Academic literature on the topic 'Enzymologie structurale'
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Journal articles on the topic "Enzymologie structurale"
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Pearson, Arwen R., Andrea Mozzarelli, and Gian Luigi Rossi. "Microspectrophotometry for structural enzymology." Current Opinion in Structural Biology 14, no. 6 (December 2004): 656–62. http://dx.doi.org/10.1016/j.sbi.2004.10.007.
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Einsle, Oliver, and Douglas C. Rees. "Structural Enzymology of Nitrogenase Enzymes." Chemical Reviews 120, no. 12 (June 15, 2020): 4969–5004. http://dx.doi.org/10.1021/acs.chemrev.0c00067.
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Schneider, Gunter, and Ylva Lindqvist. "Structural enzymology of biotin biosynthesis." FEBS Letters 495, no. 1-2 (April 19, 2001): 7–11. http://dx.doi.org/10.1016/s0014-5793(01)02325-0.
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Muretta, Joseph M., Yonggun Jun, Steven P. Gross, Jennifer Major, David D. Thomas, and Steven S. Rosenfeld. "The structural kinetics of switch-1 and the neck linker explain the functions of kinesin-1 and Eg5." Proceedings of the National Academy of Sciences 112, no. 48 (November 16, 2015): E6606—E6613. http://dx.doi.org/10.1073/pnas.1512305112.
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Kinesins perform mechanical work to power a variety of cellular functions, from mitosis to organelle transport. Distinct functions shape distinct enzymologies, and this is illustrated by comparing kinesin-1, a highly processive transport motor that can work alone, to Eg5, a minimally processive mitotic motor that works in large ensembles. Although crystallographic models for both motors reveal similar structures for the domains involved in mechanochemical transduction—including switch-1 and the neck linker—how movement of these two domains is coordinated through the ATPase cycle remains unknown. We have addressed this issue by using a novel combination of transient kinetics and time-resolved fluorescence, which we refer to as “structural kinetics,” to map the timing of structural changes in the switch-1 loop and neck linker. We find that differences between the structural kinetics of Eg5 and kinesin-1 yield insights into how these two motors adapt their enzymologies for their distinct functions.
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Buschiazzo, Alejandro, and Pedro M. Alzari. "Structural insights into sialic acid enzymology." Current Opinion in Chemical Biology 12, no. 5 (October 2008): 565–72. http://dx.doi.org/10.1016/j.cbpa.2008.06.017.
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Long, Tao, Erik W. Debler, and Xiaochun Li. "Structural enzymology of cholesterol biosynthesis and storage." Current Opinion in Structural Biology 74 (June 2022): 102369. http://dx.doi.org/10.1016/j.sbi.2022.102369.
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Kupitz, Christopher, Jose L. Olmos, Mark Holl, Lee Tremblay, Kanupriya Pande, Suraj Pandey, Dominik Oberthür, et al. "Structural enzymology using X-ray free electron lasers." Structural Dynamics 4, no. 4 (December 15, 2016): 044003. http://dx.doi.org/10.1063/1.4972069.
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Johnson, Louise N., and Gregory A. Petsko. "David Phillips and the origin of structural enzymology." Trends in Biochemical Sciences 24, no. 7 (July 1999): 287–89. http://dx.doi.org/10.1016/s0968-0004(99)01423-1.
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Patel, S., M. Martı́nez-Ripoll, Tom L. Blundell, and A. Albert. "Structural Enzymology of Li+-sensitive/Mg2+-dependent Phosphatases." Journal of Molecular Biology 320, no. 5 (July 2002): 1087–94. http://dx.doi.org/10.1016/s0022-2836(02)00564-8.
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Schnell, Robert, and Gunter Schneider. "Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis." Biochemical and Biophysical Research Communications 396, no. 1 (May 2010): 33–38. http://dx.doi.org/10.1016/j.bbrc.2010.02.118.
Full textDissertations / Theses on the topic "Enzymologie structurale"
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Contet, Alicia. "Caractérisation biochimique et biophysique des deux cytidylyltransférases de Plasmodium falciparum, enzymes clés du métabolisme des phospholipides." Thesis, Montpellier, 2015. http://www.theses.fr/2015MONTS085.
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Le paludisme est causé par l'infection et la destruction des érythrocytes par les parasites protozoaires appartenant au genre Plasmodium. Au cours de son développement dans l'érythrocyte,Plasmodium falciparum requiert la biosynthèse massive de membranes dont les principaux constituants lipidiques sont des phospholipides. La phosphatidylcholine (PC) et la phosphatidyléthanolamine (PE) représentent à elles deux environ 80 % des lipides membranaires et l'inhibition de leur biosynthèse est létale pour le parasite. La PC et la PE sont synthétisées par le parasite, principalement via les voies de novo dépendantes de la CDP-choline et de la CDP-éthanolamine (ou voies de Kennedy) en utilisant respectivement la choline et l'éthanolamine comme précurseurs. Ces travaux de thèse se focalisent sur les deux enzymes CTP:phosphocholine etCTP:phosphoéthanolamine cytidylyltransférase (PfCCT et PfECT, respectivement), catalysant les étapes limitantes des voies de Kennedy. Chez Plasmodium, les CCT et ECT possèdent deux domaines cytidylyltransférases (CT) portant l'activité catalytique, séparés par une longue région de liaison. Pour la CCT, cette duplication est retrouvée seulement chez trois organismes, tous faisant partie du phylumdes Apicomplexes : Babesia, Theileria et Plasmodium, alors que la présence de deux domaines CT estune caractéristique retrouvée chez toutes les ECT étudiées à ce jour. La première partie de ce travail de thèse concerne la caractérisation biochimique et l'inhibition la PfCCT Nous avons montré que les deux domaines CT de la PfCCT sont actifs à l'inverse de la PfECT pour laquelle seul le domaine CTN-terminal est catalytiquement actif. A la suite d'un criblage virtuel basé sur la structure de l'enzyme,nous avons identifié un composé princeps capable d'inhiber l'activité de la PfCCT in vitro, la synthèse de PC et la croissance parasitaire. Ce premier composé actif (haut µM) représente une base pour l'optimisation future de nouveaux composés plus efficaces. Dans la deuxième partie de cette thèse,nous avons déterminé le mécanisme catalytique, la spécificité de liaison des ligands et l'organisation structurale de la PfECT grâce à la combinaison d'approches biochimiques et biophysiques. L'ensemble des résultats présentés dans ce manuscrit apportent un éclairage important concernant le fonctionnement de ces deux cibles potentielles et constituent des étapes essentielles à l'élaboration d'une approche thérapeutiqueMalaria is caused by the infection and destruction of red blood cells by protozoan parasitesbelonging to the genus Plasmodium. During its intra-erythrocytic development, Plasmodiumfalciparum requires massive biosynthesis of membranes which are mainly composed of phospholipids.Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) together represent about 80% of thetotal membrane lipids and inhibition of their biosynthesis leads to parasite death. PC and PE aresynthesized by the parasite's machinery mainly through the de novo CDP-choline and CDPethanolamine(Kennedy) pathways using respectively choline and ethanolamine as precursors. Thisstudy focuses on the rate limiting steps of these pathways catalyzed by CTP:phosphocholine andCTP:phosphoethanolamine cytidylytransferases (PfCCT and PfECT, respectively). In Plasmodiumspecies, both CCT and ECT contain two catalytic cores (CT domains) separated by a long linker.Interestingly, for CCT this feature is found only in three organisms, all from the phylum ofApicomplexa: Babesia, Theileria and Plasmodium, whereas the presence of two CT domains is ageneral feature in all ECTs known so far. The first part of this work consists in the biochemicalcharacterization of PfCCT and the investigation of its druggability. We showed that both PfCCT CTdomains are active and display similar kinetic parameters while only the N-terminal CT domain wasactive in PfECT. Subsequent to an in silico structure-based screening of compounds libraries, weidentified a PfCCT inhibitor able to inhibit PC synthesis as well as P. falciparum growth in vitro in thehigh µM range. This compound represents a first step toward the optimization of future more potentcompounds. In the second part of this study, we investigated the catalytic mechanism of PfECT anddeciphered its interactions with its ligands using biochemical, biophysical and structural approaches.Collectively, these results bring new insights into the biochemical and structural properties of thesetwo keys enzymes of the phospholipid metabolism in P. falciparum and pave the way for their futuredevelopment as potential drug target
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Bou, Nader Charles. "Structural and Functional characterization of flavoenzymes involved in posttranscriptional modification of tRNA." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066205/document.
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La modification posttranscriptionnelle des acides ribonucléiques (ARNs) est une étape de maturation conservée dans tous les domaines du vivant. Mes travaux de thèse ont porté sur la caractérisation fonctionnelle et structurale de flavoenzymes impliquées dans la modification des ARN de transfert (ARNt) : les dihydrouridines synthases (Dus) dictant la formation de dihydrouridine via la flavine mononucléotide (FMN) et TrmFO responsable de la méthylation en C5 de l'uridine 54 via la flavine adénosine dinucléotide (FAD) ainsi que le methylènetétrahydrofolate. Afin d'élucider le mécanisme de TrmFO, nous avons élaboré une apoenzyme grâce à une simple mutation qui est efficacement reconstituée in vitro. Nous avons chimiquement synthétisé l'intermédiaire catalytique qui consiste en un FAD-iminium comportant un methylène sur le N5 de l'isoalloxazine. Cette espèce synthétique a été caractérisée par spectrométrie de masse et absorption UV-visible. La reconstitution de TrmFO avec cette molécule restore l'activité in vitro sur un ARNt transcrit prouvant le rôle du FAD comme agent méthylant via une méthylation réductrice. Dus2 réduit spécifiquement U20 et est constituée d'un Dus domaine néanmoins, chez les mammifères un double-stranded RNA-binding domaine (dsRBD) est présent. Afin de comprendre la fonction de cette organisation modulaire, nous avons montré que seule l'enzyme sauvage est active contrairement aux domaines isolés. Nous avons résolu les structures cristallographiques des deux domaines suggérant une redistribution des charges positives en surface. Ce dsRBD dicte la reconnaissance de l'ARNt en se fixant à la tige acceptrice/Tpsi. Ceci est régulé par une extension N-terminal, mis en évidence par des mutations, des titrations RMN ainsi qu’une structure cristallographique en complexe avec un ARN de 22 nucléotides. Ce travail illustre l’acquisition d’un dsRBD au cours de l’évolution dont la fonction est étendu à la reconnaissance des ARNtsPosttranscriptional modification of ribonucleic acids (RNAs) is a crucial maturation step conserved in all domains of life. During my thesis, I have brought structural and functional insights on flavoenzymes involved in transfer RNA (tRNA) modifications: dihydrouridine synthase (Dus) responsible for dihydrouridine formation using flavin mononucleotide (FMN) and TrmFO responsible for C5 methylation of uridine position 54 relying on flavin adenosine dinucleotide (FAD) and methylenetetrahydrofolate. To elucidate the chemical mechanism of TrmFO we designed an apoprotein via a single mutation that could be reconstituted in vitro with FAD. Furthermore, we chemically synthesized the postulated intermediate active species consisting of a flavin iminium harboring a methylene moiety on the isoalloxazine N5 that was further characterized by mass spectrometry and UV-visible spectroscopy. Reconstitution of TrmFO with this molecule restored in vitro activity on a tRNA transcript proving that TrmFO uses FAD as a methylating agent via a reductive methylation.Dus2 reduces U20 and is comprised of a canonical Dus domain however, mammals have an additional double-stranded RNA-binding domain (dsRBD). To bring functional insight for this modular organization, we showed that only full length human Dus2 was active while its isolated domains were not. tRNA recognition is driven by the dsRBD via binding the acceptor and TΨ stem of tRNA with higher affinity then dsRNA as evidenced by NMR. We further solved the X-ray structures for both domains showing redistribution of surface positive charges justifying the involvement of this dsRBD for tRNA recognition in mammalian Dus2. This was attributed to a peculiar N-terminal extension proven by mutational analysis and an X-ray structure of dsRBD in complex with 22-nucleotide dsRNA. Altogether our work illustrates how during evolution, Dus2 enzymes acquired an engineered dsRBD for efficient tRNA binding via a ruler mechanism
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Moissonnier, Loïck. "Etude fonctionnelle et structurale du transporteur de multiple drogues, BmrA, en condition d’équilibre et en temps résolu. Caractérisation structurale de BmrA en liposome par cryoEM." Electronic Thesis or Diss., Lyon 1, 2024. http://www.theses.fr/2024LYO10213.
