Rozprawy doktorskie na temat „GH15”

La paroi cellulaire fongique est une couche externe et robuste composée principalement de polysaccharides, qui protège la cellule fongique de son environnement, médie l'interaction cellule-cellule et est responsable de la forme de la cellule. La paroi cellulaire du pathogène opportuniste Aspergillus fumigatus est essentiellement composée de ß(1,3)glucane qui est synthétisé au niveau de la membrane plasmique par un complexe transmembranaire puis modifié dans l'espace pariétal par des activités de ramification, de réticulation et de dégradation. La paroi cellulaire est une structure hautement dynamique qui subit des changements constants au cours de la morphogenèse fongique. Dans cette étude, nous avons étudié le rôle de trois familles de glycoside-hydrolases (GH16, GH17 et GH55) dans le remodelage du ß(1,3)glucane la paroi cellulaire de ce pathogène fongique par différentes approches. Les activités transglycosidase et glucanase respectivement de AfCrh5p et AsScw11p ont été étudiées en utilisant des protéines recombinantes et la première structure cristallographique de la famille Crh a été résolue. D’autre part, une délétion complète des gènes de chaque famille a été réalisée pour étudier la fonction biologique de ces enzymes et a mis en évidence un rôle à de multiples facettes de ces glycosides-hydrolases dans la morphogenèse du champignon filamenteux, A. fumigatus
The fungal cell wall is an outer and robust layer mainly composed of polysaccharides, which protects the fungal cell from its environment, mediates cell-cell interaction, and is responsible for the shape of the cell. The cell wall of the opportunistic pathogen Aspergillus fumigatus is essentially composed of ß(1,3)glucan which is synthesized at the plasma membrane by a transmembrane complex and then modified in the cell wall space by branching, cross-linking and degradating activities. The cell wall is a highly dynamic structure, which undergoes constant change during cell division, growth and morphogenesis. In this study, we investigated the role of three glycoside-hydrolases families (GH16, GH17 and GH55) in cell wall remodeling of this fungal pathogen by different approaches. Transglycosidase and glucanase activities of respectively AfCrh5p and AsScw11p have been studied by using recombinant proteins and the first crystal structure of the Crh family have been resolved. Furthermore, complete deletion of each family's genes has been performed to study the biological function of these enzymes and highlighted a multi-faceted role of this glycosides-hydrolases in the morphogenesis of the filamentous fungus, A. fumigatus

Ces travaux de thèse s’intègrent dans le cadre des études et des recherches pour améliorer les performances conjointes de puissance, de gain, de rendement et stabilité d’impulsion à impulsion des amplificateurs de puissance à base de transistor en technologie GaN pour la mise en œuvre de RADAR à antennes actives en bande S qui constituent actuellement un enjeu primordial au niveau académique comme au niveau industriel. La conception de ces amplificateurs de puissance pour la détection précise et fiable de cibles représente un défi majeur pour les entreprises du domaine, lorsqu’il est associé à des rendements énergétiques ambitieux avec un objectif supérieur à 65%. Une méthode de conception d’amplificateur de puissance en technologie Q-MMIC en boîtier plastique DFN fondé sur l’utilisation des transistors compacts GH15 EU a été développée et utilisée pour concevoir un amplificateur de puissance fonctionnant en bande S [2.9 - 3.3] GHz. L’amplificateur de puissance réalisé a été caractérisé en termes de rendement en puissance ajoutée, de puissance délivrée, de gain et de stabilité d’impulsion à impulsion en présence de signaux radar. L’amplificateur de puissance compact montre des performances très intéressantes comparées à celles obtenue dans la littérature. En effet, à une puissance moyenne disponible du générateur égale à 26dBm, dans la bande [2.8 - 3.3] GHz, la PAE est comprise entre 59% et 66%, la puissance délivrée varie entre 45W et 52W sur la bande considérée et elle est associée à un gain supérieur à 20dB et une stabilité d’impulsion à impulsion calculée égale à -52dB par la méthode RMS. Les résultats de caractérisations de l’amplificateur de puissance à très haut rendement/ haute puissance à base de transistors compact GH15 EU ont démontré l’intérêt de son utilisation dans les nouvelles générations des systèmes radar en termes de performances RF, de stabilité P2P, d’intégration et de coût
This thesis work is part of the studies and research to improve the joint performance of power, gain, efficiency and pulse-to-pulse stability of transistor-based power amplifiers in GaN technology for the implementation of RADAR with active antennas in S-band, which is currently a major issue at the academic and industrial levels. The design of these power amplifiers for accurate and reliable detection of targets represents a major challenge for companies in the field, when associated with ambitious energy yields with an objective greater than 65%. A design method for a power amplifier in Q-MMIC technology in a DFN plastic package based on the use of GH15 EU compact transistors has been developed and used to design a power amplifier operating in the S-band [2.9 - 3.3] GHz. The realized power amplifier has been characterized in terms of added power efficiency, delivered power, gain and pulse-to-pulse stability in the presence of radar signals. The compact power amplifier shows very interesting performances compared to those obtained in the literature. Indeed, at an average available power of the generator equal to 26dBm, in the band [2.8 - 3.3] GHz, the PAE is between 59% and 66%, the delivered power varies between 45W and 52W on the considered band and it is associated with a gain higher than 20dB and a pulse-to-pulse stability calculated equal to - 52dB by the RMS method The results of the characterization of the GH15 EU compact transistor based high efficiency/high power power amplifier have demonstrated the interest of its use in the new generation of radar systems in terms of RF performance, P2P stability, integration and cost

A celulose é o biopolímero de maior abundância no mundo e tem potencial para se tornar fonte de energia renovável através de sua transformação em açúcares fermentáveis, que por sua vez serão transformados em etanol. A recalcitrância da celulose, principal dificuldade encontrada no processo, pode ser superada com o auxílio de enzimas (celulases). Ao menos três enzimas celulolíticas são necessárias para a degradação total da celulose, incluindo as celobioidrolases, que hidrolisam as ligações glicosídicas das extremidades redutoras e não redutoras da cadeia, as endoglucanases, que clivam a cadeia de celulose amorfa randomicamente, e as beta-glicosidases, que produzem glicose através dos celo-oligômeros. Mas para que esse processo se torne financeiramente viável é necessário conhecer o funcionamento, otimizar a atividade e aumentar a produção dessas celulases. Com o intuito de avançar na compreensão da função e estrutura dessas enzimas, o presente trabalho teve como objetivo o estudo estrutural de beta-glicosidases da família GH1 e endoglucanases da família GH5. Na primeira parte do trabalho, a expressão da endoglucanase II de Trichoderma reesei não foi alcançada, mesmo utilizando diferentes organismos e condições de expressão. Porém, na segunda etapa, foi obtida a expressão, purificação e os primeiros ensaios de cristalização de 11 beta-glicosidases bacterianas da família GH1 e 8 endoglucanases bacterianas da família GH5. Dentre elas, três beta-glicosidases e uma endoglucanase de Bacillus licheniformis foram cristalizadas e tiveram sua estrutura resolvida. As beta-glicosidases, apesar de possuírem o enovelamente similar, apresentaram variações no tamanho e posição das alças formadoras da fenda catalítica e divergem em relação a um dos aminoácidos importantes para a estabilização do substrato. Essas diferenças podem ajudar a explicar o mecanismo dessas enzimas para reconhecer substratos distintos. A endoglucanase da família GH5, possuindo dois módulos acessórios, foi cristalizada tanto na forma apo quanto complexada ao substrato celotetraose. O segundo módulo acessório possivelmente é um domínio de ligação à celulose (CBM) e seus resíduos aromáticos, que são responsáveis pela interação com o substrato, parecem complementar o sítio catalítico, sendo assim um novo mecanismo de auxílio enzimático de um CBM. O primeiro módulo acessório não possui um aparente sítio de interação com carboidratos e provavelmente funciona como um conector entre domínio catalítico e o CBM. O posicionamento do substrato no sítio de ligação é parecido com outras estruturas já determinadas, porém, suscita algumas dúvidas sobre a função dos resíduos catalíticos que é conservada na família. O carbono anomérico do substrato possui uma densidade eletrônica contínua com o glutamato da fita β4 (que deveria ser o ácido/base) e está mais próximo dele que do glutamato da fita β7 (que deveria ser o nucleófilo).
Cellulose is the most abundant biopolymer in the world and can become a renewable energy source through its transformation in fermentable sugars, which will be converted in bioethanol. The cellulose recalcitrance, main difficulty in the process, can be overcome with the aid of enzymes (cellulases). At least three cellulolytic enzymes are required for complete hydrolysis of cellulose, including cellobiohydrolases for hydrolyzing the glycosidic linkages from the reducing and non-reducing chain ends, endoglucanases for randomly cleaving cellulose chains in the amorphous regions, and beta-glucosidases for producing glucose from the solubilized cello-oligomers. But, to become a financially viable process it is necessary to know the mechanism, optimize the activity and improve the production of these cellulases. In order to advance the understanding of the structure and function of these enzymes, the present work intended to study the structure of beta-glucosidases from family GH1 and endoglucanases from family GH5. In the first part of the work, the expression of endoglucanase II from Trichoderma reesei was not achieved, even using different organisms and expression conditions. However, in the second part, the expression, purification and the crystallization first trials of eleven bacterial beta-glucosidases and eight bacterial endoglucanases were achieved. Among them, three beta-glucosidases and one endoglucanase from Bacillus licheniformis were crystallized and had their structures solved. Beta-glucosidases, although having a similar folding, showed variations in the length and position of the loops that form the catalytic cleft and diverge in relation to one of the amino acids that are important in substrate stabilization. These differences may help explain the mechanism of these enzymes to recognize distinct substrates. The endoglucanase, which has two accessory modules, was crystallized in the apo form and complexed with the substrate celotetraose. The second accessory module probably is a cellulose binding domain (CBM) and its aromatic residues, which are responsible for the substrate interaction, seem to complement the catalytic site. Therefore it can be a new mechanism of CBM assistance in the enzymatic activity. The first accessory module has no apparent interaction site with carbohydrates and probably works as a connector between the catalytic domain and CBM. The positioning of the substrate in the binding site is similar to other structures already solved but raises some questions about the role of the catalytic residues, that are conserved in the family. The anomeric carbon of the substrate has a continuous electron density with glutamate from sheet-β4 (which should be the acid/base) and is closer to it than to glutamate from sheet-β7 (which should be the nucleophile).

