, in addition to ligand binding experiments, the rapid-equilibrium random mechanism was determined. The treatment with EDTA abolished completely the activity of DHQS, and the addition of Co+2 and Zn+2 leads to full and partial recovery of enzyme activity, respectively. Excess of Zn+2 inhibits the DHQS activity, and the ITC data revealed two sequential binding sites, which is consistent with the existence of a secondary inhibitory site. The crystallization protocol was established and ongoing experiments will provide the three-dimensional structure of mtDHQS, which will benefit both the design of novel inhibitors as well as detailed analysis of domain rearrangements of protein. Taken together, these results represent an essential step for the rational design of specific inhibitors that can provide a promising alternative to a new, effective, and shorter treatment for TB.
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Pocquet, Lucrèce. "Ancrage supramoléculaire de complexes organométalliques dans la béta-lactoglobuline pour la catalyse asymétrique dans l'eau. Effet des ligands prochiraux hémilabiles." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066322/document.
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Ce travail de thèse est consacré à la conception de métalloenzymes artificielles. Un tel concept permet de combiner les avantages des catalyseurs enzymatiques et organométalliques, tels que la sélectivité catalytique élevée et l'efficacité des systèmes enzymatiques et la large portée de substrats des catalyseurs des métaux de transition. Notre approche repose sur l’utilisation de complexes de métaux de transition avec un ligand prochiral hémilabile, qui une fois insérés dans la protéine hôte sera forcé d’adopter une configuration spécifique. La chiralité sera ainsi amenée au plus près du centre métallique et permettra d’augmenter l’énantioselectivité des réactions catalysées. Dans cette thèse, nous rapportons la synthèse de nouveaux complexes de palladium à ligands pinces NCN hémilabiles prochiraux et l’étude de leurs propriétés structurales. De plus, l’ancrage supramoléculaire de ces complexes dans la β-lactoglobuline (β-LG) bovine a été étudié expérimentalement et théoriquement par modélisation moléculaire. Ces constructions ont montré une activité catalytique dans la condensation d’aldol dans l’eau, et permettent d’obtenir, dans certains cas, le produit Cis. Cette diastéréosélectivité inhabituelle résulte de la seconde sphère de coordination apportée par l'hôte protéique. Dans une deuxième partie, on rapporte la synthèse de nouveaux complexes semi-sandwich de ruthénium avec des ligands β-aminothioéther hémilabiles, ainsi que l'étude de leur insertion dans la protéine. Les hybrides catalysent l'hydrogénation par transfert d'arylcétones avec une énantiosélectivité élevée. L'amélioration de la sélectivité a été attribuée au transfert de chiralité de la protéine vers le complexe et à son tour vers le produit de réactionThis PhD work focused on the development of artificial metalloenzymes. Such a concept allows to combine typical advantages of both enzymatic and organometallic catalysts, such as high catalytic selectivity and efficiency of enzymatic systems and wide substrate scope of transition metals catalysts. Our approach consists in the utilization of transition metal complexes with a prochiral hemilabile ligand, once embedded within the protein host, could be forced to adopt a specific stereoconfiguration. This would in turn make possible to bring the chirality centers closer to the catalytic metal center and, therefore, to increase the enantioselectivity of catalyzed reactions.In this thesis, we report the synthesis of new palladium complexes of prochiral hemilabile NCN pincer ligands and the study of their structural properties. Furthermore, the supramolecular anchoring of these complexes to bovine β-lactoglobulin (β-LG) was studied both experimentally and theorically by computational calculation. These constructs were shown to catalyze aldol condensation reactions in aqueous media, affording, in some cases, the less-favorable cis product. This unusual diastereoselectivity was ensued by the second sphere of coordination brought by the protein host. In a second part, the synthesis of new half sandwich ruthenium complexes of prochiral hemilabile β-aminothioether ligands is reported as well as the study of their insertion in the protein. The hybrids catalyzed the transfer hydrogenation of arylketones with high enantioselectivity. The enhancement of selectivity was attributed to chirality
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Esmieu, Charlène. "Etude de la relation structure-activité de complexes bio-inspirés de la réductase de l'oxyde nitreux." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV044/document.