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Selon l’organisation mondiale de la santé, la résistance aux antibiotiques est un problème majeur pour l’humanité en raison de l’émergence de bactérie multi-résistantes. L’émergence de ces résistances chez les bactéries provient du fait qu’elles sont capables de mettre en place de nombreuses stratégies pour empêcher les antibiotiques de fonctionner. En particulier, la première ligne de défense de ces bactéries est la surexpression de transporteurs ABC (ATP-Binding Cassette) qui expulsent les antibiotiques en dehors de la cellule bactérienne, diminuant leurs concentrations sous leurs seuils de cytotoxicité. Plus de 50 ans d’étude sur ces transporteurs ont permis à la communauté scientifique d’établir un mécanisme global, notamment grâce à l’acquisition de structures 3D de plus en plus nombreuses. Ceci a été intimement lié par l’évolution technologique et méthodologique de la biologie structurale ces dernières années avec notamment l’émergence de la cryoEM. Plus les connaissances avancent plus les questions deviennent précises, et il demeure toujours de nombreuses questions sur la compréhension de leur fonctionnement. Dans le cadre de mon projet, j’ai étudié BmrA, l’un de ces transporteur ABC exprimé chez Bacillus subtilis qui lui confère une résistance à la cervimycine C, un antibiotique sécrété par Streptomyces tendae son compétiteur naturel dans le même biotope. De plus, ce transporteur est capable de fixer et de transporter une grande variété de molécules dont de nombreux antibiotiques en adoptant à la fois une conformation prenant en charge le ligand (IF, conformation ouverte vers l’intérieur de la cellule), et une conformation ouverte vers l’extérieur de la cellule (OF) pour le relarguer. Cette capacité de manipuler plusieurs molécules reste une question très discutée, surtout dans la compréhension du mécanisme de transport au niveau moléculaire. Au cours de ma thèse, j’ai participé à une étude d’enzymologie structurale sur un mutant inactif E504A en présence de ligands (Rhodamine 6G, Hœchst 33342) afin d’améliorer les connaissances sur ce mécanisme. Ces ligands jouent un rôle d’effecteur allostérique sur la fixation d’ATP de BmrA, impactant la transition entre les conformations IF et OF. La résolution de plusieurs structures 3D par cryoEM a été réalisé en variant la concentration d’ATP. Une analyse de la flexibilité de chacune de ces conformations a mis en lumière les réarrangements moléculaires que BmrA peut adopter pour assurer sa poly-spécificité. De plus, j’ai apporté de nombreuses informations fonctionnelles en ce qui concerne le couplage entre le transport du ligand et l’activité ATPasique de ce transporteur. La deuxième partie de mes travaux repose sur l’étude de la transition conformationnelle se produisant chez BmrA après la fixation d’ATP à l’aide de techniques dites « en temps résolu ». L’objectif a été de suivre ces changements conformationnels au cours du temps grâce à la fluorescence intrinsèque de BmrA couplée à la cryoEM. J’ai développé et optimisé les conditions expérimentales pour réaliser cette étude, et notamment acquis des informations cinétiques et dynamiques sur des mutants ainsi que la protéine sauvage.Enfin, la dernière partie du manuscrit a consisté à reconstituer BmrA dans un environnement amphipathique plus natif que les détergents afin d’acquérir sa structure 3D par cryoEM. J’ai optimisé ce protocole de reconstitution pour obtenir le meilleur échantillon possible à déposer sur grille. Au cours de ce processus, j’ai caractérisé la formation du protéoliposome à chaque étape du protocole en l’observant par cryoEM. Grâce à cette étude, j’ai pu obtenir les premières classes 2D de BmrA en bicouche lipidique. En conclusion, cette thèse offre une nouvelle façon d’étudier la relation structure-fonction des protéines en développant des outils et une méthodologie d’enzymologie structurale pour visualiser la dynamique de ce transporteur ABC, ainsi qu’une première approche pour l’étudier en liposomeAccording to the World Health Organization, antibiotic resistance is a major problem for humanity due to the emergence of multiresistant bacteria. The emergence of these resistances in bacteria is due to their ability to implement numerous strategies to prevent antibiotics from working. In particular, the first line of defense of these bacteria is the overexpression of ABC (ATP-Binding Cassette) transporters, which expel antibiotics out of the bacterial cell, reducing their concentrations below their cytotoxic thresholds. Over 50 years of study on these transporters have enabled the scientific community to establish a global mechanism, particularly thanks to the increasing acquisition of 3D structures. This has been closely linked to the technological and methodological evolution of structural biology in recent years, especially with the emergence of cryoEM. As knowledge advances, the questions become more precise, and many questions remain about understanding their functioning. As part of my project, I studied BmrA, one of these ABC transporters expressed in Bacillus subtilis, which confers resistance to cervimycin C, an antibiotic secreted by Streptomyces tendae, its natural competitor in the same biotope. Additionally, this transporter is capable of binding and transporting a wide variety of molecules, including many antibiotics, by adopting both a conformation that takes up the ligand (IF, inward-facing conformation) and an outward-facing conformation (OF) to release it. This ability to handle multiple molecules remains a highly debated question, especially in understanding the transport mechanism at the molecular level. During my Ph.D., I participated in a structural enzymology study on an inactive E504A mutant in the presence of ligands (Rhodamine 6G, Hoechst 33342) to improve knowledge of this mechanism. These ligands act as allosteric effectors on the ATP binding of BmrA, impacting the transition between IF and OF conformations. The resolution of several 3D structures by cryoEM was achieved by varying the concentration of ATP. An analysis of the flexibility of each of these conformations highlighted the molecular rearrangements that BmrA can adopt to ensure its polyspecificity. Moreover, I provided numerous functional insights regarding the coupling between ligand transport and the ATPase activity of this transporter. The second part of my work focused on studying the conformational transition occurring in BmrA after ATP binding using so-called "time-resolved" techniques. The objective was to monitor these conformational changes over time using the intrinsic fluorescence of BmrA coupled with cryoEM. I developed and optimized the experimental conditions to conduct this study, particularly acquiring kinetic and dynamic information on mutants as well as the wild-type protein. Finally, the last part of the manuscript involved reconstituting BmrA in a more native amphipathic environment than detergents to obtain its 3D structure by cryoEM. I optimized this reconstitution protocol to obtain the best possible sample for grid deposition. During this process, I characterized the formation of the proteoliposome at each stage of the protocol by observing it with cryoEM. Thanks to this study, I was able to obtain the first 2D classes of BmrA in a lipid bilayer. In conclusion, this thesis offers a new way to study the structure-function relationship of proteins by developing structural enzymology tools and methodology to visualize the dynamics of this ABC transporter, as well as a first approach to studying it in liposomes
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Kubiak, Xavier. "Etude fonctionnelle et structurale d'arylamine N-acetyltransferases atypiques chez Legionella pneumophila et Bacillus cereus." Paris 7, 2012. http://www.theses.fr/2012PA077080.