Fungos de podridão branca e fungos de podridrão parda são capazes de degradar a biomassa lignocelulósica utilizando diferentes mecanismos. Os fungos de podridão parda degradam a celulose e a hemicelulose enquanto modificam a lignina, sendo, portanto, as enzimas hidrolíticas ativas em substrato rico em lignina. O arsenal enzimático diferenciado desses fungos torna-os alvos para prospecção de enzimas que podem ser aplicadas em diversos processos biotecnológicos. O fungo Gloeophyllum trabeum é uma das espécies mais compreendidas deste grupo, e sabe-se que é capaz de degradar a celulose sem produzir CBHs. Além disso já foi reportado uma GH5 proveniente desse organismo com caraterísticas processivas. Nossa tentativa de clonagem dessa enzima (GtGH5) em A. nidulans A773 não foi bem sucedida, todavia nosso grupo esta repetindo os passos de clonagem e expressão. No genoma desse organismo esta presente um gene que codifica uma GH45. A GtGH45 foi a clonada e expressa com alto rendimento em A. nidulans cepa A773. A expressão, secreção e acumulaçao da GtGH45 foi positiva após 72 h de incubação em meio líquido (maltose como indutor), confirma por eletroforese SDS-PAGE do extrato enzimático concentrado e dialisado. A massa molar de 18,4 kDa foi consistente com a predição da sequência de 204 amino-ácidos obtída apartir da sequência gênica e corrobora com a caracterização por espectometria de massas (18,9 kDa). A análise filogenética é consistente com estudos anteriores, agrupando a proteína alvo com outras GH45s de basidiomicetos na subfamília C. A enzima purificada foi testada para determinação da especificidade pelo substrato apresentando maior atividade em arabinoxilana, xilana, arabinoxilana e CMC e foi capaz de gero celo-oligômeros a partir da hidrólise de PASC. O pH ótimo em CMC foi 2,5, além se demonstrar ser temoestável em ensaio de Thermoflour. A hidrólise enzimática de substratos lignocelulósicos derivados de cana-de-açúcar mostrou que a GtGH45 foi eficiente para suplementar coqueteis enzimáticos comerciais, principalmente na conversão de xilana.
White-rot and brown-rot basidiomycetes are able to transform lignocellulosic materials through different mechanisms. The brown-rot fungi are able to degrade cellulose and hemicellulose meanwhile modifies lignin. The hydrolytic enzymes of these fungi act on a lignin-enriched substrate, which makes them targets to prospect new enzymes for polysaccharides hydrolysis. Gloeophyllum trabeum is one of the best understood fungal species in this group. An interesting processive GH5-endoglucanase (EG) has been described in this species suggesting an unusual pathway for lignocellulosic degradation without cellobiohydrolases (CBH). Our first attempt to clone this GtGH5 was default but our grup is trying a new attempt of heterologous expression of this protein. Furthermore, G. trabeum genome points out for a low molar mass GH45-EG. Here we report on a recombinant high-yield G. trabeum ATCC 11539 GtGH45 production system. Expression and secretion of endoglucanase GtGH45 was positive after 72 h incubation of the transformed A. nidulans stain A773 in stationary liquid cultures (maltose as inductor). The SDS-PAGE electrophoresis of ultra-filtrated extract showed a single-band GtGH45 over expressed band. The molar mass of 18,4 KDa was consistent with the predicted 204 amino acids sequence derived from gene sequence analyses and corroborates with mass spectometry characterization (18,9 KDa). Even more, the phylogenetic analyze is consistent to previews studies, clustering the target protein with others GH45 from basidiomycetes at subfamily C. The purified protein was assayed for substrate specificity showing activity against xylan, arabinoxylan and PASC. GtGH45 was able to produce cello-oligomers from PASC. The optimum pH was 2.5 with CMC as the substrate. Xylan conversion was enhanced when GtGH45 was mixed with commercial enzymes in enzymatic hydrolysis of alkaline-sulfite pretreated sugar cane bagasse.

A degradação enzimática rápida, eficiente e robusta de polissacarídeos derivados de biomassa lignocelulósica é atualmente um grande desafio na produção de biocombustíveis e considerada uma alternativa viável e promissora para se enfrentar a crise energética mundial e diminuir a dependência das fontes fósseis de energia. O bagaço de cana-de-açúcar no Brasil é a principal matéria lignocelulósica sustentável de grande potencial para a produção do etanol de 2ª geração. O principal requisito para a consolidação dessa abordagem é a disponibilidade de enzimas que hidrolisam a celulose, hemicelulose e outros polissacarídeos em açúcares fermentescíveis e em condições adequadas para a utilização industrial. O presente estudo visou à caracterização molecular, estrutural e funcional da endoglucanase GH12 do fungo Aspergillus terreus (AtGH12) por diferentes técnicas. O gene que codifica para essa enzima foi clonado e expressado no fungo filamentoso A. nidulans linhagem A773. A cepa com maior secreção foi selecionada e a sequência da enzima confirmada por espectrometria de massas MALDI TOF MS. Posteriormente, através de estudos funcionais de parametrização enzimática como pH e temperatura ótimos, estabilidade térmica, efeitos supressores e potencializadores de aditivos, a enzima AtGH12 foi caracterizada bioquímica e fisicamente. A espectrometria de massas do substrato hidrolisado pela catálise enzimática foi tomada como uma forma de investigar o padrão de clivagem da hidrólise e estudo do reconhecimento enzima/substrato para a AtGH12. As caracterizações estruturais das enzimas recombinantes obtidas utilizando as técnicas de espalhamento dinâmico de luz, dicroísmo circular, espalhamento de raios X a baixo ângulo e gel nativo serviram para determinação do enovelamento e estado oligomérico em solução da AtGH12. Com o intuito de fornecer subsídios para o desenvolvimento de coquetéis enzimáticos mais eficazes para hidrólise da biomassa lignocelulósica, a atividade da AtGH12 foi avaliada frente ao bagaço de cana-de-açúcar pré-tratados pelos processos hidrotérmicos e organossolve. Posteriormente, o seu grau de sinergismo nesse tipo de substrato foi determinado com o coquetel enzimático comercial Acellerase®.
Fast, more efficient and robust enzymatic degradation of lignocellulosic biomassderived polysaccharides is currently a major challenge in the production of biofuels and considered a feasible and promising alternative to confront the global energy crisis and reduce the dependence on fossil energy resources. The sugarcane bagasse in Brazil is the most abundant and sustainable lignocellulosic material for the production of 2nd generation ethanol. The main requirement for the consolidation of this approach is the availability of enzymes that hydrolyze cellulose, hemicelluloses and other polysaccharides into fermentable sugars suitable for industrial use. The present study was aimed at molecular, structural and functional characterization of an endoglucanase from the fungus Aspergillus terreus (AtGH12) using different techniques. The gene encoding this enzyme has been cloned and expressed in the filamentous fungus Aspergillus nidulans strain A773. The strain with increased secretion was selected and the enzyme sequence was confirmed by mass spectroscopy MALDI TOF MS. Later, functional studies such as analysis of optimal pH and temperature, thermal stability, suppression and enhance effects of additives were applied to the AtGH12 characterization. The mass spectrometry of hydrolyzed substrate from the enzyme catalysis was acquired as a way to investigate the cleavage pattern of hydrolysis and the study of the enzyme/substrate interaction. Structural characterization of the recombinant enzymes was obtained using techniques such as dynamic light scattering, circular dichroism as well as small angle X-ray scattering and native gel, aided to determine the folding and oligomeric state of AtGH12 in solution. In order to provide support for the development of more effective enzyme cocktails for hydrolysis of lignocellulosic biomass, the activity of AtGH12 was evaluated using sugarcane bagasse pretreated by hydrothermal and organosolv processes. Subsequently, the degree of synergism in this type of substrate was measured using a commercial enzyme cocktail Acellerase®.

A dinâmica estrutural fundamentando a função das xilanases GH11 ainda não está clara. Novo conhecimento sobre a dinâmica catalítica dessas enzimas é crucial para a engenharia de novas enzimas melhoradas beneficiando, assim, diversas indústrias biotecnológicas e de química verde. Com base nesse fato, esse trabalho teve por objetivo obter novas informações acerca da dinâmica catalítica de uma xilanase GH11, através do uso de um conjunto de diversas técnicas avançadas de biofísica molecular em nível bulk e em nível de molécula única (inglês single molecule ou sm). Para isso, foram projetadas xilanases GH11 de Bacillus subtilis ssp. subtilis 168 (XynA) com mutações únicas de cisteína para a marcação dos resíduos D119 e R122 no domínio polegar, do resíduo N54 no domínio dedos, e do resíduo N151 na alfa-hélice, seguidas pela sua construção e produção por métodos de biologia molecular. Esses mutantes foram marcados em seus respectivos grupos tióis com a sonda fluorescente sensível à polaridade Acrylodan, com a sonda de spin MTSSL, e com a sonda fluorescente fotoestável AttoOxa11. A xilanase tipo selvagem for marcada em seu N-terminal com a sonda fotoestável Alexa Fluor® 488 5-SDP Ester. Foram utilizados ensaios de espectrofotometria de fluorescência em nível bulk e de espectroscopia de ressonância paramagnética eletrônica para investigar como a dinâmica do domínio polegar da xilanase GH11, temperatura, e ligação ao substrato se correlacionam um com o outro. Os resultados atestaram que um estado do domínio polegar controlado por temperatura, aberto, dinâmico e flexível tem mais chances de se ligar efetivamente ao substrato de uma maneira produtiva, o que está em completo acordo com estudos anteriores de simulação de dinâmica molecular, cristalografia, desnaturação térmica, e análise funcional por desenho racional de mutantes de domínio polegar de xilanases GH11. Com base nas evidências adquiridas e em estudos anteriores, nós propomos um conjunto de hipóteses e modelos para a dinâmica catalítica da xilanase, focando no papel do domínio polegar nesse processo. No intuito de determinar a constante de afinidade da xilanase por seu substrato e os tempos de relaxamento e constantes de velocidade dos movimentos do domínio polegar, foram feitas medidas de espectroscopia de correlação de fluorescência simples e combinada com transferência eletrônica fotoinduzida, usando as xilanases marcadas com as sondas fluorescentes fotoestáveis, na presença e na ausência de substrato. Os resultados mostraram tempos de difusão muito maiores para as xilanases na presença de substrato, como efeito da afinidade da enzima pelo mesmo. Entretanto, não foi verificada nenhuma curva de decaimento como efeito de supressão dinâmica da sonda por PET. Esses mesmos conjugados foram aplicados com sucesso em microscopia por imagem de tempo de vida de fluorescência, no intuito de analisar sistematicamente a afinidade da xilanase por partículas insolúveis e filmes de substrato, e por fragmentos insolúveis de frações de processos de deslignificação e desestruturação de bagaço de cana-de-açúcar, assim como para a análise da composição, estrutura e topologia desses materiais. Foi possível verificar a presença de xilano na maioria das frações desse bagaço tratado, mas em quantidades variáveis
The structural dynamics underlying the function of GH11 xylanases is still unclear. New insights into the catalytic dynamics of these enzymes are crucial for engineering novel improved enzymes benefiting biotechnological and green chemistry industries. The objective of this work was to obtain new information concerning the catalytic dynamics of a GH11 xylanase, by using a combination of advanced molecular biophysics techniques, both at the bulk level and at the single molecule level (sm). Mutant GH11 xylanases from Bacillus subtilis ssp. subtilis 168 (XynA) were designed with single point cysteine mutations for labeling the residues D119 and R122 on the thumb domain, N54 on the fingers domain, and N151 on the alpha helix, followed by their construction and production by molecular biology methods. These mutants were labeled at their respective thiol groups by the polarity sensitive fluorescent probe Acrylodan, by the electron spin probe MTSSL, and by the photostable fluorescent probe AttoOxa11. The wild-type xylanase was labeled at its N-terminus by the photostable fluorescent probe Alexa Fluor® 488 5-SDP Ester. Bulk fluorescence spectrophotometry and electron paramagnetic resonance assays were used to investigate how the thumb domain dynamics of the GH11 xylanase, temperature and substrate binding were correlated. These results demonstrated that a temperature controlled, open, dynamical and flexible thumb domain state is more likely to effectively bind the substrate in a productive way, which is in complete agreement with previous studies from molecular dynamics simulations, crystallography, thermal denaturation, and function analysis by the rational design of thumb mutants for GH11 xylanases. Based on this evidence and previous studies, we proposed a hypothesis for the xylanase catalytic dynamics, focusing on the role of the thumb domain. In order to determine the xylanase affinity constant for its substrate and the relaxation times and rate constants of the thumb domain movements, fluorescence correlation spectroscopy measurements were performed. Both simple and combined measurements with photoinduced electron transfer were performed, using the xylanases labeled with photostable fluorescent probes, in the presence and absence of substrate. The results have shown longer diffusion times for the xylanases in the presence of substrate, as an effect of the enzyme affinity for it. However, it was not verified any decay curve as an effect of the dynamic suppression of the probe via PET. The same conjugates were successfully applied to fluorescence-lifetime imaging microscopy, aiming to systematically analyze the affinity for xylanase of substrates in the form of insoluble particles and films, and for water insoluble fractions from sugarcane bagasse delignification processes. In addition, the composition, structure and topology of these materials was examined. It was possible to verify the presence of xylan in most fractions of this treated bagasse, although in variable quantities

