Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第13793号 / 工博第2897号 / 新制||工||1428(附属図書館) / 26009 / UT51-2008-C709 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 濵地 格 / 学位規則第4条第1項該当

Depuis la découverte des sources hydrothermales en 1977, un petit nombre d'études ont permis l'isolement et la caractérisation de micro-organismes pouvant résister à de hautes pressions et températures. Parmi ceux-ci, Pyrococcus yayanosii, une archée hyperthermophile de l'ordre des Thermococcales est issue du site Ashadze (dorsale medio-atlantique) à 4100 m de profondeur. Cette espèce représente le premier organisme à la fois hyperthermophile et piézophile strict décrit à ce jour. Elle ne peut en effet se diviser à des pressions inférieures à 20 MPa et sa pression optimale de croissance est de 52 MPa. Afin d'étudier les mécanismes que met en oeuvre cette espèce pour se développer sous hautes pressions, des expériences de transcriptomique (puces à ADN) et protéomique (LC-MS/MS) ont été entreprises à différentes pressions notamment sub- et supraoptimales. La distinction entre les effets « stress » et « adaptations » à la pression a été effectué en comparant les résultats obtenus chez une autre Thermoccocale proche, Pyrococcus furiosus, qui est piézosensible. La détermination des pressions sub et supra optimales a été préalablement effectuée sur une large gamme de pression hydrostatique. Des analyses génomiques ont aussi été effectuées sur les Thermococcales en général et ses deux espèces en particulier et montrent des différences importantes au niveau des voies de biosynthèse des acides aminés ainsi que des transporteurs membranaires. Les analyses transcriptomiques et protéomiquesmontrent que P. yayanosii joue essentiellement sur ses mécanismes de production d'énergie (métabolisme del'hydrogène), de mobilité (chimiotactisme), de traduction (protéines ribosomales) ainsi que sur ses mécanismes de défense (CRISPR/cas). P. furiosus met en place des mécanismes se basant aussi sur la traduction et la mobilité (archaellum). Il semble que ces derniers puissent ainsi être considérés comme des réponses aux stress, alors que la modulation énergétique uniquement présente chez P. yayanosii soit plus un « shift » métabolique permettant à la cellule de s'adapter aux différentes conditions de pression de son environnement. / No

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Les archées sont principalement connues pour leur capacité à croître et survivre dans des conditions extrêmes de température, pression, pH, etc. qui sont hostiles à l’homme. Néanmoins, il est désormais clair que les archées sont aussi présentes de manière ubiquitaire dans divers environnements. L’étude détaillée des différents aspects de la biologie de ces microorganismes a amené à des découvertes pour le moins inattendues comme celle de la virosphère associée aux archées qui est unique. En effet, plusieurs virus infectant les archées ont été isolés et présentent une incroyable diversité tant au niveau morphologique que génomique et ne ressemblent aucunement aux virus connus de bactéries ou d’eucaryotes. Récemment, l’analyse en détails du cycle viral a mis à jour de nouveaux mécanismes d’interactions avec la cellule hôte. Au cours de mes travaux de thèse, nous nous sommes intéressés aux systèmes virus-hôtes présents dans les milieux hyperthermiques et acidophiles en sélectionnant les virus fusiforme et filamenteux SSV1 et SIRV2 en tant que modèles d’étude. Tout d’abord, nous avons défini une nouvelle classification des virus fusiformes en basée sur l’analyse comparative des protéines structurales et des génomes viraux. L’ensemble des virus considérés forme un réseau global malgré le fait qu’ils ont été isolés dans des environnements distincts ; qu’ils infectent des hôtes qui sont distant phylogénétiquement parlant et que certains de leurs virions présentent une certaine pléomorphicité. Ensuite, la caractérisation en détails de l’architecture des virions fusiformes de SSV1 a révélé qu’ils étaient enveloppés, composés de protéines de capside glycosylées et contenaient le complexe nucléoprotéique. Finalement, nous nous sommes concentrés sur la manière