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Les arylamine N-acétyltransférases (NATs) sont des enzymes du métabolisme des xénobiotiques ayant un rôle essentiel dans la biotransformation de composés de type aminé aromatique (polluants, médicaments, pré-carcinogènes, etc. ). La structure tridimensionnelle des NATs caractérisées révèle des caractéristiques structurales communes, dont une triade catalytique Cys-His-Asp strictement conservée. Legionella pneumophila et Bacillus cereus sont deux bactéries pathogènes constituant un problème majeur de santé publique. L pneumophila est l'agent étiologique de la légionellose, une pneumopathie sévère, tandis que B. Cereus est responsables de très nombreuses intoxications alimentaires. Ces deux organismes sont ubiquitaires respectivement des milieux aquatiques et des sols, niches qui représentent des lieux d'exposition à des aminés aromatiques. Ces travaux de thèse révèlent l'existence chez L, pneumophila et B, cereus de NATs aux propriétés atypiques en comparaison des isoformes connues à ce jour. L'étude des séquences de NATs issues de trois souches cliniques de L pneumophila montre l'existence d'une hétérogénéité génétique inhabituelle qui affecte les propriétés catalytiques et structurales des trois variants enzymatiques. L'étude in vivo d'une souche delétée du gène nat révèle l'existence d'une voie de détoxication NAT-dépendante des aminés aromatiques chez L. Pneumophila. Chez B. Cereus, nous avons identifié une nouvelle isoforme NAT dont la triade catalytique contient un glutamate au lieu de Paspartate canonique. Nos résultats montrent contre toute attente que cette isoforme est active et correctement repliée. La résolution de la structure 3D de cette nouvelle isoforme révèle une grande similarité de topologie et d'orientation de la triade avec celles de NATs classiques. L'ensemble de ces données suggère l'existence d'une diversité fonctionnelle et structurale bien plus importante que prévue au sein de cette famille d'enzymeArylamine N-acetyltransferases (NATs) are xenobiotic metabolizing enzymes involved in the biotransformation of a wide range of aromatic amine chemicals (pollutants, drugs, pre-carcinogens). The 3D structure of NATs has been recently solved and all NATs characterized to date share the same structural features, including a strictly conserved Cys-His-Asp catalytic triad. Legionella pneumophila and Bacillus cereus are two bacterial pathogens that constitute a public health issue both in France and in the world. L. Pneumophila is the etiologic agent of legionellosis, a severe pneumonia, while B, cereus is responsible for a high number of foodborne intoxications. These two organisms are ubiquitous of aquatic environments and soils, respectively, which provide a risk of exposure to aromatic amine compounds. Our work is focused on L. Pneumophila and B. Cereus NAT isoforms that exhibit atypical features compared to isoforms characterized so far. The study of NATs sequences from three clinical strains of L. Pneumophila reveals several amino acid variations between strains. This unusual sequence heterogeneity leads to variations in catalytic and structural properties in the three variants. The characterization of a nat knock-out strain reveals that L pneumophila possesses in vivo a NAT-dependent detoxification pathway of aromatic amines chemicals. We also demonstrate the existence of a new NAT isoform in B. Cereus that lacks the canonical catalytic triad. Indeed, (BACCR)NAT3 has a glutamate instead of an aspartate at the catalytic position. Against ail expectations this isoform is active and correctly folded. Interestingly, the 3D structure of the enzyme has been solved and shows a classic NAT fold and catalytic triad geometry compared to classical NAT enzymes. Taken together, these results suggest a greater functional and structural diversity than expected in this enzyme family
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Bou, Nader Charles. "Structural and Functional characterization of flavoenzymes involved in posttranscriptional modification of tRNA." Electronic Thesis or Diss., Paris 6, 2017. http://www.theses.fr/2017PA066205.