In 1990, the classification of carbohydrate-active enzymes (CAZymes) was introduced by the scientist Bernard Henrissat. According to sequence similarity, these enzymes were separated into families with conserved structures and reaction mechanisms. One interesting class of CAZymes is the group of glycoside hydrolases (GHs) containing more than 138000 modules divided into 131 families as of February 2013. One of the most versatile and the largest of these GH families, containing enzymes with numerous biomass-deconstructing activities, is glycoside hydrolase family 5 (GH5). However, for large and diverse families like the GH5 family, another layer of classification is required to get a better understanding of the evolution of diverse enzyme activities. In Paper I, a new subfamily classification of GH5 is presented in order to sort the family members into distinct groups with predictive power. In total, 51 subfamilies were defined. Despite the fact that several hundred GH5 enzymes have been characterized, 20 subfamilies lacking biochemically characterized enzymes and 38 subfamilies without structural data were identified. These highlighted subfamilies contain interesting targets for future investigation. The GH5 family includes endo-β-mannanases catalyzing the hydrolysis of the β-1,4-linked backbone of mannan polysaccharides, which are common hemicelluloses found as storage and structural polymers in plant cell walls. Mannans are commonly utilized as raw biomaterials in food, feed, paper, textile and cosmetic industries, and mannanases are often applied for modifying and controlling the property of mannan polysaccharides in such applications. The overwhelming majority of characterized mannanases are from microbial origin. The situation for plant mannanases is quite different, as the catalytic properties for only a handful have been determined. Paper II describes the first characterization of a heterologously expressed Arabidopsis β-mannanase.
År 1990 introducerade forskaren Bernard Henrissat en klassificering av kolhydrataktiva enzymer (CAZymer), enligt vilken enzymerna - baserat på sekvenslikhet - delades in i familjer med konserverade strukturer och reaktionsmekanismer. En intressant CAZym-klass är glykosidhydrolaserna (GH), en klass som i februari 2013 innehöll fler än 138000 katalytiska moduler indelade i 131 olika familjer. En av de största och mest varierade av GH-familjerna är glykosidhydrolasfamilj 5 (GH5), vilken innehåller en mångfald av identifierade enzymaktiviteter relevanta för nedbrytning av biomassa. För stora och diversifierade familjer som GH5 krävs det dock ytterligare en klassificeringsnivå för att bättre förstå evolutionen och uppkomsten av de många förekommande enzymaktiviteterna. I manuskript I presenteras en ny uppdelning av GH5 enzymer i subfamiljer med syfte att dela upp familjemedlemmarna i distinkta grupper som representerar olika funktioner. Utifrån denna klassificering kan sedan ett enzyms funktion förutsägas baserat på vilken subfamilj det tillhör. Totalt definierades 51 subfamiljer. Trots att hundratals GH5 enzymer har karaktäristerats så visade det sig att 20 av subfamiljerna helt saknar biokemiskt karaktäriserade enzymer och 38 av dem saknar publicerade proteinstrukturer. Dessa subfamiljer är särskilt intressanta för framtida studier. GH5-familjen inkluderar endo-β-mannanaser som katalyserar hydrolysen av den β-1,4-länkade huvudkedjan i mannanpolysackarider. Dessa växtpolymerer som ingår i hemicellulosagruppen är vanligt förekommande i cellväggarna, där de fungerar som energilagringsmolekyler eller har en strukturell funktion. Mannaner används ofta som råmaterial för industriell livs- och djurfodersproduktion, papper, textilier och kosmetika. I dessa processer behövs ofta mannanaser för modifiering och kontroll av egenskaperna hos dessa polysackarider. Den överväldigande majoriteten av alla karaktäriserade mannanaser kommer från mikroorganismer. Endast för ett fåtal växtmannanaser har de katalytiska egenskaperna analyserats. Manuskript II beskriver den första karaktäriseringen av ett heterologt uttryckt β-mannanas från Arabidopsis.

QC 20130506

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A conversão enzimática dos polissacarídeos da biomassa é um fator chave no desenvolvimento de bioetanol de segunda geração. A recalcitrância da lignocelulose para a degradação enzimática e o custo elevado de enzimas hidrolíticas necessárias para a despolimerização de polissacarídeos encontrados na parede celular da planta são barreiras significativas para a produção em larga escala e para a comercialização de biocombustíveis e bioprodutos derivados da biomassa vegetal. A fim de aumentar rapidamente a produção de biocombustíveis celulósicos e bioprodutos, existe a necessidade de desenvolver coquetéis enzimáticos mais eficientes e de menor custo para a conversão de biomassa em açúcares fermentáveis. Nesse contexto, o estudo de enzimas degradadoras da parede celular é essencial. No presente trabalho a enzima endo-1,4-?-D-glucanase de Aspergillus terreus (ATEG_09894) foi clonada para expressão em A. nidulans. O teste de expressão mostrou a expressão solúvel da proteína. Sua identidade foi confirmada por espectrometria de massas. O pH ótimo e a temperatura ótima para sua atividade enzimática foram de 5,0 e 55ºC, respectivamente. A desnaturação térmica mostrou que a partir de 60ºC a enzima começa a perder estrutura. Interessantemente a enzima apresenta atividades ?-glucanase e xiloglucanase, tendo preferência por ?-glucano. A caracterização estrutural mostrou que ATEG_09894 foi expressa corretamente e os resultados de SEC e SAXS mostram que a proteína é monomérica em solução e o modelo de sua estrutura tridimensional indica que a superfície eletrostática da molécula contribui para um monômero estável. Os dados apresentados nesse trabalho são importantes pois identificam peculiaridades da enzima ATEG_09894, que atua na degradação da biomassa, sugerem os determinantes de sua seletividade enzimática e apresenta a degradação, não usual, de ?-glucanos...
The enzymatic conversion of polysaccharides from biomass is a key factor in the development of second generation bioethanol. The recalcitrance of lignocellulose to enzymatic degradation and the high cost of hydrolytic enzymes necessary for the depolymerization of polysaccharides found in plant cell wall are significant barriers to large-scale production and commercialization of biofuels and bioproducts derived from plant biomass. In order to rapidly increase the production of biofuel and byproducts cellulosic, a need exists to develop more efficient and lower cost enzymatic cocktails for the conversion of biomass into fermentable sugars. In this context, the study of cell wall degrading enzymes is essential. In this work the enzyme endo-1,4-?-D-glucanase from Aspergillus terreus (ATEG_09894) was cloned for expression in A. nidulans. The expression test showed the expression of a soluble protein. Its identity was confirmed by mass spectrometry. The optimum pH and optimum temperature for the enzyme activity were 5.0 and 55 ºC, respectively. The thermal denaturation showed that above 60 ºC the enzyme starts to lose structure. Interestingly, the enzyme has ?-glucanase and xiloglucanase activities, preferring ?-glucan. Structural characterization showed that ATEG_09894 was expressed correctly folded and the results of SEC and SAXS show that the protein is monomeric in solution and the model of its three dimensional structure indicates that the electrostatic surface molecule contributes to a stable monomer. The data presented in this study are important because they identify peculiarities of ATEG_09894, which acts in the degradation of biomass, suggest the determinants of its selectivity and enzymatic degradation presents, unusual, of ?-glucans and xyloglucans, promising feature for degradation of lignocellulosic material

Orientador: Eleni Gomes
Coorientador: Fábio Márcio Squina
Banca: Roberto da Silva
Banca: Henrique Ferreira
Banca: Leandro Cristante de Oliveira
Banca: André Ricardo de Lima Damásio
Resumo: A conversão enzimática dos polissacarídeos da biomassa é um fator chave no desenvolvimento de bioetanol de segunda geração. A recalcitrância da lignocelulose para a degradação enzimática e o custo elevado de enzimas hidrolíticas necessárias para a despolimerização de polissacarídeos encontrados na parede celular da planta são barreiras significativas para a produção em larga escala e para a comercialização de biocombustíveis e bioprodutos derivados da biomassa vegetal. A fim de aumentar rapidamente a produção de biocombustíveis celulósicos e bioprodutos, existe a necessidade de desenvolver coquetéis enzimáticos mais eficientes e de menor custo para a conversão de biomassa em açúcares fermentáveis. Nesse contexto, o estudo de enzimas degradadoras da parede celular é essencial. No presente trabalho a enzima endo-1,4-β-D-glucanase de Aspergillus terreus (ATEG_09894) foi clonada para expressão em A. nidulans. O teste de expressão mostrou a expressão solúvel da proteína. Sua identidade foi confirmada por espectrometria de massas. O pH ótimo e a temperatura ótima para sua atividade enzimática foram de 5,0 e 55ºC, respectivamente. A desnaturação térmica mostrou que a partir de 60ºC a enzima começa a perder estrutura. Interessantemente a enzima apresenta atividades β-glucanase e xiloglucanase, tendo preferência por β-glucano. A caracterização estrutural mostrou que ATEG_09894 foi expressa corretamente e os resultados de SEC e SAXS mostram que a proteína é monomérica em solução e o modelo de sua estrutura tridimensional indica que a superfície eletrostática da molécula contribui para um monômero estável. Os dados apresentados nesse trabalho são importantes pois identificam peculiaridades da enzima ATEG_09894, que atua na degradação da biomassa, sugerem os determinantes de sua seletividade enzimática e apresenta a degradação, não usual, de β-glucanos...
Abstract: The enzymatic conversion of polysaccharides from biomass is a key factor in the development of second generation bioethanol. The recalcitrance of lignocellulose to enzymatic degradation and the high cost of hydrolytic enzymes necessary for the depolymerization of polysaccharides found in plant cell wall are significant barriers to large-scale production and commercialization of biofuels and bioproducts derived from plant biomass. In order to rapidly increase the production of biofuel and byproducts cellulosic, a need exists to develop more efficient and lower cost enzymatic cocktails for the conversion of biomass into fermentable sugars. In this context, the study of cell wall degrading enzymes is essential. In this work the enzyme endo-1,4-β-D-glucanase from Aspergillus terreus (ATEG_09894) was cloned for expression in A. nidulans. The expression test showed the expression of a soluble protein. Its identity was confirmed by mass spectrometry. The optimum pH and optimum temperature for the enzyme activity were 5.0 and 55 ºC, respectively. The thermal denaturation showed that above 60 ºC the enzyme starts to lose structure. Interestingly, the enzyme has β-glucanase and xiloglucanase activities, preferring β-glucan. Structural characterization showed that ATEG_09894 was expressed correctly folded and the results of SEC and SAXS show that the protein is monomeric in solution and the model of its three dimensional structure indicates that the electrostatic surface molecule contributes to a stable monomer. The data presented in this study are important because they identify peculiarities of ATEG_09894, which acts in the degradation of biomass, suggest the determinants of its selectivity and enzymatic degradation presents, unusual, of β-glucans and xyloglucans, promising feature for degradation of lignocellulosic material
Doutor