dont les virus d’archées interagissent avec la cellule hôte. Alors que les virions de SIRV2 semblent utiliser une stratégie pour l’entrée qui est similaire aux bactériophages dits flagellotrophiques ; on observe que les virions de SSV1 emploient un mécanisme de sortie qui rappelle le bourgeonnement des virus eucaryotes enveloppés. L’ensemble de ces recherches participent à une meilleure compréhension de la biologie des archées ainsi que de leurs virus et permettent de définir des cibles intéressantes pour de futures études. / Although, archaea were initially regarded as exotic microorganisms capable of growing in conditions which are hostile to humans, it became clear that they are ubiquitous and abundant in various environments. Detailed studies focusing on different aspects of archaeal biology have led to many unexpected discoveries, including the unique virosphere associated with archaea. Indeed, highly diverse viruses characterized by uncommon virion shapes and mysterious genomic contents have been isolated that typically do not resemble viruses of either bacteria or eukaryotes. Recent analysis of the sequential events of the viral cycle resulted in major breakthroughs in the field. In the framework of my PhD studies, I have focused on two model hyperthermo-acidophilic virus-host systems, the spindle-shaped SSV1 and rod-shaped SIRV2, both infecting organisms of the genus Sulfolobus. Initially, we defined structure-based lineages for all known spindle-shaped viruses isolated from highly divergent hosts and residing in very different environments. Then, we provided insights into the architecture of spindle-shaped viruses by showing that SSV1 virions are composed of glycosylated structural proteins and contain a lipid envelope. Finally, we focused on virus-host interplay. Whereas SIRV2 virions appear to use a similar entry strategy as flagellotrophic bacteriophages, SSV1 virions employ an exit mechanism reminiscent of the budding of eukaryotic enveloped viruses. Collectively, these studies shed light on the biology of archaeal viruses and help to define interesting targets that should be the focus of intensive research in the next future.

Kyoto University (京都大学) / 0048 / 新制・論文博士 / 博士(工学) / 乙第12200号 / 論工博第3989号 / 新制||工||1438(附属図書館) / 26272 / UT51-2008-C970 / (主査)教授 今中 忠行, 教授 青山 安宏, 教授 森 泰生 / 学位規則第4条第2項該当

Orientadores: Glaucia Maria Pastore, Fábio Márcio Squina / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos / Made available in DSpace on 2018-08-22T10:16:22Z (GMT). No. of bitstreams: 1 Silva_JunioCota_D.pdf: 6755717 bytes, checksum: 8ce3ba1fb6b01179b70462bf26349499 (MD5) Previous issue date: 2013 / Resumo: Atualmente há uma crescente demanda para o desenvolvimento de combustíveis não-fósseis alternativos. Assim, como a biomassa lignocelulósica é uma das fontes de energia mais abundantes na natureza, pode ser estabelecida uma economia verde e sustentável, com o objetivo de processar a grande quantidade de energia estocada nessas matérias-primas. O etanol de cana-de-açúcar é uma das melhores opções em biocombustíveis e sua produção pode mais que dobrar, se os açúcares constituintes da parede celular vegetal forem utililizados. No entanto, o alto custo de produção das enzimas para hidrolisar e processar os materiais lignocelulósicos é um fator altamente limitante para o uso de tecnologias verdes. Este trabalho se propôs a avaliar novos biocatalisadores e construir uma enzima quimérica na tentativa de obter glicosidases com melhor desempenho que as já relatadas. Enzimas despolimerizadoras de ß-1,3-glucanos têm consideráveis aplicações biotecnológicas, incluindo produção de biocombustíveis, insumos químicos e farmacêuticos. No segundo capítulo, mostramos a caracterização funcional e a estrutura de baixa resolução da laminarase hipertermofílica de Thermotoga petrophila (TpLam), além de seu modo de operação por eletroforese capilar de zona, mostrando que ela cliva especificamente ligações ß-1,3-glucosídicas internas. O dicroismo circular (CD) UV-distante demonstrou que TpLam é formada principalmente por elementos estruturais do tipo beta, e a estrutura secundária é preservada após incubação