Full textAbstract:
La modification posttranscriptionnelle des acides ribonucléiques (ARNs) est une étape de maturation conservée dans tous les domaines du vivant. Mes travaux de thèse ont porté sur la caractérisation fonctionnelle et structurale de flavoenzymes impliquées dans la modification des ARN de transfert (ARNt) : les dihydrouridines synthases (Dus) dictant la formation de dihydrouridine via la flavine mononucléotide (FMN) et TrmFO responsable de la méthylation en C5 de l'uridine 54 via la flavine adénosine dinucléotide (FAD) ainsi que le methylènetétrahydrofolate. Afin d'élucider le mécanisme de TrmFO, nous avons élaboré une apoenzyme grâce à une simple mutation qui est efficacement reconstituée in vitro. Nous avons chimiquement synthétisé l'intermédiaire catalytique qui consiste en un FAD-iminium comportant un methylène sur le N5 de l'isoalloxazine. Cette espèce synthétique a été caractérisée par spectrométrie de masse et absorption UV-visible. La reconstitution de TrmFO avec cette molécule restore l'activité in vitro sur un ARNt transcrit prouvant le rôle du FAD comme agent méthylant via une méthylation réductrice. Dus2 réduit spécifiquement U20 et est constituée d'un Dus domaine néanmoins, chez les mammifères un double-stranded RNA-binding domaine (dsRBD) est présent. Afin de comprendre la fonction de cette organisation modulaire, nous avons montré que seule l'enzyme sauvage est active contrairement aux domaines isolés. Nous avons résolu les structures cristallographiques des deux domaines suggérant une redistribution des charges positives en surface. Ce dsRBD dicte la reconnaissance de l'ARNt en se fixant à la tige acceptrice/Tpsi. Ceci est régulé par une extension N-terminal, mis en évidence par des mutations, des titrations RMN ainsi qu’une structure cristallographique en complexe avec un ARN de 22 nucléotides. Ce travail illustre l’acquisition d’un dsRBD au cours de l’évolution dont la fonction est étendu à la reconnaissance des ARNtsPosttranscriptional modification of ribonucleic acids (RNAs) is a crucial maturation step conserved in all domains of life. During my thesis, I have brought structural and functional insights on flavoenzymes involved in transfer RNA (tRNA) modifications: dihydrouridine synthase (Dus) responsible for dihydrouridine formation using flavin mononucleotide (FMN) and TrmFO responsible for C5 methylation of uridine position 54 relying on flavin adenosine dinucleotide (FAD) and methylenetetrahydrofolate. To elucidate the chemical mechanism of TrmFO we designed an apoprotein via a single mutation that could be reconstituted in vitro with FAD. Furthermore, we chemically synthesized the postulated intermediate active species consisting of a flavin iminium harboring a methylene moiety on the isoalloxazine N5 that was further characterized by mass spectrometry and UV-visible spectroscopy. Reconstitution of TrmFO with this molecule restored in vitro activity on a tRNA transcript proving that TrmFO uses FAD as a methylating agent via a reductive methylation.Dus2 reduces U20 and is comprised of a canonical Dus domain however, mammals have an additional double-stranded RNA-binding domain (dsRBD). To bring functional insight for this modular organization, we showed that only full length human Dus2 was active while its isolated domains were not. tRNA recognition is driven by the dsRBD via binding the acceptor and TΨ stem of tRNA with higher affinity then dsRNA as evidenced by NMR. We further solved the X-ray structures for both domains showing redistribution of surface positive charges justifying the involvement of this dsRBD for tRNA recognition in mammalian Dus2. This was attributed to a peculiar N-terminal extension proven by mutational analysis and an X-ray structure of dsRBD in complex with 22-nucleotide dsRNA. Altogether our work illustrates how during evolution, Dus2 enzymes acquired an engineered dsRBD for efficient tRNA binding via a ruler mechanism
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Nusbaum, Julien. "Caractérisation structurale et fonctionnelle de la peptide déformylase du phage Vp16T." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLS510/document.
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Les protéines en cours de synthèse subissent des modifications très précoces de leur extrémité N-terminale, dès lors que celle-ci émerge du tunnel de sortie du ribosome. La première modification est l’excision de la méthionine initiatrice, assurée par une méthionine aminopeptidase (MetAP), précédée de sa déformylation par une enzyme peptide déformylase (PDF) chez les bactéries et dans les mitochondries et chloroplastes. Ce processus est ubiquitaire et essentiel, et a été décrit dans tout le règne du vivant. Chez les bactéries, les PDFs de type 1B se fixeraient au ribosome à proximité de l’extrémité du tunnel de sortie du peptide naissant, via son hélice α C-terminale. Or des analyses métagénomiques récentes ont révélé la présence insoupçonnée de gènes codant des PDFs putatives chez des virus marins. De manière inattendue, toutes les PDF virales présentent des séquences C-terminales très courtes et dépourvues de l’hélice α3. L’identification de ces PDFs atypiques soulève alors de nouvelles questions quant à leur possible interaction au ribosome et à leur fonction biologique. L’objectif de ma thèse a donc été de réaliser la caractérisation complète et intégrée de la peptide déformylase du bactériophage Vp16T, dont la séquence est l’une des plus courtes connues à ce jour. J’ai montré que le phage Vp16T code une protéine active, in vivo et in vitro, et qu’elle peut se lier au ribosome malgré l’absence d’hélice α C-terminale. La caractérisation structure-fonction de Vp16PDF a révélé des caractéristiques uniques qui pourraient alors expliquer sa fonction au cours de la réplication du phage. Ainsi j’ai montré que l’expression de Vp16PDF chez E. coli modifie la structure de l’enveloppe, induit l’accumulation d’agrégats et finalement inhibe la croissance bactérienne. De plus, l’étude de souches bactériennes mutantes a montré que Vp16PDF interfère spécifiquement avec le repliement et l’adressage de protéines membranaires. Cette dernière fonction pourrait permettre de déstabiliser la membrane de l’hôte et ainsi favoriser la libération des particules viralesBeing synthesized proteins undergo very early changes in their N-terminal end, since it emerges from the outlet channel of the ribosome. The first modification is the excision of the initiator methionine, provided by a methionine aminopeptidase (MetAP), preceded by its deformylating enzyme peptide deformylase (PDF) in bacteria and in mitochondria and chloroplasts. This process is ubiquitous and essential, and has been described in the kingdom of life. In bacteria, Type 1B PDFs would bind to the ribosome near the end of the outlet tunnel of the nascent peptide via its C-terminal helix α. But recent metagenomic analyzes revealed the unexpected presence of genes encoding putative PDFs in marine viruses. Unexpectedly, all viral PDF have very short C-terminal sequences and lacking the α3 helix. The identification of these atypical PDFs then raises new questions about their possible interaction with ribosome and their biological function. The aim of my thesis was therefore to achieve the complete and integrated characterization of peptide deformylase bacteriophage Vp16T, the sequence is one of the shortest known to date. I showed that the phage Vp16T code an active protein in vivo and in vitro, and can bind to the ribosome despite the absence of the C-terminal helix α. The structure-function characterization Vp16PDF revealed unique features that could then explain its function in the replication of the phage. Thus I have shown that expression in E. coli Vp16PDF modifies the envelope structure, induces accumulation of aggregates and ultimately inhibits bacterial growth. In addition, the study of mutant bacterial strains showed that Vp16PDF specifically interfere with the folding and addressing of membrane proteins. This latter function could help destabilize the membrane of the host and thereby promote release of viral particles
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Bazeille, Nicolas. "Caractérisation structurale et fonctionnelle de l’hélicase du syndrome de Bloom et analyse de la toxicité du cadmium sur cette enzyme." Thesis, Paris 11, 2011. http://www.theses.fr/2011PA112340.