Els quitosans i quitooligosacàrids (COS), obtinguts per enzims modificadors de la quitina, presenten un elevat interès biotecnològic a causa de l’important nombre d’aplicacions en àrees tant diverses com són l’agricultura, el tractament d’aigües, la indústria alimentària, la biomedicina i la cosmètica, entre d’altres. D’entre les funcions biològiques dins la indústria medicofarmacèutica destaquen activitats antiinflamatòries, immunoestimulants, antimicrobianes, antitumorals, de prevenció de l’obesitat i control del colesterol, com a vectors per a teràpia gènica i de promoció de la cicatrització i regeneració de la pell. Aquestes propietats biològiques dels COS no només són dependents del grau de polimerització i acetilació sinó que possiblement també depenen del patró d’acetilació d’aquests. Actualment la síntesi de COS presenta dos problemes principals: poca reproductibilitat entre lots i l’origen animal dels productes, que dificulten la seva utilització en la indústria medicofarmacèutica. Amb l’objectiu de sobreposar-se a aquestes limitacions s’ha pretès desenvolupar una plataforma biotecnològica per a la producció de COS de baix pes molecular amb seqüències definides. El projecte pretén utilitzar l’activitat transglicosidasa de les quitinases com a eina sintètica per a obtenir oligòmers de quitina i quitosà definits amb patrons d’acetilació repetitius. Les quitinases són glicosil hidrolases que catalitzen la hidròlisi d’enllaços glicosídics β1,4 de polímers de quitina i quitosà. Algunes quitinases presenten també activitat de transglicosidació (TG) mitjançant la qual són capaces d’introduir nous enllaços glicosídics entre un molècula donadora i una acceptora amb la conseqüent generació de COS oligomèrics. Aquestes quitinases poden utilitzar-se per a la polimerització in vitro de COS, però l’activitat hidrolítica que presenten tendeix a despolimeritzar els productes de TG ràpidament. Amb l’objectiu d’augmentar l’activitat de TG de quitinases per a la obtenció de nous COS estructuralment definits, en aquesta tesi s’ha aplicat l’estratègia glicosintasa (GS) sobre diferents quitinases de la família GH18 per mutació del residu assistent i l’ús d’un derivat oxazolina com a donador, amb les que s’ha aconseguit una important disminució de l’activitat hidrolítica i un increment de l’activitat de TG. Tot i l’increment de l’activitat de TG, l’activitat hidrolítica residual que presenten dona lloc a la hidròlisi dels productes GS i de TG formats que impedeix augmentar el rendiment. Amb aquest propòsit s’ha optat per la modificació d’un dels enzims seleccionats per enginyeria de proteïnes. Mitjançant mutagènesi dirigida s’ha aconseguit incrementar el rendiment en polímer fins a un 60% (p/p) amb l’ús d’un derivat oxazolina d’un COS de quitina (el 60% del qual correspon al producte de la reacció GS), i també la obtenció d’oligòmers/polímers de quitosà amb l’ús de derivats oxazolina de quitooligosacàrids parcialment desacetilats, estructuralment definits.
Los quitosanos y quitooligosacáridos (COS), obtenidos por enzimas modificadores de la quitina, presentan un elevado interés biotecnológico debido al importante número de aplicaciones en áreas tan dispares como son la agricultura, el tratamiento de aguas, la industria alimenticia, la biomedicina y la cosmética, entre otras. Entre las funciones biológicas dentro de la industria médico-farmacéutica destacan actividades antiinflamatorias, inmunoestimulantes, antimicrobianas, antitumorales, de prevención de la obesidad y control del colesterol, como vectores de terapia génica y de promoción de la cicatrización y regeneración de la piel. Estas propiedades biológicas de los COS no solo son dependientes del grado de polimerización y acetilación sino que posiblemente también dependen del patrón de acetilación de estos. Actualmente la síntesis de COS presenta dos problemas principales: poca reproducibilidad entre lotes y el origen animal de los productos, que dificultan su utilización en la industria médico-farmacéutica. Con el objetivo de sobreponerse a estas limitaciones se ha pretendido desarrollar una plataforma biotecnológica para la producción de COS de bajo peso molecular con secuencias definidas. El proyecto pretende utilizar la actividad transglicosidasa de las quitinasas como herramienta sintética para obtener oligómeros de quitina y quitosano definidos con patrones de acetilación repetitivos. Las quitinasas son glicosil hidrolasas que catalizan la hidrólisis de enlaces glicosídicos β1,4 de polímeros de quitina y quitosano. Algunas quitinasas presentan también actividad de transglicosidación (TG) mediante la cual son capaces de introducir nuevos enlaces glicosídicos entre una molécula donadora y una aceptora con la consecuente generación de COS oligoméricos. Estas quitinasas pueden utilizarse para la polimerización in vitro de COS, pero la actividad de hidrólisis que presentan tiende a despolimerizar los productos de TG rápidamente. Con el objetivo de aumentar la actividad de TG de quitinasas para la obtención de nuevos COS estructuralmente definidos, en esta tesis se ha aplicado la estrategia glicosintasa (GS) sobre diferentes quitinasas de la familia GH18 por mutación del residuo asistente y el uso de un derivado oxazolina como donador, con las que se ha logrado una importante disminución de la actividad de hidrólisis y un incremento de la actividad de TG. A pesar del incremento de la actividad de TG, la actividad hidrolasa residual que presentan da lugar a la hidrólisis de los productos GS y de TG formados que impide el aumento del rendimiento. Con este propósito se ha optado por la modificación de una de las enzimas seleccionadas por ingeniería de proteínas. Mediante mutagénesis dirigida se ha logrado incrementar el rendimiento en polímero hasta un 60% (p/p) con el uso de un derivado oxazolina de un COS de quitina (el 60% del cual corresponde al producto de la reacción GS), y también la obtención de oligómeros/polímeros de quitosano con el uso de derivados oxazolina de quitooligosacáridos parcialmente desacetilados, estructuralmente definidos.
Chitosan and chitooligosaccharides (COS), obtained by chitin-modifying enzymes, are of interest to a wide variety of areas such as agriculture, water treatment, food industry, biomedicine and cosmetics, among others, due to their important number of applications. Among the biological roles on the medical-pharmaceutical industry are amply demonstrated anti-inflammatory, immunostimulants, antimicrobians and antitumorals activities, obesity prevention and cholesterol control, ability of gene and drug delivery, wound healing and skin regeneration activities. These biological properties are not only related to their degree of polymerization and acetylation, but possibly they are also dependent on their pattern of acetylation. Nowadays the synthesis of COS has two main hurdles: a poor reproducibility between batches and the animal origin of the products, which difficult their usage in the medical-pharmaceutical industry. With the goal of overcome these limitations, we were aiming at the development of a biotechnological platform for the production of sequence-defined low molecular weight chitosans. The project addresses the use of the transglicosidase activity of chitinases as a synthetic tool to obtain well-defined oligomers with repeating patterns of acetylation. Chitinases are glycoside hydrolases that catalyse the hydrolysis of β1,4 glycosidic bonds of chitin and chitosan polymers. Some chitinases have also transglycosydase activity (TG), allowing them to introduce new glycosidic bonds between donor and acceptor sugar molecules with the consequent generation of oligomeric COS. Such transglycosylating chitinases can be used for the in vitro polymerization of COS, but the hydrolytic activity of these enzymes tends to depolymerise the TG products quickly. With the main goal of increase TG activity of chitinases to obtain new well-defined COS, in the present work we use the glycosynthase technology (GS) on different GH18 chitinases by mutation of the assisting residue and the use of an activated glycosyl donor (an oxazoline derivative), with which an important diminish of the hydrolytic activity and an increase of the TG activity have been obtained. Despite the higher TG activity, the residual hydrolytic activity of the assisting residue mutants results in the hydrolysis of the GS and TG products that not allow the increase of the polymer yield. Protein engineering (rational approach) was used to modify one of the selected enzymes. By site-directed mutagenesis it has been possible to increase the polymer yield up to 60% (w/w) using a chitin oligomer oxazoline derivative (the 60% of which corresponds to the product of the GS reaction), and it has also been possible to obtain chitosan oligomers/polymers using structurally defined partially deacetylated COS oxazoline derivatives.