por 16 horas a 90 º C. A forma determinada pelo pequeno espalhamento de raios-X a baixo ângulo revelou uma arquitetura de multi-domínio da enzima, com um arranjo de envelope em forma de V, no qual os dois módulos de ligação de carboidrato estão ligados ao domínio catalítico. A engenharia de enzimas multifuncionais pode melhorar coquetéis enzimáticos para tecnologias emergentes de biocombustíveis. Dinâmica molecular através de modelos baseados em estrutura (SB) é uma ferramenta eficaz para avaliar a disposição tridimensional das enzimas quiméricas, bem como para inferir a viabilidade funcional antes da validação experimental. No terceiro capítulo, descrevemos a montagem computacional de uma quimera bifuncional xilanase-liquenase (XylLich), usando os genes xynA e bglS de Bacillus subtilis. As análises in silico da área de superfície acessível ao solvente (SAS) e da raiz quadrada média das flutuações (RMSF) previram uma quimera completamente funcional, ou seja, uma enzima cujo substrato tem acesso ao seu sítio catalítico com pequenas flutuações e variações ao longo das cadeias polipeptídicas. A quimera preservou as características bioquímicas das enzimas parentais, com exceção de uma pequena variação na temperatura de operação e na eficiência catalítica (kcat / Km). Também foi verificado ausência de mudanças significativas no modo de operação catalítico. Além disso, a produção de enzimas quiméricas pode ser mais rentável do que a produção de uma única enzima separadamente, comparando-se o rendimento da produção de proteína recombinante e a atividade hidrolítica da enzima quimérica com as enzimas parentais. ß-Glicosidases (BGLs) são enzimas muito úteis e com grande potencial para serem empregadas em diversos processos industriais. Entretanto, algumas características são essenciais para tornar viáveis as aplicações, como por exemplo estabilidade à temperatura e ao pH, bem como baixa inibição por íons e outros compostos químicos. Assim, no quarto capítulo buscamos estudar três BGLs dos organismos extremófilos Pyrococcus furiosus e Thermotoga petrophila. Os genes PfBgl1, TpBgl1 and TpBgl3 foram clonados no vetor pET28a e as proteínas expressadas em Escherichia coli e posteriormente purificadas em duas etapas cromatográficas. As enzimas purificadas foram avaliadas quanto ao pH e temperatura de atividade, sendo que as BGLs da família GH1 (PfBgl1 e TpBgl1) apresentaram faixas mais largas de pH e temperatura de operação do que a família GH3 (TpBgl3). As BGLs mostraram grande estabilidade ao pH e o maior tempo de meia-vida (a 99 ° C) foi verificado no pH 6, e além disso, não foram significativamente afetadas pela presença de EDTA ou de íons, exceto a TpBgl1 que foi inibida por Hg2+ e Fe2+. As atividades específicas para um conjunto de diferentes substratos sugeriram que TpBgl3 é mais específica que as BGLs GH1. O kcat e kcat / Km em 4-nitrofenol-ß-D-glicopiranosídeo (pNPG) indicam que TpBgl3 é a mais eficiente para hidrólise do substrato, embora seja a enzima que foi inibida com a menor concentração de glicose (30.1 mM). Além disso, as BGLs foram analisadas quanto à influência de seis monossacarídeos na catálise, e demonstraram serem fracamente inibidas pela maioria dos açúcares testados. Os ensaios de CD UV-distante revelaram que a estrutura secundária das BGLs não é afetada pelas variações de pH, e os estudos de desnaturação térmica evidenciaram que as BGLs são proteínas hipertermofílicas / Abstract: There is an increasing demand for the development of alternative non-fossil fuels. Thus, since the lignocellulosic biomass is the most abundant source in nature, it may be settled a green and sustainable economy, aiming to process the great amount of energy stocked in these raw materials. The ethanol from sugarcane is one of the best options concerning biofuels and its productivity could be raised more than double if the use of sugars constituents of plant cell wall is considered. However the high production cost of the enzymes to hydrolyze and process lignocellulose is a great limiting factor for green technologies. In this way, this work proposed to evaluate new enzymes and engineer a chimeric enzyme in the