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La double hélice d’ADN est une structure stable qui assure à la fois la sauvegarde et la transmission de l’information génétique. Pour accéder à cette information, une vaste famille d’enzymes multifonctionnelles appelées hélicases réalise la séparation des bases complémentaires de l’ADN. Certaines de ces hélicases sont associées chez l’homme à des syndromes de prédisposition au cancer. C’est le cas du syndrome de Bloom (BS), une maladie génétique à transmission récessive qui se traduit par une augmentation de l’instabilité génétique mais où aucun phénomène d’haplo-insuffisance ou de dominance négative n’est constaté chez les porteurs hétérozygotes. On reconnait pourtant que la protéine du syndrome de Bloom (BLM) adopte une structure multimérique in vitro mais sans que l’expression chez certains hétérozygotes d’une enzyme inactive ne soit considérée comme un facteur à risque. Pour expliquer ce paradoxe, nous avons étudié la structure de l’enzyme BLM et constater qu’elle fonctionne sous la forme d’un monomère, un résultat nouveau qui justifie mieux pourquoi ces formes inactives n’influence pas le degré de prédisposition au cancer. D’autre part, la toxicité cadmium est susceptible d’avoir un lien direct avec l’inactivation de l’hélicase BLM car les cellules exposées au cadmium présentent des analogies avec celles des patients atteints du syndrome de Bloom. Effectivement, nous avons observé qu’in vitro, de faibles concentrations de cadmium réduisent les activités de cette hélicase en induisant son oligomérisation. Ces travaux apportent des informations nouvelles sur le mécanisme moléculaire de l’hélicase BLM et soulignent son importance dans le maintien de l’intégrité du génomeThe DNA double helix is a stable structure that ensures both the protection and transmission of genetic information. To access this information, a large family of multifunctional enzymes called helicases performs the separation of complementary bases of DNA. Some of these helicases in humans are associated with cancer predisposition syndromes. This is the case of Bloom syndrome (BS), a recessive genetic disease that results in an increase in genetic instability but where no phenomenon of haploinsufficiency or dominant negative is found in carriers heterozygotes. Yet we recognize that the Bloom syndrome protein (BLM) adopts a multimeric structure in vitro, but the expression among some heterozygotes of an inactive enzyme is not considered as a risk factor. To explain this paradox, we studied the structure of the BLM and find that it works as a monomer, a new result which justifies why most inactive forms does not influence the degree of cancer predisposition. On the other hand, cadmium toxicity is potentially linked to the inactivation of the BLM helicase as cells exposed to cadmium present analogies with those of patients with Bloom syndrome. Indeed, we observed in vitro, that low concentrations of cadmium reduce helicase activity by promoting its oligomerization. These studies provide new information on the molecular mechanism of the BLM helicase and emphasize its importance in maintaining genome integrity
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Reymann, Jean-Marc. "Aldose reductase de cristallin de porc : enzymologie et structure." Université Louis Pasteur (Strasbourg) (1971-2008), 1993. http://www.theses.fr/1993STR13085.
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L'aldose reductase (ar) est un enzyme qui catalyse la reduction par le nadph du glucose en sorbitol, et dont l'inhibition chez les sujets diabetiques presente un interet pharmacologique. Une revue faisant le point des connaissances acquises sur l'ar depuis sa decouverte jusqu'a nos jours est exposee. Une procedure originale et performante de l'isolement de l'ar a partir de cristallin de porc est decrite. Le developpement d'une autre forme de l'enzyme resultant de l'oxydation de l'ar au cours de la purification et du stockage est mis en evidence. Les resultats d'etudes biochimiques et cristallographiques suggerant l'existence in vivo de l'ar sous la forme holo-enzyme sont presentes. La formation d'oligomeres est montree dans le cas de la forme apo-enzyme de l'ar, la forme (native) holo-enzyme etant de nature strictement monomerique. Des cristaux d'ar sous la forme apo- et holo-enzyme sont obtenus. La structure tridimensionnelle de l'ar de cristallin de porc est resolue a partir de l'analyse aux rayons x de ces cristaux. La connaissance de cette structure devrait aider a la conception d'inhibiteurs beaucoup plus specifiques de l'ar que ceux actuellement disponibles
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Rahman, Pour Rahman. "Enzymology and structural enzymology of dye-decolorizing peroxidases and a primary study of encapsulin." Thesis, University of Warwick, 2015. http://wrap.warwick.ac.uk/73395/.