La création de nouvelles enzymes pour l’hydrolyse de la biomasse est une stratégie clé pour ledéveloppement du bioraffinage. Dans ce contexte, les xylanases de la famille GH11 sont déjàdéployées dans de nombreux procédés industriels et donc bien positionnées pour jouer un rôleimportant dans ces procédés. La cible de cette étude, la xylanase GH11 (Tx-Xyl) de la bactérieThermobacillus xylanilyticus, est une enzyme thermostable et donc une bonne candidate pour destravaux d’ingénierie visant l’amélioration de son activité sur des substrats ligno-cellulosiques.Dans cette étude, deux stratégies d’ingénierie des enzymes ont été employées afin d’obtenir denouvelles informations portants sur les relations structure-fonction au sein de Tx-Xyl. La premièrestratégie a consisté en l’utilisation d’une approche de mutagenèse aléatoire, couplée à l’emploi deméthodes de recombinaison in vitro. Ces travaux avaient pour objectif d’améliorer la capacitéhydrolytique de Tx-Xyl sur la paille de blé. La deuxième stratégie mise en oeuvre s’est appuyée surune approche semi-rationnelle visant la création d’une enzyme chimérique, qui bénéficierait d’uneamélioration des interactions enzyme-substrat au niveau du sous-site -3.Le premier résultat majeur de cette thèse concerne le développement d’une méthode de criblagequi permet l’analyse à haut débit de banques de mutants pour la détection de variants quiprésentent une activité hydrolytique accrue directement sur paille de blé. A l’aide de ce crible, nousavons pu analyser plusieurs banques de mutants, représentant un total de six générations demutants, et identifier une série de combinaisons de mutations différentes. D’un côté, un variant,comportant deux mutations silencieuses, permet une meilleure expression de Tx-Xyl, alors qued’autres enzymes mutées présentent des modifications intrinsèques de leurs aptitudes catalytiques.Comparés à l’enzyme parentale Tx-Xyl, certains mutants solubilisent davantage les arabinoxylanes dela paille et, lorsqu’ils sont déployés avec un cocktail de cellulases, participent à une réactionsynergique qui permet un accroissement du rendement des pentoses et du glucose libérés.A l’aide d’une approche semi-rationnelle, une séquence de 17 acides aminés en provenance d’unexylanase GH11 fongique a été ajoutée à l’extrémité N-terminale de Tx-Xyl, afin de créer de nouveauxbrins β. L’enzyme chimérique a pu être exprimée avec succès et caractérisée. Néanmoins, l’analysede ses propriétés catalytiques a révélé que celle-ci ne présente pas davantage d’interactions avec sonsubstrat dans le sous-site -3, mais les résultats obtenus fournissent de nombreux renseignements surles relations structure-fonction au sein de l’enzyme. De plus, ces travaux nous permettent depostuler que Tx-Xyl posséderait un site de fixation secondaire pour les xylanes, un élement jusqu’iciinsoupçonné dans cette enzyme. Par ailleurs, l’analyse de nos résultats nous permet de proposer uneexplication rationnelle pour l’échec de notre stratégie initiale
Engineering new and powerful enzymes for biomass hydrolysis is one area that will facilitate thefuture development of biorefining. In this respect, xylanases from family GH11 are already importantindustrial biocatalysts that can contribute to 2nd generation biorefining. The target of this study, theGH11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus is thermostable, and is thus an interestingtarget for enzyme engineering, aiming at increasing its specific activity on lignocellulosic biomass,such as wheat straw. Nevertheless, the action of xylanases on complex biomass is not yet wellunderstood, and thus the use of a rational engineering approach is not really feasible.In this doctoral study, to gain new insight into structure-function relationships, two enzymeengineering strategies have been deployed. The first concerns the development of a randommutagenesis and in vitro DNA shuffling approach, which was used in order to improve the hydrolyticpotency of Tx-Xyl on wheat straw, while the second strategy consisted in the creation of a chimericenzyme, with the aim of probing and improving -3 subsite binding, and ultimately improvinghydrolytic activity.The first key results that has been obtained is the development of a novel high-throughputscreening method, which was devised in order to reliably pinpoint mutants that can better hydrolyzewheat straw. Using this screening method, several generations of mutant libraries have beenanalyzed and a series of improved enzyme variants have been identified. One mutant, bearing silentmutations, actually leads to higher gene expression, while others have intrinsically altered catalyticproperties. Testing of mutants has shown that some of the enzyme variants can improve thesolubilization of wheat straw arabinoxylans and can work in synergy with cellulose cocktails torelease both pentose sugars and glucose.Using a semi-rational approach, 17 amino acids have been added to the N-terminal of Tx-Xyl, withthe aim of adding two extra β-strands coming from a GH11 fungal xylanase. A chimeric enzyme hasbeen successfully expressed and purified and its catalytic properties have been investigated.Although this approach has failed to create increased -3 subsite binding, the data presented revealsimportant information on structure-function relationships and suggest that Tx-Xyl may possess ahitherto unknown secondary substrate binding site. Moreover, a rational explanation for the failureof the original strategy is proposed

Plant primary cell walls are hydrated extracellular complexes composed largely of polysaccharides: cellulose, hemicellulose and pectin. Cell wall constituents and composition vary in cell-, environment-, and species-dependent manners. For example, within land plant hemicelluloses xyloglucan is ubiquitous while mixedlinkage (1→3),(1→4)-β-D-glucan (MLG) is found only in the Poales and Equisetum. Glycosyl hydrolase 16 (GH16) enzyme family members include numerous enzymes with pertinence to the understanding of the ‘lives’ of cell wall hemicelluloses. However, despite this, the details of the interactions between GH16 enzymes and their substrates have often not been elucidated. Likewise, the true preferences of many of these enzymes and the range of substrates which they can utilise remain to be fully explored. By providing a greater wealth of information for the correlation of enzyme structure with reaction catalysed, such an understanding would enable better predictions of the activities of novel enzymes. Crucially, this would also allow better identification of roles performed by these enzymes in planta as well as of the potential applications of these enzymes. This work sought to further our understanding of the interactions between GH16 enzymes and their substrates by the study of five activities exhibited by GH16 enzymes – xyloglucan endotransglucosylase (XET), xyloglucan endoglucanase/hydrolase (XEG/XEH), mixed-linkage glucan : xyloglucan endotransglucosylase (MXE), lichenase and cellulose : xyloglucan endotransglucosylase (CXE). All of the analysed activities act on xyloglucan and/or MLG. Of particular focus is the novel enzyme MXE from the evolutionarily isolated genus Equisetum (horsetail), which acts on both. Notable findings include: identification of MXE/CXE gene; determination of the substrate specificity of MXE; defining of the sites of attack of lichenase, XEG, XET and MXE; discovery of novel xyloglucan structures and discrepancies between the xyloglucan present in different barley organs.

Dans le contexte de la bioéconomie, la découverte et la caractérisation des enzymes capables de dégrader la paroi végétale est particulièrement intéressante pour l’utilisation de la biomasse lignocellulosique dans l’industrie. A cet égard, la métagénomique fonctionnelle est un outil puissantpour découvrir de nouvelles enzymes à partir d’écosystèmes microbiens variés, comme l’illustrent les travaux sur le tube digestif du termite Pseudacanthotermes militaris. Cette étude a fourni une mine d’informations et identifié un hypothétique locus d’utilisation du xylane (XUS), codant pour cinq glycosides hydrolases (GH) et une carbohydrate esterase (CE) de Bacteroidales.Le XUS du métagénome de Pseudacanthotermes militaris contient une xylanase de la famille GH10 qui possède une organisation modulaire complexe dans laquelle la séquence du domaine GH10 est interrompue par une insertion de deux carbohydrate binding modules (CBM). Des travaux préliminaires ont montré que cette enzyme modulaire, désignée Pm25, est active sur xylane. Par conséquent, un des objectifs de cette étude a été la caractérisation détaillée des propriétés biochimiques et catalytiques de Pm25. Le rôle des CBM a également été examiné en quantifiant les interactions protéines-sucres et permettant ainsi une meilleure compréhension du rôle spécifique de ces modules, les résultats obtenus permettent de cerner l’impact de la modularité de Pm25 sur ses propriétés fonctionnelles.Dans une deuxième partie de l’étude, nous avons entrepris d’étudier la fonction de Pm25 dans le contexte du cluster XUS. Pour ce faire, nous avons étudié les enzymes adjacentes à Pm25 sur le locus,une autre GH10, une GH11, une GH115 et une GH43. La comparaison des paramètres cinétiques et une étude détaillée des produits d’hydrolyse ont été analysés par spectrométrie de masse et ont révélé que la GH10 et la GH11 étaient les enzymes clefs de la dépolymérisation en étant 20 fois plus efficaces que Pm25. En parallèle, nous avons développé un protocole pour l’utilisation de la micro-thermophorèse (MST) pour quantifier les interactions CBM-sucres, une approche intéressante qui nécessite peut d’échantillon et de ligand contrairement à d’autres méthodes biophysiques. Dans l’ensemble, cette étude a révélé le rôle important de Pm25 et ses homologues dans les locus d’utilisation des xylanes chez les Bacteroidetes et a permis d’identifié le sens de cette architecture particulière
In the context of bioeconomy, the discovery and study of plant-cell wall degrading enzymes is particularly relevant for the use of lignocellulosic biomass for industrial purposes. In this respect, functional metagenomics has proven to be a powerful tool to discover new enzymes from a variety of microbial ecosystems, as exemplified by work performed on the gut of the termite Pseudacanthotermes militaris. This study provided a wealth of information and identified an interesting hypothetical xylan utilization system, encoding five glycoside hydrolases (GH) and one carbohydrate esterase (CE) annotated from bacteroidales. The Pseudacanthotermes militaris-derived putative XUS cluster contains a GH10 xylanase that displays a quite complex modular arrangement wherein the GH10 catalytic module contains two insertional carbohydrate binding modules (CBM). During the preliminary work, this modular enzyme, designated Pm25, was shown to be active on xylan, thus in the present research we set out to more thoroughly characterize its biochemical and catalytic properties.The role of the CBM was also investigated, quantifying protein-carbohydrate interactions and thus providing better insight into the specific role of the modules. Taken together, the results obtained provide insight into how Pm25 modularity translates into functional properties. In second part of our study, we set out investigate the function of Pm25 in the context of the XUS cluster. To achieve this we studied a xylan utilization system, which is constituted by another GH10, GH11, GH115 and GH43. The comparison of kinetic parameters and a detailed end product analysis by mass spectrometry showed that GH10 and GH11 outweigh over 20 fold Pm25 catalytic efficiency. In parallel, we developed the use of MicroScale Thermophoresis (MST) to quantify CBM-carbohydrates interactions, an interesting approach requiring smaller concentration of proteinsand ligands compared to other biophysical methods. Overall this study highlighted the important role of Pm25 homologs in the xylan utilization system in Bacteroidetes, and pinpointed the meaning of its unusual architecture

Orientador: Roberto Ruller
Coorientador: Leticia Maria Zanphorlin Murakami
Banca: Mário Tyago Murakami
Banca: Clelton Santos
Resumo: O processamento da biomassa lignocelulósica para a produção do etanol de segunda geração (E2G) e outras aplicações requer enzimas termoestáveis capazes de operar sob altas temperaturas e longos períodos de incubação. Nesse contexto, reportamos aqui a validação experimental preliminar de uma abordagem in silico para a obtenção enzimas hidrolíticas termoestáveis. Simulações computacionais da endoxilanase GH11 de Bacillus subtilis (XBS) usando a metodologia de otimização de interações eletrostáticas (EIO) indicaram três resíduos (K99, R122 and K154) críticos para o aumento da termoestabilidade através da substituição de cargas. Assim, analisamos os efeitos da mutação K99E em diversas propriedades da XBS selvagem (XBSWT) e também de uma XBS obitida in vitro através de evolução dirigida (XBSM6). Após expressão heteróloga em células de Escherichia coli BL21 e purificação através de cromatografia de afinidade, realizamos a caracterização bioquímica e biofisica das enzimas. Além disso, conduzimos estudos de termotolerância incubando as enzimas em condições industriais (pH 4,8 e 50 °C). Finalmente, realizamos testes de suplementação de um coquetel enzimático comercial esperando obter melhoras no processo de hidrólise da biomassa. Os resultados mostraram que a mutação K99E foi capaz de aumentar a temperatura de desnaturação térmica (Tm) das enzimas em aproximadamente 1 °C e melhorar a termotolerância de XBSWT e XBSM6 sem alterar suas condições ótimas. Somado a isso, a enzima...
Abstract: Processing of lignocellulosic biomasses for second-generation bioethanol (SGB) production and others applications requires thermostable enzymes able of operating under high temperatures and long incubation periods. In this context, we report here the preliminary experimental validation of an in silico approach for engineering hydrolytic enzymes towards thermostability. Previous computational simulations of GH11 endo-xylanase from Bacillus subtilis (XBS) using the electrostatic interaction optimization (EIO) methodology indicated three amino acid residues (K99, R122 and K154) critical for increasing thermostability through charge replacement. Thus, we analyzed the effects of the K99E mutation on several proprietes of two XBS enzymes: the wild-type XBS (XBSWT) and an XBS obitited in vitro by directed evolution (XBSM6). After heterologous expression in Escherichia coli BL21 cells and purification by affinity chromatography, we performed the biochemical and biophysical characterization of enzymes. We also conducted thermotolerance studies, incubating the enzymes under industrial conditions (pH 4.8 and 50 ° C). Finally, we performed supplementation assays of a commercial enzyme cocktail hoping to obtain improvements in the biomass hydrolysis process. The results showed that the K99E mutation was able to increase the enzymes thermal denaturation temperature (Tm ) by approximately 1 °C and improve thermotolerance of XBSWT and XBSM6 without alter its optimum conditions. In addition, ...
Mestre