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Etude de la relation structure-activité de complexes bio-inspirés de la réductase de l'oxyde nitreux N2O est un puissant gaz à effet de serre et est impliqué dans la destruction de la couche d'ozone, ce qui rend sa dégradation très intéressante. Il s'agit d'un intermédiaire du cycle catalytique de la nitrification bactérienne. En effet, en biologie une métalloenzyme est capable de réduire N2O à deux électrons en N2 et H2O. Le site actif de la réductase de l'oxyde nitreux, le centre CuZ, renferme l'unique association de quatre ions cuivre pontés par un ion sulfure. Afin d'obtenir des complexes capables d'activer N2O et d'approfondir la compréhension du mécanisme catalytique de l'enzyme nous avons élaboré des modèles inspirés du centre CuZ. Il s'agit de complexes dinucléaires de cuivres possédant le motif {Cu2(µ-S)} supposé indispensable à l'activation de N2O. Les complexes à valence mixtes décrits dans ces travaux ont été complétements caractérisés et leur activité vis-à-vis de la réduction de N2O a été évaluée. Ces complexes constituent le premier modèle de ce type capable de réduire N2O. Des études spectroscopiques, électrochimiques et théoriques nous ont également permises de proposer un mécanisme réactionnel, passant par la formation d'un adduit complexe-N2O. Nous avons également pu mettre en évidence le rôle crucial de la molécule d'eau, ligand exogène des complexes, dans ce mécanisme. En parallèle, la stabilité en solution de différentes liaisons disulfures présentes au sein de ligands tétranucléants, en présence de CuII, a été évaluée. La réactivité de la liaison disulfure est dépendante de la fixation des ions cuivre à proximité des atomes de soufres. Trois ligands possédants des substituants aminés différents ont été testés, chacun présentant une réactivité particulière. Nous avons montré pour l'un de ces ligands que l'oxydation de la liaison disulfure pouvait être réalisée en absence d'oxydant fort, l'eau jouant le rôle de nucléophileStructure-activity relationships in copper complexes bio-inspired from nitrous oxide reductase N2O is a powerful greenhouse gas and is involved in the ozone layer destruction, which makes it degradation very interesting. N2O is an intermediate of the catalytic cycle of bacterial nitrification. Indeed, in biology a metalloenzyme can reduce N2O with two electrons to N2 and H2O. The active site of nitrous oxide reductase, the CuZ center, contains a unique combination of four copper ions bridged by a sulfide ion. In order to obtain complexes able to activate N2O and deepen the understanding of the catalytic mechanism of the enzyme we have developed models based on the CuZ center. Binuclear copper containing the {Cu2(μ-S)} pattern that is supposed essential to N2O activation have been synthetized. Mixed valent complexes described in this work were fully characterized and their activity toward N2O was evaluated. These complexes are the first model like this capable of N2O reduction. Spectroscopic, electrochemical and theoretical studies have also allowed us to propose a reaction mechanism, which passes through the formation of an adduct complex-N2O. We were also able to highlight the crucial role of the exogenous water molecule in this mechanism. In parallel, the solution stability of different disulfide bonds present in tetranucleating ligands in the presence of CuII was evaluated. The reactivity of the disulfide bond is dependent upon the binding of copper ions near sulfur atoms. Three ligands with different amino groups were tested, each having a specific reactivity. We have shown for one of them that the oxidation of the disulfide bond could be carried out in the absence of strong oxidizer, water acting as the nucleophil
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Alonso, Cotchico Lur. "Computational design of artificial metalloenzymes." Doctoral thesis, Universitat Autònoma de Barcelona, 2018. http://hdl.handle.net/10803/664006.
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El diseño enzimático es el área basada en el descubrimiento y/o optimización de biomoléculas para el desarrollo de reacciones químicas no naturales. Es un área que se encuentra en su mayor crecimiento y constituye uno de los puntos clave en la transición de la química hacia alternativas más ecológicas.
Una manera elegante de sintetizar nuevos biocatalizadores es mediante la inclusión de cofactores organometálicos en estructuras biológicas, dando lugar a lo que se conoce como Metaloenzimas Artificiales (ArMs). Estos híbridos combinan la versatilidad catalítica de los compuestos organometálicos con la especificidad de los receptores biológicos. El diseño de ArMs ha crecido exponencialmente en las últimas dos décadas gracias al desarrollo de áreas como la biología estructural y la catálisis organometálica. La modelización molecular trata de guiar los diseños proporcionando información estructural para la construcción de catalizadores óptimos. Sin embargo, a pesar de los avances en rendimiento computacional y de nuevas técnicas computacionales, la complejidad del trato de sistemas que contienen metales de transición hace que el área de los ArMs no sea propiamente explorado por los modelizadores.