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The aim of this project is to provide a detailed comparative study in enzymology of dye-decolorising peroxidases, DyP, from Pseudomonas fluorescens and from Thermobifida fusca, a thermophile bacterium. Another objective is a primary study of encapsulin, a recently discovered icosahedral nanocompartment protein from Rhodococcus jostii Rha1. Three peroxidase genes from P. fluorescens and one from T. fusca were cloned, expressed, and their products were purified and the enzymes kinetically characterized with different substrates, lignin model compounds and lignocellulose. In addition, encapsulin has been purified, its assembly/disassembly under different pH conditions was studied and finally, its presence or absence in the extracellular fraction of R. jostii investigated. DyP type peroxidases from Gram-positive bacteria have been studied recently and showed oxidation activity toward Mn (II) and lignin model compounds. Gram-negative pseudomonads, also show activity for lignin oxidation and contain DyP-type peroxidase genes. P. fluorescens Pf-5 contains three DyP-type peroxidases (35, 40 and 47 kDa). In this study each of them was overexpressed in Escherichia coli, purified, and characterised by different substrates, Kraft lignin and lignocellulose. Each of the aforementioned enzymes shows activity for oxidation of most of the substrates, but the 35 kDa DyP1B and 40 kDa DyP2B enzymes show activity for oxidation of Mn (II). Only in the presence of Mn (II) and hydrogen peroxide, incubation of finely powdered lignocellulose with DyP1B leads to the release of a low molecular weight lignin fragment that was identified by mass spectrometry as a ß-aryl ether lignin dimer that contains one G unit and one H unit bearing a benzylic ketone. A mechanism for releasing of the -aryl ether lignin dimer fragment from the lignin molecule via oxidation is proposed. A DyP-type peroxidase enzyme from the thermophilic cellulose degrader Thermobifida fusca was investigated for its catalytic ability for lignin oxidation. TfuDyP was found to oxidise a ß-aryl ether lignin model compound, forming an oxidised tetramer. A crystal structure of TfuDyP was determined, to 1.7 Å resolution, which was found to contain a diatomic oxygen ligand bound to the heme centre, hydrogen-bonded to active site residues Asp-203 and Arg-315. For three amino acid residues present in distal heme pocket, site directed mutagenesis was performed and the effect of each mutation on enzyme activity was measured by three different substrates. Recently DyPB peroxidase from Rhodococcus jostii RHA1 has been recognised as a bacterial lignin peroxidase enzyme. The dypB gene is next to a gene that encodes an encapsulin protein that previously was shown in Thermotoga maritima to assemble and form into a nano-compartment comprised of 60-subunits. DyPB protein contains a C-terminal sequence motif that is supposed to lead the protein to the encapsulin nanocompartment. In this study, R. jostii RHA1 encapsulin gene was overexpressed in R. jostii RHA1, and the encapsulin protein was extracted as a high molecular weight native assembly (Mr >106 kDa). It was shown that by treatment of the purified nanocompartment at pH 3.0, it is able to be denatured to form a low molecular weight species and most importantly it is able to be re-assembled to form the native nanocompartment at pH 7.0. Dynamic light scattering showed that DyPB peroxidase in vitro could be assembled with encapsulin in a monomeric state to form an assembly of encapsulated DyPB in similar size and shape compared to the encapsulin-only nanocompartment. By using a nitrated lignin UV-Vis assay method, it was shown that the assembled complex of DyPB-encapsulin exhibited enhanced lignin degradation activity per mg DyPB present, compared with native DyPB. The stoichiometry of encapsulin/µmol DyPB in the assembled complex was measured, 8.6 mol encapsulin/mol DyPB, that was comparable to the predicted value of 10 obtained from the crystal structure.
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Jansson, Anna. "Structural enzymology of the biosynthesis of polyketide antibiotics /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-916-1/.
Full textBooks on the topic "Enzymologie structurale"
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Park, Kwan-Hwa. Carbohydrate-active enzymes: Structure, function and applications. Cambridge: Woodhead Publishing Ltd, 2008.
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Miller, Justin M., ed. Mechanistic Enzymology: Bridging Structure and Function. Washington, DC: American Chemical Society, 2020. http://dx.doi.org/10.1021/bk-2020-1357.
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Agricultural Biotechnology Symposium on "Carbohydrate-Active Enzymes: Structure, Function, and Applications" (2008 Seoul National University). Carbohydrate-active enzymes: Structure, function and applications. Boca Raton: CRC Press, 2008.
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Copeland, Robert Allen. Enzymes: A practical introduction to structure, mechanism, and data analysis. New York: VCH Publishers, 1996.
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I, Mackness M., Clerc M, North Atlanitic Treaty Organization. Scientific Affairs Division., and NATO Advanced Research Workshop on Esterases, Lipases, and Phospholipases (1993 : Bordeaux, France), eds. Esterases, lipases, and phospholipases: From structure to clinical significance. New York: Plenum, 1994.
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R, Acharya K., ed. Glycogen phosphorylase b: Description of the protein structure. Singapore: World Scientific, 1991.
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Castellano, Immacolata. Gamma-glutamyl transpeptidases: Structure and function. Basel: Springer, 2013.
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Dugas, Hermann. Bioorganic chemistry: A chemical approach to enzyme action. 3rd ed. New York: Springer, 1996.
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Dugas, Hermann. Bioorganic chemistry: A chemical approach to enzyme action. 2nd ed. New York: Springer-Verlag, 1989.
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Copeland, Robert A. Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis. Vch Pub, 1996.
Find full textBook chapters on the topic "Enzymologie structurale"
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Korman, Tyler Paz, Brian Douglas Ames, and Shiou-Chuan Tsai. "Structural Enzymology of Aromatic Polyketide Synthase." In ACS Symposium Series, 167–84. Washington, DC: American Chemical Society, 2007. http://dx.doi.org/10.1021/bk-2007-0955.ch012.
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Robyt, John F. "Starch: Structure, Properties, Chemistry, and Enzymology." In Glycoscience, 1437–72. Berlin, Heidelberg: Springer Berlin Heidelberg, 2008. http://dx.doi.org/10.1007/978-3-540-30429-6_35.
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Champoux, James J. "Human DNA Topoisomerase I: Structure, Enzymology and Biology." In Cancer Drug Discovery and Development, 53–69. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4614-0323-4_2.
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"Methods in Enzymology." In G Protein Coupled Receptors - Structure, xix—liv. Elsevier, 2013. http://dx.doi.org/10.1016/b978-0-12-391861-1.09989-5.
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Karabencheva, Tatyana, and Christo Christov. "Structural and Computational Enzymology." In Structural and Mechanistic Enzymology - Bringing Together Experiments and Computing, 1–4. Elsevier, 2012. http://dx.doi.org/10.1016/b978-0-12-398312-1.00001-9.