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
The genus Trichoderma includes species that have the ability to antagonize plant pathogens in a complex process ranging from antibiosis, competition for nutrients, mycoparasitism and induction of defense mechanisms in plants or a combination of these. Trichoderma species are known for their ability to produce lytic enzymes such as exoglucanases, endoglucanases, chitinases, and proteases that is essential for phytopathogen cell wall degradation. In the development and differentiation processes of Trichoderma, β-glucanases contributes significantly to the morphogenetic-morphological process that lead to the presence of β-glucans as the main component of fungi wall. In this work, we studied the functional role of the gluc31 gene that encodes an endo β-1,3-glucanase of the GH16 family of Trichoderma harzianum ALL42 through the deletion of this gene. This study demonstrated that the absence of Δgluc31 gene did not affect the in vivo mycoparasitism ability of the mutant T. harzianum ALL42, however the involvement of this gene in cell wall remodeling and synthesis were demonstrated. In the absence of the gluc31 gene, a higher deposition of chitin polymers on the cell wall of the mutant hyphae was observed. The absence of the gluc31 gene in T. harzianum also demonstrated an effect on the expression of other genes belonging to the family 16 of glycosyl hydrolases, due to the function redundancy found among the glucanases.
O gênero Trichoderma inclui espécies que possuem habilidade de antagonizar patógenos de plantas em um processo complexo que vão desde antibiose, competição por nutrientes, micoparasitismo, indução de mecanismos de defesa em plantas ou ainda uma combinação desses. Espécies de Trichoderma são conhecidas por sua capacidade de produzir enzimas líticas tais como exoglucanases, endoglucanases, quitinases e proteases que desempenham papéis importantes na degradação da parede de fitopatógenos. Nos processos de desenvolvimento e diferenciação de Trichoderma, as β-glucanases contribuem de forma significativa no processo morfogenéticomorfolítico uma vez que a β-glucanas é o componente principal da sua parede. Nesse trabalho, realizou-se o estudo do papel funcional do gene gluc31 que codifica uma endo β-1,3-glucanase da família GH16 de Trichoderma harzianum ALL42, através da deleção deste gene. Observamos que a ausência do gene gluc31 não afetou a capacidade de micoparasitismo, in vitro, da espécie mutante de T. harzianum ALL42, entretanto, o envolvimento deste gene na síntese e remodelamento da parede celular foi demonstrado. Na ausência do gene gluc31, uma maior quantidade de polímero de quitina na parede celular das hifas da linhagem mutante Δgluc31 foi observado. A ausência do gene gluc31 em T. harzianum demonstrou ainda um efeito sobre a expressão dos outros genes pertencentes à família 16 de glicosil hidrolases em ensaios de RT-qPCR, devido a redundância de função entre as glucanases.

Introdução e objetivos: β-glicosidases da família GH1 das glicosil-hidrolases possuem um dobramento do tipo barril (β/α)8. Propõe-se que proteínas com este dobramento, que usualmente classificam-se como tendo um único domínio, na verdade são compostas por dois domínios, cada um deles correspondendo a um \"meio barril\" (β/α)4. Assim, as proteínas com dobramento barril (β/α)8 seriam provenientes de uma duplicação e fusão gênica de um ancestral \"meio barril\" (β/α)4. O objetivo geral deste projeto é investigar a existência de dois domínios (β/α)4, as metades N- e C-terminal, na estrutura (β/α)8 barril da β-glicosidase A de Thermotoga maritima (bglTm) e β-glicosidase B de Paenibacillus polymyxa (bglB) por meio de análise da desnaturação térmica e química dessas enzimas. Resultados: Para atingir esse objetivo foram introduzidas mutações que rompem contatos não covalentes entre os supostos domínios destas β-glicosidases. Os segmentos de DNA que codificam as enzimas selvagem e mutantes foram clonados em plasmídeo de expressão pLATE51 e as enzimas recombinantes expressas em Escherichia coli BL21(DE3). Foram purificadas com sucesso as enzimas selvagens e duas mutantes de bglTm, denominadas T1 e T2, que possuem 2 e 4 mutações respectivamente em resíduos na interface inter-metades. Para confirmar o enovelamento destas proteínas recombinantes foi empregada a análise de estruturas secundárias por dicroísmo circular, também o espectro de fluorescência intrínseca de triptofano e sua supressão por acrilamida e finalmente foram determinados parâmetros cinéticos. Observou-se que não houve mudanças significativas na exposição dos triptofanos das proteínas recombinantes, sugerindo que se encontram enoveladas, o que está de acordo com a observação de que as proteínas recombinantes mantêm sua atividade catalítica. Já pela análise de dicroísmo circular concluiu-se que a mutante T1 possui dobramento semelhante à selvagem bglTm e que T2 apresenta diferenças significativas, sendo as porcentagens de α-hélice de 21%, 22% e 10% para bglTm, T1 e T2, respectivamente. Em seguida demonstrou-se que T1 mantém a termo estabilidade semelhante à selvagem, enquanto que T2 tem termo estabilidade reduzida, apresentando kobs de 0,3 min-1 em 80°C e de 0,06 min-1 em 75 °C.. O cálculo de Tm através de Differential Scanning Fluorimetry foi feito para bglB e T2 (42 e 81.7 °C respectivamente), enquanto que bglTm e T1 mantiveram-se estáveis na faixa de temperatura analisada (até 95°C). Na análise do efeito da temperatura sobre a estrutura das mutantes T1 e T2 não se observou nenhuma evidência da presença dos dois supostos domínios (β/α)4. A análise da desnaturação por cloreto de guanidina mostrou que o c50 diminuiu para T2 (2,4 M), mas não mostrou alteração para T1 (4,5 M) em comparação à bglTm (4,3 M). Coerentemente, a estabilidade da enzima selvagem e de T1 na ausência de desnaturante é a mesma (ΔGH2O = 5,2 kcal/mol), mas se reduziu para a T2 (ΔGH2O = 3,5 kcal/mol). Os cálculos do parâmetro m mostraram uma cooperatividade semelhante na desnaturação de bglTm, T1 e T2, não evidenciando independência entre os dois supostos domínios (β/α)4. Em conclusão, a análise estabilidade da β-glicosidase bglTm frente à temperatura e ao cloreto de guanidina não revelaram a presença dos domínios (β/α)4 que correspondem às metades N- e C-terminal desta β-glicosidase.
Introduction and Aims: β-glucosidases from the family GH1 of the glycosil-hidrolases presents a (β/α)8 barrel folding. These proteins are usually classified as single domain, however it has been alternatively proposed that they actually are formed by two \"half barrel\" (β/α)4. Thus, (β/α)8 barrel proteins had evolved from an \"half barrel\" ancestor that underwent a duplication-fusion event. The general goal of this project is the search for the two putative (β/α)4 domains, which form the N- and C-terminal ends of the β-glucosidase A from Thermotoga maritima (bglTm) and β-glucosidase B from Paenibacillus polymyxa (bglB), detecting their presence through the thermal and chemical stability of these (β/α)8 barrel proteins. Results: Site-directed mutagenesis was employed to replace residues forming non-covalent interaction between the putative (β/α)4 domains. DNA segments coding for bglB, bglTm and mutant bglTm were cloned into the pLATE51 expression vector and produced as recombinant proteins in E. coli BL21(DE3). The bglB, bglTm and two mutant bglTm, hereafter called T1 and T2, with 2 and 4 mutations respectively on residues in the interface between the protein halves, were purified. They were stable folded as shown by detecting their catalytic activity upon two different substrates and also by circular dichroism (CD) and tryptophan fluorescence analysis. Nevertheless, T2 showed a decrease in the α-helix content (10 %) in the CD analysis, whereas bglTm and T1 are similar (22 %). The wild-type bglTm and T1 are thermostable, whereas T2 was inactivated after pre-incubation at high temperature (kobs = 0,3 min-1 at 80 °C and 0,06 min-1 at 75 °C). The Differential Scanning Fluorimetry experiments revealed Tm of 42 e 81.7 °C for bglB and T2, respectively, whereas wild-type bglTm and mutant T1 did not showed any thermal transition up to 95 °C. Indeed, the analysis of the thermal stability of T1 and T2 did not reveal any evidence of the putative (β/α)4 domains. Following that, the analysis of the protein denaturation by guanidine hydrochloride showed that the c50 for T2 was reduced (2.4 M), whereas no modification was observed for bglTm and T1 (4,3 and 4,5 M, respectively). In agreement the stability of bglTm and T1 (ΔGH2O = 5,2 kcal/mol) is similar, but it was reduced for T2 (ΔGH2O = 3,5 kcal/mol). The m parameter showed a similar cooperative denaturation for wild-type bglTm and mutants T1 and T2, but no evidence of the independent unfolding of the putative (β/α)4 domains was found. Conclusion: In conclusion, the analysis of the thermal and chemical stability of the bglTm did not reveal the presence of the putative (β/α)4 domains that form the N- and C-terminal end of these β-glucosidase.

La chitotriosidase (CHIT1) est une chitinase humaine appartenant à la famille glycosyl hydrolase 18 (GH18) qui hydrolyse la chitine. CHIT1 présente plusieurs caractéristiques enzymatiques conservées dans la famille GH18 qui ne sont pas complètement comprises. Pour renforcer nos connaissances sur le mécanisme catalytique de CHIT1 et de la famille GH18, j'ai amélioré la résolution des structures obtenues par diffraction de rayon-X du domaine catalytique de CHIT1. Ces structures correspondent à la forme apo de CHIT1, pseudo-apo ainsi qu’en complexe avec la chitobiose ont été obtenues à des résolutions comprises entre 0.95Å et 1.10Å. Mes résultats m’ont permis de proposer un nouveau mécanisme d’hydrolyse des chaines chito-oligosaccharidiques. En outre, grâce à une nouvelle stratégie de cristallisation, la première structure cristalline de CHIT1 complète a pu être obtenue à une résolution de 1.95Å. Mon étude donne de nouvelles perspectives sur le mode d'action de CHIT1 et les caractéristiques enzymatiques conservées dans la famille GH18
Chitotriosidase (CHIT1) is a human chitinase belonging to the glycosyl hydrolase family 18 (GH18), a highly conserved enzyme family. GH18 enzymes hydrolyze chitin, a N-acetyl glucosamine polymer. CHIT1 is characterized by many enzymatic features that are conserved in GH18 and not completely understood. To increase our knowledge on the catalytic mechanism in CHIT1 and GH18 family, I improved the X-ray resolution crystal structure of CHIT1 catalytic domain in apo and pseudo apo forms as well as in complex with a synthetic substrate to a resolution range between 0.95Å and at 1.10Å. My results allow me to suggest a new mechanism for chito-oligosaccharide chains hydrolysis. Moreover, thanks to a new a crystallogenesis strategy, I obtained the first crystal structure of full length CHIT1 at 1.95Å resolution. My study presents many structural and mechanistic aspects of CHIT1 which gives new insights onto its mode of action and shed light into the conserved enzymatic features in GH18 chitinase family