El grupo InSiliChem, dónde se ha desarrollado este Ph.D., se centra en el desarrollo de estrategias para el estudio y el diseño de ArMs. En particular, se basa en el desarrollo de estrategias computacionales multinivel que incluyen una gran variedad de técnicas computacionales. Este Ph.D. trata de aumentar el potencial de la plataforma computacional para el diseño de ArMs 1) mediante la incorporación de distintas técnicas como la simulación de Dinámica Molecular (MD) y 2) validando el procedimiento mediante el diseño de casos enzimáticos reales.
Los resultados obtenidos se resumen en los siguientes puntos:
• Se ha descrito el mecanismo catalítico de dos nuevas ArMs. Estas son: una hidratasa basada en el receptor LmrR (Lactococcus Multidrug Resistance Regulator) que incluye un cofactor de Fenantrolina unida a cobre; y una variedad de mutantes de complejos de Streptavidina-Noyori capaces de realizar la reducción de iminas cíclicas. Este estudio revela la importancia de la contribución de las simulaciones MD como parte del protocolo computacional para la decodificación del mecanismo catalítico de los nuevos enzimas, así como el impacto de la segunda esfera de coordinación en las tendencias catalíticas. (Capítulo 4)
• Basándonos en la misma metodología, se han modelizado nuevas hidratasas basadas en la inclusión de amino acidos no naturales (UAA). Primero, aplicados a la proteína LmrR, para la que fueron computacionalmente propuestos nuevos mutantes más eficaces en cuanto a la enantioselectividad del sistema (fueron después experimentalmente validados). Después, basándonos en la experiencia adquirida, el concepto de novo fue expandido al diseño de Metalopéptides Artificiales. (Capítulo 5)
• La última parte del trabajo se centra en descifrar variables moleculares que en los estudios previos han resultado de gran relevancia. Entre ellos, la relación entre la configuración estructural del centro activo y la actividad catalítica del ArM. En particular, se ha estudiado la adaptabilidad de una variedad de complejos LmrR-hemo claves para el transcurso de la reacción de ciclopropanación. Los resultados sugieren que la flexibilidad del receptor es un punto crítico para que los ArMs basados en el grupo hemo adquieran actividad catalítica. Con el objetivo de profundizar más en la importancia de esta variable molecular, este estudio se ha expandido, además, a proteínas naturales reconocedoras del grupo hemo. (Capítulo 6)
En conclusión, este Ph.D. representa un paso adelante en el desarrollo metodológico del diseño computacional de Metaloenzimas Artificiales. El trabajo realizado clarifica la importancia de la cooperación a nivel molecular entre los compuestos basados en metales de transición y sus receptores biológicos. Ésto facilita el camino hacia nuevos diseños de biocatalizadores basados en Metaloenzimas Artificiales.Enzyme design is the scientific field that aims at discovering and/or optimizing biomolecules to reach new-to-Nature reactions. It is an area in wild expansion and constitutes one of the cornerstones of the transition of chemical practices towards greener alternatives. An elegant way to construct novel biocatalysts is through the embedding of organometallic cofactors into biological scaffolds, leading to the so-called Artificial Metalloenzymes (ArMs). These hybrids bridge the catalytic versatility of the organometallic compound with the substrate and spatial specificity of the biological host. The design of ArMs has spread increasingly during the last two decades taking clear advantage of the major expansion of structural biology and the maturity of organometallic catalysis. Molecular modelling aims to help designers to provide with structural information that could serve for constructing optimum biocatalysts. However, despite the increasing improvement of the computation performance and the exponential development of new simulation techniques, the complexity of dealing with transition metal including systems has promoted modellers not to explore the ArM constructs. The InSiliChem group, in which this Ph.D. has been performed, has focused on developing a specific framework for the study and design of ArMs. In particular, this has been based on the development of in silico multiscale strategies including standard computational methods. This Ph.D. aims at increasing the potentiality of our computational platform for ArM design by 1) including classical Molecular Dynamics simulations into the integrative computational framework and 2) testing the validity of the methodology for the design of real case ArMs. The results obtained could be summarize as follows: • The catalytic mechanism of two novel ArMs were decoded using the updated computational pipeline. These were a copper-Phenanthroline containing hydratase based on the Lactococcus Multidrug Resistance Regulator (LmrR) and a variety of novel mutants based on Streptavidine-Noyori complexes for cyclic imine reduction reaction. The study revealed the importance of the contribution of the MD simulations to decode the catalytic mechanism of these ArMs and to assess the impact of second sphere mutations on the catalytic tendencies. (Chapter 4) • Using the same approach, calculations were carried out for the in silico design of hydratases, but in this case based on the inclusion of a novel unnatural amino acid. This was