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Dey, Mishtu. "Enzymology of Microbial Dimethylsulfoniopropionate Catabolism." In Structural and Mechanistic Enzymology, 195–222. Elsevier, 2017. http://dx.doi.org/10.1016/bs.apcsb.2017.05.001.
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Gupta, Munishwar Nath, and Vladimir N. Uversky. "Enzymology: early insights." In Structure and Intrinsic Disorder in Enzymology, 1–29. Elsevier, 2023. http://dx.doi.org/10.1016/b978-0-323-99533-7.00013-3.
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Korman, Tyler Paz, Brian Ames, and Shiou-Chuan (Sheryl) Tsai. "Structural Enzymology of Polyketide Synthase: The Structure–Sequence–Function Correlation." In Comprehensive Natural Products II, 305–45. Elsevier, 2010. http://dx.doi.org/10.1016/b978-008045382-8.00020-4.
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Cohen, Fred E., and scott R. Presnell. "The combinatorial approach." In Protein Structure Prediction, 207–28. Oxford University PressOxford, 1996. http://dx.doi.org/10.1093/oso/9780199634972.003.0009.
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Abstract With the advent of protein X-ray crystallography in the 1950s and the more recent use of multidimensional NMR spectroscopy, biologists have become familiar with the detailed organization of protein structures at the atomic level. These structures have fundamentally changed our understanding of mechanistic enzymology and molecular recognition in the immune system. More recently, the ability of structural studies to influence drug discovery and design has become clear (1). In spite of these advances, the rate of pro tein sequence determination via DNA sequencing vastly exceeds the rate of structure determination. How can this gap between sequence and structure be bridged? With an increase in the number of proteins of known structure, the likelihood that a new sequence will be similar to a known structure increases. Currently, ∼20% of the sequences identified in genome sequencing efforts are related to well-characterized proteins (2).
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Yasunobu, Kerry T., and Anthony Tan. "Advances in Monoamine Oxidase Enzymology." In Structure and Functions of Amine Oxidases, 209–17. CRC Press, 2018. http://dx.doi.org/10.1201/9781351076951-20.
Full textConference papers on the topic "Enzymologie structurale"
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Davies, Gideon J. "FROM LYSOZYME AND BACK AGAIN: STRUCTURAL ENZYMOLOGY OF GLYCOSYL TRANSFER." In XXIst International Carbohydrate Symposium 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.382.
Full textReports on the topic "Enzymologie structurale"
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Morrison, Mark, Joshuah Miron, Edward A. Bayer, and Raphael Lamed. Molecular Analysis of Cellulosome Organization in Ruminococcus Albus and Fibrobacter Intestinalis for Optimization of Fiber Digestibility in Ruminants. United States Department of Agriculture, March 2004. http://dx.doi.org/10.32747/2004.7586475.bard.
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Improving plant cell wall (fiber) degradation remains one of the highest priority research goals for all ruminant enterprises dependent on forages, hay, silage, or other fibrous byproducts as energy sources, because it governs the provision of energy-yielding nutrients to the host animal. Although the predominant species of microbes responsible for ruminal fiber degradation are culturable, the enzymology and genetics underpinning the process are poorly defined. In that context, there were two broad objectives for this proposal. The first objective was to identify the key cellulosomal components in Ruminococcus albus and to characterize their structural features as well as regulation of their expression, in response to polysaccharides and (or) P AA/PPA. The second objective was to evaluate the similarities in the structure and architecture of cellulosomal components between R. albus and other ruminal and non-ruminal cellulolytic bacteria. The cooperation among the investigators resulted in the identification of two glycoside hydrolases rate-limiting to cellulose degradation by Ruminococcus albus (Cel48A and CeI9B) and our demonstration that these enzymes possess a novel modular architecture specific to this bacterium (Devillard et al. 2004). We have now shown that the novel X-domains in Cel48A and Cel9B represent a new type of carbohydrate binding module, and the enzymes are not part of a ceiluiosome-like complex (CBM37, Xu et al. 2004). Both Cel48A and Cel9B are conditionally expressed in response to P AA/PPA, explaining why cellulose degradation in this bacterium is affected by the availability of these compounds, but additional studies have shown for the first time that neither PAA nor PPA influence xylan degradation by R. albus (Reveneau et al. 2003). Additionally, the R. albus genome sequencing project, led by the PI. Morrison, has supported our identification of many dockerin containing proteins. However, the identification of gene(s) encoding a scaffoldin has been more elusive, and recombinant proteins encoding candidate cohesin modules are now being used in Israel to verify the existence of dockerin-cohesin interactions and cellulosome production by R. albus. The Israeli partners have also conducted virtually all of the studies specific to the second Objective of the proposal. Comparative blotting studies have been conducted using specific antibodies prepare against purified recombinant cohesins and X-domains, derived from cellulosomal scaffoldins of R. flavefaciens 17, a Clostridium thermocellum mutant-preabsorbed antibody preparation, or against CbpC (fimbrial protein) of R. albus 8. The data also suggest that additional cellulolytic bacteria including Fibrobacter succinogenes S85, F. intestinalis DR7 and Butyrivibrio fibrisolvens Dl may also employ cellulosomal modules similar to those of R. flavefaciens 17. Collectively, our work during the grant period has shown that R. albus and other ruminal bacteria employ several novel mechanisms for their adhesion to plant surfaces, and produce both cellulosomal and non-cellulosomal forms of glycoside hydrolases underpinning plant fiber degradation. These improvements in our mechanistic understanding of bacterial adhesion and enzyme regulation now offers the potential to: i) optimize ruminal and hindgut conditions by dietary additives to maximize fiber degradation (e.g. by the addition of select enzymes or PAA/PPA); ii) identify plant-borne influences on adhesion and fiber-degradation, which might be overcome (or improved) by conventional breeding or transgenic plant technologies and; iii) engineer or select microbes with improved adhesion capabilities, cellulosome assembly and fiber degradation. The potential benefits associated with this research proposal are likely to be realized in the medium term (5-10 years).