O sistema hormônio de crescimento (GH) / fator de crescimento insulina- símile tipo 1 (IGF-1) é o principal determinante e regulador do crescimento linear pósnatal. O GH é codificado pelo gene Growth Hormone 1 (GH1). Mutações no GH1 com efeito dominante negativo e herança autossômica dominante são as principais causas monogênicas de deficiência isolada de hormônio de crescimento (DIGH), enquanto deleções ou mutações de ponto no GH1 causam formas raras autossômicas recessivas de DIGH. No grupo de pacientes com DIGH do ambulatório de Endocrinologia do Desenvolvimento do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, foram identificadas apenas deleções em homozigose no GH1 mesmo após estudo criterioso deste gene. Esta diferença em relação aos dados descritos na literatura poderia ser justificada pelo critério diagnóstico para a DIGH adotado pelo nosso grupo, sendo utilizado pico de GH em teste de estímulo inferior a 3,3 ug/L, em contraste com os valores de corte descritos na literatura que variam de 7 a 10 ug/L. Devido a esse fator, pacientes com mutações no GH1 com herança autossômica dominante poderiam estar sendo erroneamente diagnosticados como portadores de baixa estatura familiar ou idiopática (BEI) em nosso serviço. Adicionalmente, mutações que originam moléculas de GH biologicamente inativas também poderiam estar presentes nestes pacientes. Pelos fatores acima apresentados, expandimos o estudo do GH1 para um grupo de crianças classificadas como BEI. Foram selecionadas 98 de 487 crianças avaliadas em nosso serviço com baixa estatura utilizando os seguintes critérios: peso e comprimento normais para idade gestacional ao nascimento, escore-Z da altura < -2, escore-Z do IGF-1 < -1 e pico de resposta de GH >= 3,3 ug/L no teste de estímulo. DNA foi extraído de leucócitos periféricos desses pacientes para rastreamento de mutações no gene GH1. Realizamos estudo molecular por reação em cadeia da polimerase e sequenciamento automático de toda a região codificadora do GH1. Segregação familiar foi realizada para as variantes alélicas identificadas. Em nossa casuística, foram identificadas 10 variantes alélicas nos éxons 4 e 5 e no íntron 4 do GH1, sendo três variantes ainda não descritas na literatura (c.407G > A/p.Val122Ile, c.507C > T/p.Tyr169Tyr e c.456+19G > T). A análise in silico de todas as variantes identificadas indicou ausência de predição de efeito deletério sobre a proteína do GH. Estudo complementar realizado pelo nosso grupo identificou em crianças diagnosticadas com DIGH grave apenas uma paciente com mutação no GH1 responsável pela forma dominante desta doença. Em conclusão, mutações no GH1 causadoras da forma autossômica dominante de DIGH ou Tipo II não foram encontradas em nossa casuística, o que sugere que estas mutações sejam infrequentes em nossa população
The growth hormone (GH) / insulin-like growth factor-1 (IGF-1) axis is the most important hormonal regulator of post-natal linear growth. GH is encoded by the Growth Hormone 1 gene (GH1). Mutations in GH1 with dominant inheritance, which exerts a dominant negative effect on the bioactive GH isoforms, are the main causes of monogenic isolated deficiency of growth hormone (IGHD), while deletions or point mutations in GH1 are responsible for a rare autosomal recessive form of IGHD. However, only homozygous deletions were identified in patients with IGHD from Unidade de Endocrinologia do Desenvolvimento do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, even after detailed investigation of GH1. This difference regarding to literature can be caused by different criteria used to diagnose IGHD in our group, which adopted the cutoff value of peak GH < 3.3ug/L in response to stimulation test, in contrast to literature that describes other groups that use the cutoff peak value of the 7 - 10ug/L. Consequently, patients with autosomal dominant inheritance mutations in GH1 could be being erroneously diagnosed, as having idiopathic short stature (ISS) in our group. Additionally, mutations that cause biologically inactive GH can also be responsible for short stature in these patients. Due to the factors described above, we decided to screen mutations in GH1 in a group of children classified as ISS. We selected 98 of 487 children followed in our department with short stature according to the following criteria: normal birth weight and length for gestational age, height SDS <= -2, IGF-1 SDS < -1 and peak GH in stimulation test >= 3.3 ug/L. Genomic DNA was extracted from peripheral blood leucocytes of the patients to screen for mutations in GH1. We performed molecular analysis by polymerase chain reaction and automated sequencing of the entire coding region of the GH1. Segregation analysis was performed in the presence of allelic variations. In our casuistic, we identified 10 allelic variants in exon 4, exon 5 and intron 4 of GH1, three of which have not been described (c.407G > A/p.Val122Ile, c.507C > T/p.Tyr169Tyr and c.456+19G >T). In silico analysis predicted that none of the mutant alleles would result in deleterious effect on the GH protein. An additional study in children diagnosed with severe IGHD, identified just one patient with the pathogenic GH1 mutation responsible for the dominant form of this disease. In summary, defects in GH1 responsible for the autosomal dominant form of IGHD or Type II were not found in our cohort of Brazilian patients, suggesting that these mutations are infrequent in our population

Glicosídeos essenciais a vida podem ser sintetizados por métodos enzimáticos com altíssima especificidade. O mecanismo de reação de β-glicosidases GH1 por dupla-substituição envolve a formação de intermediário covalente (glicosil-enzima) que pode seguir duas rotas. Uma envolve sua hidrólise na ligação β-glicosídica entre glicone e aglicone do substrato, liberando o monossacarídeo da extremidade não-redutora; enquanto na outra rota, transglicosilação ou síntese por controle cinético, o intermediário é atacado por um aceptor glicosídico (2ª molécula de substrato), gerando um novo glicosídeo. BglB possui resíduos (W e H) que formam um \"canal\" por onde a água ataca o intermediário covalente no sítio ativo, sendo alvos de potenciais mutações que alteram o balanço entre as rotas e ampliando a eficiência de transglicosilação. Para caracterizar as bases moleculares da razão entre essas duas rotas catalíticas, β-glicosidases da família GH1 Spodoptera frugiperda (Sfβglu; AF052729) e Tenebrio molitor (Tmβglu; AF312017) foram produzidas como enzimas recombinantes em leveduras Pichia pastoris GS115, concentradas com 90% (p/v) de sulfato de amônio e diálise reversa com PEG 10000, e dialisadas em tampão CP 100 mM pH 6. A pureza foi confirmada por banda única com tamanho semelhante a 50 kDa (Sfβgly) e 56 kDa (Tmβgly) em SDS-PAGE. A atividade recuperada após purificação é 152% (Sfβgly) e 171% (Tmβgly) e a concentração protéica de 0,134 µg/µL (Sfβgly) e 0,217 µg/µL (Tmβgly). A razão entre as reações de hidrólise e transglicosilação (vH/vT) foi analisada utilizando os substratos pNPβ-gluco (0,1 mM a 8 mM) e MUβ-gluco (0,1 mM a 40 mM), a partir da velocidade de formação dos produtos pNP ou MU e glicose, respectivamente. Tmβgly catalisa reações de transglicosilação com ambos, mas vH/vT depende da [S] variando de +∞ (vH>>vT) a 1,5 para pNPβ-gluco e +∞ a 1 para MUβ-gluco. Sfβgly, ao contrário, não transglicosila com substrato MUβ-gluco. Além disso, vH/vT para pNPβ-gluco é 2 e independe da [S]. Já Sfβgly K201F conjuga propriedades de ambas, pois não transglicosila com MUβ-gluco, mas para pNPβ-gluco há ocorrência de transglicosilação dependente da [S], variando de +∞ (vH>>vT) a 1,25. Os parâmetros cinéticos (Vmax e Km) foram ajustados no Enzifitter e por simulação numérica na equação do modelo cinético ping-pong para dois substratos, e mostram maior (Km2) em Tmβgly e Sfβgly K201F do que em Sfβgly. Também foi avaliado o efeito da adição de aceptores alternativos que substituem a água em ensaios com pNPβ-gluco 4mM na presença de alcóois. Para as três enzimas a adição de metanol torna a vglc menor do que de vpNP, indicando ocorrência de transglicosilação. Com adição de n-propanol vH/vT diminui cerca de 7 vezes em Sfβgly e 2 vezes em Tmβgly. Os efeitos destes alcoóis sobre Sfβgly são maiores do que para Tmβgly e Sfβgly K201F, sugerindo acesso mais fácil destes ao intermediário covalente em Sfβgly, indicando diferenças entre as enzimas na configuração desta região. Portanto, notamos que vH/vT não está ligada à afinidade pela segunda molécula de substrato, podendo ser modulada por mutação do resíduo K201 de Sfβgly, posição relacionada ao acesso da água ou aceptor alternativo ao sítio ativo.
Glycosides are essential to life and can be synthesized by enzymatic methods with high specificity. The reaction mechanism of GH1 β-glucosidases by double-substitution involves the formation of covalent intermediate (glycosyl-enzyme) which may follow two routes. One of them involves hydrolysis in β-glycosidic bond between aglycone glucone and the substrate, releasing a monosaccharide from the non-reducing end, whereas in the other route, transglycosylation or synthesis by kinetic control, the intermediate is attacked by a glucosyl acceptor (second substrate molecule), generating a new glucoside. BglB has two residues (W and H) that form a \"channel\" through which water attacks the covalent intermediate on the active site. Thus, the residues are potential targets for mutations that alter the balance between routes, increasing the efficiency of transglycosylation. To characterize the molecular basis of the ratio between these two catalytic routes, two β-glycosidases from GH1 family, Spodoptera frugiperda (Sfβgly; AF052729) and Tenebrio molitor (Tmβgly; AF312017) were produced as recombinant enzymes in Pichia pastoris GS115, concentrated using 90% (w/v) ammonium sulfate and reverse dialysis with PEG 10000, and dialyzed in 100 mM CP buffer pH 6. SDS-PAGE confirmed that Sfβgly (~50 kDa) and Tmβgly (~56 kDa) were pure after that procedure. The activity recovered after purification were 152% (Sfβgly) and 171% (Tmβgly) and protein concentration were 0.134 mg/mL (Sfβgly) and 0.217 mg/mL (Tmβgly). The ratio between the hydrolysis and transglycosylation (vH/vT) was analyzed using pNPβ-gluco substrates (0.1 mM to 8 mM) and MUβ-gluco (0.1 mM to 40 mM), the rate of formation of pNP or MU and glucose. Tmβgly catalyzes transglycosylation reactions with both substrates, but vH/vT depends on [S] ranging from +∞ (vH>>vT) to 1.5 for pNPβ-gluco and + ∞ to 1 for MUβ-gluco. In contrast, Sfβgly did not catalyses transglycosilation with MUβ-gluco. Moreover, vH/vT for pNPβ-gluco-is 2 and is independent of [S]. Sfβgly K201F combines properties of both, because it does not catalyse transglycosylation with MUβ-gluco, but for pNPβ-gluco the occurrence of transglycosylation is dependent on [S], ranging from + ∞ (vH>>vT) to 1.25. The kinetic parameters (Vmax e Km) were adjusted in Enzfitter and numerical simulation showing that Km2is higher for Tmβgly and Sfβgly K201F than Sfβgly. We also evaluated the effect of adding alternative acceptors that replace the water in pNPβ gluco-4 mM. For the three enzymes the addition of methanol makes vglc less than pNP, indicating the occurrence of transglycosylation. Addition of n-propanol decreases vH/vT about 7 times in Sfβgly and 2 times Tmβgly. The effects on Sfβgly are higher than on Tmβgly and Sfβgly K201F, suggesting easier access to the covalent intermediate in Sfβgly. We observe that vH/vT is not related to affinity for the second substrate molecule and may be modulated by mutation of residue K201 Sfβgly, position related to the access of water or alternative acceptor to the active site.