first applied to the LmrR scaffold, for which mutants suggested via computation for optimum enantiomeric excess (ee) were then experimentally assessed with success. Next, based on the experience obtained, we expanded the de novo exercise towards the design of Artificial Metallopeptides. (Chapter 5) • The final part of the work focused on deciphering molecular variables that our previous studies showed to be far more complex than expected. This was the impact of the active site configuration to define the catalytic activity of the ArMs. In particular, we decoded the rearrangement of a variety of LmrR-heme complex for the cyclopropanation reaction to proceed. From this study it clearly appeared that the flexibility of the receptor is key for the porphyrin based ArMs to reach their catalytic activity. To further assess the importance of this molecular variable, we expanded this work to the study of distinct naturally occurring heme binding proteins. (Chapter 6) Overall, this Ph.D. represents a step forward on the methodological development of the computer based enzyme design. Furthermore, it sheds light on how transition metal compounds could cooperate with biological scaffolds at the molecular level with the focus on the de novo design of new biocatalysts.
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Montel, Sonia. "La 1-Deoxy-D-Xylulose-5-Phosphate Reductoisomerase, une métalloenzyme cible pour l'élaboration d'inhibiteurs chélatants." Thesis, Montpellier, Ecole nationale supérieure de chimie, 2012. http://www.theses.fr/2012ENCM0013.
Full textAbstract:
La voie non-mévalonate est fortement présente chez les plantes et les bactéries mais est absente chez les mammifères. C'est pourquoi inhiber la synthèse des isoprénoïdes et identifier un inhibiteur de cette voie enzymatique contribuera grandement à la recherche de nouveaux antibiotiques, antifongiques et herbicides. Les propriétés uniques de la 1-deoxy-D-xylulose 5-phosphate reductoisomérase (DXR), l'enzyme centrale de cette voie enzymatique, en font une cible très intéressante pour la synthèse de nouveaux composés. La Fosmidomycine agit comme un inhibiteur de la DXR et reste aujourd'hui, avec son homologue acétylé FR90098, la référence en termes d'inhibiteur même si de nombreux efforts ont été faits pour la synthèse d'analogues depuis plusieurs années comme expliqué dans le premier chapitre avec la mise en relation de la structure des composés et leur activité. L'analyse de la diffraction des rayons X de la DXR avec la Fosmidomycine où le substrat naturel montre que la fonction phosphonate ou phosphate interagit avec une poche polaire hautement spécifique dans le site actif de l'enzyme permettant peu de modifications. Par comparaison, la fonction acide hydroxamique qui chélate le cation de l'enzyme offre la possibilité de modifications par l'introduction d'autres fonctions complexantes. Dans ce contexte, de nombreuses modifications comme l'introduction de fonctions carbamoylphosphinate, amidoxime, N-hydroxyurée et dérivées d'uraciles comme unités complexantes ont été synthétisées pour trouver des nouvelles familles d'inhibiteurs de la DXR. Toutes ces fonctions possèdent des propriétés de chélation intéressantes. En effet, elles ont déjà conduit à de puissants inhibiteurs de différentes métalloenzymesThe non-mevalonate pathway is highly present in higher plants, protozoa and bacteria but as no equivalent in mammals. That is why shut down isoprenoid biosynthesis and identify a non-mevalonate pathway inhibitor would greatly contribute to the search for safer antibiotics, antimalarials and for our concern herbicides. The unique properties of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), the central enzyme of this pathway, make it a remarkable and attractive target for drug design. Fosmidomycin acts as an inhibitor of DXR and still remains, along with its N-acetyl homologue FR90098, one of the most potent inhibitor ever known even if extensive work on the development of Fosmidomycin analogue derivatives have been developed since the last decade as demonstrated in the first chapter with the development of a structure activity relationship of all the potential inhibitors of this enzyme already reported in the literature. The X-ray diffraction analysis of the co-crystals of DXR and Fosmidomycin or substrate shows that the phosphonic/phosphate group interacts with a highly specific polar pocket in the enzyme site, allowing only few structural modifications. By contrast, the cation chelating subunit represented by the hydroxamic acid function offers fine tuning possibilities for the complexation abilities as well as potential secondary interactions with the NADPH cofactor or directly with the enzyme. In this context, several modifications such as the introduction of carbamoylphosphinate, amidoxime, N-hydroxyurea and uracil complexing subunits have been made in order to find new families of DXR inhibitors. All of these functions show promising chelation capabilities as they already led to potent inhibitors of different metalloenzymes
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Ceccaldi, Pierre. "Identification des déterminants moléculaires de la réactivité d'une molybdoenzyme modèle : La nitrate réductase A d' Escherichia coli." Thesis, Aix-Marseille, 2013. http://www.theses.fr/2013AIXM4016/document.