attempt to prospect glycosyl hydrolases with better performance than those reported up to date. 1,3-ß-Glucan depolymerizing enzymes have considerable biotechnological applications including the production of biofuels, feedstock-chemicals and pharmaceuticals. In the first chapter we showed the comprehensive functional characterization and low-resolution structure of hyperthermophilic laminarase from Thermotoga petrophila (TpLam), besides its mode of operation through capillary zone electrophoresis, which specifically cleaves internal ß-1,3-glucosidic bonds. Far-UV circular dichroism demonstrated that LamA is formed mainly by beta structural elements, and the secondary structure is maintained after incubation up to 16 hours at 90ºC. The structure determined by small angle X-ray scattering revealed a multi-domain structural architecture of the enzyme with a V-shape envelope arrangement of the two carbohydrate binding modules in relation to the catalytic domain. Multifunctional enzyme engineering can improve enzyme cocktails for emerging biofuel technology. Molecular dynamics through structure-based models (SB) is an effective tool for assessing the tridimensional disposal of chimeric enzymes as well as for inferring the functional practicability before experimental validation. In the second chapter we describe the computational design of a bifunctional xylanase-lichenase chimera (XylLich) using the xynA and bglS genes from Bacillus subtilis. In silico analysis of the average surface accessible area (SAS) and the root mean square fluctuation (RMSF) predicted a fully functional chimera, i.e. the substrate has access to the catalytic pocket with minor fluctuations and variations along the polypeptide chains. The chimera preserved the biochemical characteristics of the parental enzymes, with the exception of a slight variation in the temperature of operation and the catalytic efficiency (kcat/Km). The absence of substantial shifts in the catalytic mode of operation was also verified. Furthermore, the production of chimeric enzymes could be more profitable than producing a single enzyme separately, based on comparing the recombinant protein production yield and the hydrolytic activity achieved for XylLich with that of the parental enzymes. ß-Glucosidases (BGLs) are very useful enzymes with a great potential to be employed in several industrial processes. However, some features are required to become viable the enzyme applications, such as temperature and pH stability as well, low ions and chemicals inhibition. Thus this work aimed to study three BGLs from the extremophiles organisms Pyrococcus furiosus and Thermotoga petrophila. The genes PfBgl1, TpBgl1 and TpBgl3 were cloned into pET28a vector and the proteins were expressed in Escherichia coli and further purified in two chromatographic steps. The purified enzymes were evaluated for pH and temperature of activity, which showed that BGLs from the glycosyl hydrolases family 1 (PfBgl1, TpBgl1) presented a wider range of pH and temperature operation than BGL from family 3 (TpBgl3). The BGLs showed great stability to a range of pH (4-10) and the highest time of half-life (at 99 °C) was at pH 6, besides they were not significantly affected by the presence of EDTA or ions, except TpBgl1 that was inhibited by Hg2+ and Fe2+. The specific activities in a set of different substrates suggested that TpBgl3 is more specific than GH1 BGLs. The kcat and kcat/Km in pNPG indicate that TpBgl3 is the most efficient among BGLs characterized herein, although this enzyme is inhibited with the lowest glucose concentration (30.1 mM). Furthermore, the BGLs were assayed for influence of six monosaccharides in catalysis, which the results suggested a weak inhibition by the most of those carbohydrates tested. The CD experiments revealed that the secondary structure of BGLs is not affected by the pH variations and the denaturation studies evidenced that the BGLs are indeed hyperthermophilic / Doutorado / Ciência de Alimentos / Doutor em Ciência de Alimentos

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The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates. / Chemistry