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Les molybdoenzymes (MoEs) à bisPGD sont des métalloprotéines dont le site actif est constitué d'un cofacteur à molybdène (Mo) mononucléaire. Elles sont impliquées dans les cycles biogéochimiques de l'azote, du carbone et du soufre et catalysent essentiellement des réactions d'oxydoréduction à 2 e-/2 H+ envers une grande variété de substrats. En dépit de la connaissance de la structure cristallographique de nombreuses MoEs, leur fonctionnement reste encore largement incompris. Ces enzymes présentent un intérêt biotechnologique car certains de leurs substrats sont des composés toxiques notoires, tels que les oxydes de sélénium ou d'arsenic. L'objectif de ma thèse a été d'identifier quels facteurs structuraux gouvernent la réactivité d'une MoE à bisPGD, la nitrate réductase A d'E. coli. Le premier axe de mes travaux de thèse a consisté à étudier l'activité de l'enzyme envers différents substrats et examiner le rôle du ligand protéique du Mo dans sa réactivité, en combinant des approches de mutagenèse dirigée, de biochimie et de spectroscopie RPE. J'ai montré que le ligand protéique du Mo est impliqué dans une étape clé du cycle catalytique. Le second axe a consisté à identifier les relations existantes entre la structure atomique du site actif et ses signatures spectrales. Pour augmenter la résolution et permettre d'identifier les transitions structurales mises en jeu lors de l'interconversion entre les différentes formes spectrales, j'ai utilisé la spectroscopie RPE impulsionnelle, qui permet de détecter les noyaux magnétiques (1H et 14N, …). Mes résultats constituent un pré-requis nécessaire pour l'étude structurale à haute résolution du site actif de la nitrate réductaseMolybdenum (Mo) is a rare transition metal that is indispensable to most living organisms. In particular, it makes part of the active site of metalloenzymes involved in the biogeochemical cycles of carbon, nitrogen and sulphur. In this context, prokaryotic molybdoenzymes (MoEs) with the bisPGD cofactor at their active site essentially catalyze oxidoreduction reactions with 2 e-/2 H+ towards a wide range of substrates. Given that some MoEs can activate substrates that are well-known pollutants, understanding the mechanism of these enzymes accounts for a major prerequisite for future enzymatic engineering strategies aimed at optimizing enzyme reactivity towards bioremediation processes. To identify the molecular determinants of the reactivity of MoEs, we have explored the importance of the Mo proteic ligand aspartate in the respiratory Nitrate Reductase from E. coli. We have combined biochemistry and EPR spectroscopy to analyze the impact of the Mo-ligand substitution on both the enzymatic and the structural properties of the molybdenum cofactor. Our results show that the nature of the proteic ligand plays a critical role in the reactivity of the active site. A second part of my thesis work consisted in establishing the link between spectroscopic data on the MoV centre and its atomic structure. To get a high level of resolution and to identify which kind of structural modification is responsible for the spectroscopic differences between every Mo(V) signature, pulsed EPR spectroscop is most promising. Our results constitute a pre-requisite for structural studies of every species of the MoV center of the NRA
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Träff, Annika. "Asymmetric transformation of ß- and γ-functionalized alcohols : Study of combined ruthenium-catalyzed racemization and enzymatic resolution." Doctoral thesis, Stockholms universitet, Institutionen för organisk kemi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-56947.
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The major part of this thesis describes the asymmetric synthesis of β- and γ-amino alcohols through the combination of ruthenium catalyzed racemization and enzymatic kinetic resolution. The dynamic kinetic resolution, DKR, protocol for chlorohydrins was improved by employing Bäckvall’s catalyst, which is a base activated racemization catalyst, in combination with Burkholderia cepacia lipase. These optimized conditions broadened the substrate scope and improved the yields and ee’s of the obtained chlorohydrin acetates. The utility of the method was demonstrated in the synthesis of (S)-salbutamol. In the second part of the thesis, DKR was utilized in the enantio-determining step of the total synthesis of (R)-duloxetine. Optimized DKR conditions, combining Bäckvall’s catalyst together with Candida antarctica lipase B, afforded a β-cyano acetate in high yield and ee. (R)-Duloxetine was accessible through synthetic alterations of the enantioenriched β-cyano acetate in high overall yield. A dynamic kinetic asymmetric transformation, DYKAT, protocol to obtain enantio- and diastereomerically pure γ-amino alcohols was developed. In a first step N-Boc-aminoketones were obtained in high enantiomeric purity through a proline-catalyzed Mannich reaction. Subsequent in situ reduction coupled with a highly efficient DYKAT yielded γ-amino acetates in high dr and ee. The γ-amino alcohols were available through simple hydrolysis/deprotection with retained stereochemistry. In the final part of the thesis a heterogeneous bifunctional catalytic system is reported, which combines the catalytic properties of transition metal-catalyzed racemization with enzymatic acylation. A novel ruthenium-phosphonate complex was synthesized and then covalently anchored to the active site of solid supported Candida antarctica lipase B. The partially inhibited beads proved to be catalytically active both in racemization as well as enzymatic acylation.At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: In press. Paper 3: Epub ahead of print.
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Josse, Denis. "Vers la détermination du centre actif et de la structure de la paraoxonase humaine." Université Joseph Fourier (Grenoble), 1999. http://www.theses.fr/1999GRE10190.
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La paraoxonase humaine (ec 3. 1. 8. 1, pon1) est une metalloenzyme a calcium associee aux lipoproteines plasmatiques de type hdl. Elle est impliquee dans la prevention de l'oxydation des lipoproteines de type ldl. Notre interet pour la pon1 tient a son activite d'hydrolase d'organophosphores (opase). Notre objectif est d'utiliser une forme ingenierisee de cette enzyme dans le traitement de l'intoxication par les ops neurotoxiques de guerre et pour la decontamination. L'optimisation de l'efficacite catalytique de la pon1 implique l'elucidation prealable de son mecanisme catalytique et la determination de sa structure tridimensionnelle. La nature et la concentration du detergent associe a la pon1 purifiee determinent la stabilite et l'activite de l'enzyme. La modification chimique selective d'aminoacides et la mutagenese dirigee ont permis l'identification de residus essentiels aux activites opase et arylesterase (are) de la pon1 humaine. Ces residus sont : c41, c352, h114, h133, h154, h242, h284, e52, e194, d53, d168, d182, d268, d278 et w280. Chez le lapin, les aminoacides h114, h133, h154, e52, d53 et d268 sont essentiels a l'activite are de la pon1, mais d168, e194, h242 et w280 ne le sont pas. Afin d'identifier les ligands du calcium et de sonder son environnement, la liaison du 4 5ca, apres electrophorese en conditions non denaturantes, et du terbium, un analogue fluorescent du calcium, ont ete etudiees. L'etude de substrats suicides a ete envisagee afin d'identifier des residus du centre actif. Des electrophoreses realisees en conditions non denaturantes et des modifications chimiques par pontage avec des reactifs bifonctionnels ont montre que les pon1 de l'homme et du lapin sont dimeriques, actives et homogenes en presence d'une concentration micellaire de triton x-100. La structure quaternaire, l'activite et la stabilite de la pon1 dependent de la nature et de la concentration du detergent ainsi que de la concentration en ions calcium.