Orientador: Prof. Dr. Wanius José Garcia da Silva
Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biotecnociência, 2017.
A hidrolise enzimatica da celulose e realizada pela acao sinergica de pelo menos tres tipos de celulases distintas: endoglucanases, exoglucanases e B.glicosidases. As endoglucanases e celobiohidrolases sao frequentemente inibidas pelo aumento da concentracao de celobiose (dimero de glicose) no meio reacional. As ¿À-glicosidases sao enzimas que clivam celobiose em monomeros de glicose. Portanto, as B-glicosidases sao essenciais ao processo de hidrolise da celulose por impedirem o acumulo de celobiose e, assim, evitar a diminuicao da taxa de hidrolise. Processos de pre-tratamento da biomassa lignocelulosica sao empregados, antes da reacao de hidrolise enzimatica da celulose, a fim de retirar a fracao de lignina e aumentar a taxa de conversao da celulose em glicose. Porem, estes processos de pre-tratamento da biomassa lignocelulosica nao sao 100% eficientes na remocao da lignina. Estudos previos mostraram que a adicao de lignina a celulose pura pode causar a reducao da liberacao de acucar em valores superiores a 60%. Assim, neste estudo, nos caracterizamos a adsorcao nao produtiva da enzima ¿À-glicosidase da familia GH1 da bacteria Thermotoga petrophila (TpBGL1) em ligninas extraidas de diferentes biomassas (cana-de-acucar e eucalipto). Em pH 7 e 6, nossos resultados indicaram que a repulsao eletrostatica enfraquece a adsorcao nao produtiva de TpBGL1 em ligninas. Contudo, em pH 4 a atracao eletrostatica fortalece a adsorcao nao produtiva. Alem disso, o aumento da temperatura de 25 oC para 70 oC nao resultou em um aumento significativo da adsorcao de TpBGL1 em ligninas, provavelmente porque nao ocorre um aumento significativo de regioes hidrofobicas na estrutura da enzima expostas ao solvente. Todas as informacoes obtidas neste estudo poderao ser uteis para aplicacoes biotecnologicas no campo de conversao de polissacarideos estruturais em bioenergia.
The B-glucosidases are a group of important enzymes employed in a large number of industrial applications. In this study, we reported for the first time the photobiosynthesis of stable and functional silver nanoparticles (AgNPs) using two hyperthermostable bacterial â-glucosidases with industrial potential. The syntheses were simple and rapid processes carried out by mixing â-glucosidases and silver solution under irradiation with light (450-600 nm), therefore, compatible with the green chemistry methodology. The synthesized AgNPs were characterized using a series of physical techniques. Absorption spectroscopy showed a strong absorption band at 460 nm due to surface plasmon resonance of the AgNPs. X-ray diffraction analysis showed that the AgNPs were purely crystalline in nature. Electron microscopy showed AgNPs of variable diameter ranging from 20 to 100 nm. In addition, electron microscopy, zeta potential and Fourier transform infrared spectroscopy results confirmed the presence of â-glucosidases coating and stabilizing the AgNPs. The results also showed that the enzymatic activities were maintained in the â-glucosidases assisted AgNPs. The data described in this study should provide a useful basis for future studies of â-glucosidases assisted AgNPs, including biotechnological applications.

We used the GENECONV program, the Hsu et al. (2010) method and phylogenetic analyses to analyze the gene conversions which occurred in the growth hormone, folate receptor and trypsin gene families of six primate species. Significant positive correlations were found between sequence similarity and conversion length in all but the trypsin gene family. Converted regions, when compared to non-converted ones, also displayed a significantly higher GC-content in the growth hormone and folate receptor gene families. Finally, all detected gene conversions were found to be less frequent in conserved gene regions and towards functionally important genes. This suggests that purifying selection is eliminating all gene conversions having a negative functional impact.

Orientador: Mário Tyago Murakami
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Abstract: The abstract is available with the full electronic document
Doutorado
Bioquimica
Doutora em Biologia Funcional e Molecular

O objetivo deste estudo foi identificar SNPs na região promotora e no intron I do gene GH (região alvo) e verificar sua possível associação com o crescimento de O. niloticus, o que foi executado em duas etapas: (1) prospecção de SNPs; (2) associação de SNPs com crescimento das variedades Red-Stirling e Chitralada. As análises de associação foram realizadas por meio de metodologia estatística baseada em análise univariada de modelo linear misto considerando-se efeitos fixos e aleatórios. Nove SNPs foram identificados no promotor (GHP1 a GHP9) e um na região 5 UTR (GHP10), os quais formaram 10 blocos genotípicos (A a J). Na população de associação seis novos blocos foram identificados (K a P). Os blocos B, P, K, L e M foram associados aos melhores pesos e os SNPs GHP6 a GHP10 demonstraram associação significativa (P < 0,05) como o crescimento. Portanto, foi possível estimar um conjunto de genótipos com maior efeito genético aditivo sobre o crescimento, o qual poderia ser utilizado em futuros programas de melhoramento genético assistidos por marcadores moleculares.
The present study aimed to identify SNPs in the proximal promoter region and in the first intron of GH gene and to evaluate if there is association of SNPs variation with the O. niloticus growth rate. Firstly, SNP searching in the two targeted regions was carried out in four strains. Then, two strains, Red-Stirling and Chitralada were used in grow-out testing in cages. Association between SNPs and growth rate were statistically estimated by univariate linear mixed model taking into account fixed and random effects. Nine SNPs were found in the proximal promoter region and one in the 5 UTR region, which formed 10 genotype blocks (A to J). Five of these genotype blocks (F to J) were not found in the grow-out individuals. However, six new genotype blocks (K to P) were identified. Genotype blocks B, P, K, L and M were statistically associated to the best weights, and the SNPs GHP6 to GHP10 individually showed significant association (P < 0,05) with growth. These findings found herein may potentially be used as Marked-Assisted Selection in tilapia breeding programs.

El retard de creixement pot ser secundari en un percentatge indeterminat de pacients a un problema genètic. En aquest treball s'analitza el gen de l'hormona de creixement (GH1) i s'estudien els canvis en la seqüència que produeixin un ARN missatger anòmal o una proteïna que no sigui funcional. S'ha estudiat el mapa polimòrfic del gen GH1 en una població control (n=307), per definir el mapa de SNPs. Alhora s'ha buscat un model en el qual poder estudiar l'expressió d'aquest gen. S'ha aïllat l'ARN de leucòcits de sang perifèrica tant de pacients com de controls, i s'ha amplificat el missatger de GH1. També s'ha posat a punt un model cel·lular en el qual poder estudiar la bioactivitat de les proteïnes recombinants portadores dels canvis detectats en pacients (Phe25Tyr i Val173Leu).

Les conclusions del treball han estat les següents:

1. Els nostres resultats han confirmat i ampliat la descripció de la variabilitat estructural del gen GH1. Han permès descriure el mapa complet per primera vegada, en una població adulta amb talla normal, compresa entre -2 i +2 SDS (n=307).

2. En les poblacions amb retard crònic de creixement (n=728) s'han detectat en un 11% de pacients canvis en la seqüència del gen GH1 diferents dels SNPs del mapa control. Entre els canvis, l'11,3% correspon a canvis puntuals d'un aminoàcid (1% del total de pacients índex).

3. L'expressió de GH1 en leucòcits de controls no és constitutiva ni constant en quant a les variants de splicing observades. Aquesta metodologia no permet detectar de forma fiable mutacions en pacients. En canvi, ens ha permès confirmar l'expressió de mutants com en el cas d'una substitució predictora de canvi d'aminoàcid, així com descartar o confirmar anomalies de splicing en al·lels portadors de canvis intrònics.

4. La utilització d'eines informàtiques ha permès realitzar una aproximació teòrica a l'efecte que dos canvis d'aminoàcid (Phe25Tyr i Val173Leu) a la proteïna GH tenen sobre la seva estructura i interacció amb GHR. L'anàlisi d'homologia de les proteïnes mutants amb les dels altres gens del clúster de la GH ha permès concloure que la substitució Phe25Tyr es pot haver generat per recombinació gènica amb el gen GH2, mentre que no és el cas per a la proteïna amb el canvi Val173Leu.

5. Les proteïnes recombinants obtingudes al laboratori (WT i mutants) han estat aptes per al seu estudi in vitro (inmunoreactivitat, proliferació cel·lular i bioactivitat).

6. Les GHs mutants Phe25Tyr i Val173Leu presenten una inmunoactivitat i una bioactivitat disminuïdes. Els models cel·lulars de les línies HepG2 i C-28/I 2 no han permès obtenir resultats, mentre que el model de cultiu primari de condròcits fetals humans ha permès detectar que les dues GHs mutants estimulen amb menor intensitat la transcripció de IGF1 mentre que provoquen un augment en l'expressió de GHR i IGF1R.

7. El canvi d'aminoàcid Phe25Tyr incorporat en un al·lel de GH1 portat en heterozigosi per dues famílies no relacionades es manifesta com a retard de creixement durant la infantesa que s'acompanya de resposta deficitària de secreció de GH i disminució de IGF1. La resposta al tractament amb GH és adequada i permet recomanar el tractament fins al final del creixement. El canvi Val173Leu detectat en una pacient amb talla baixa i RIUC, sense dèficit aparent de GH, permet suggerir que la detecció precoç de mutacions al gen GH1 quan la secreció aparenta ser normal, permetria un tractament més precoç que hagués millorat el seu pronòstic de talla final. L'anàlisi dels haplotips complets de GH1 permet interpretar l'efecte de la combinació de mutacions a la proteïna amb els genotips per als SNPs.


TITLE OF THE THESIS: "MOLECULAR CARACTERITZATION OF GH1 GENE IN CONTROL AND PATHOLOGIC POPULATIONS, EXPRESSION PATTERNS IN PERIPHERAL BLOOD LEUCOCYTES AND FUNCTIONAL STUDY OF MUTANT
PROTEINS DETECTED IN PATIENTS"

SUMMARY: The growth delay can be a secondary factor in front of a genetic problem in an indeterminate percentage of patients. In this work, it is analyzed the gene of growth hormone (GH1) and it is studied the changes in the sequence that produce an anomalous messenger ARN or a protein that is not functional. The polymorphic map of gene GH1 has been studied in a control population (n=307), to define the map of SNPs. At the same time, a model has been looked for, in which it could be possible to study the expression of this gene. The ARN of leucocytes of peripheral blood of patients and controls has been isolated, and the GH1 messenger has been amplified. Moreover, we have worked in a cellular model that allows us to study the bioactivity of recombinant proteins carrying the changes detected in patients (Phe25Tyr and Val173Leu).
The conclusions of the work have been the following ones:

1. Our results have confirmed and extended the description of the structural variability of gene GH1. They have allowed us to describe the complete map for the first time, in an adult population with normal stature, between -2 and +2 SDS (n=307).

2. In the populations with chronic growth delay (n=728) in an 11% of the patients, changes have been detected in the sequence of gene GH1 different from the SNPs of the map control. Within these changes, 11.3% correspond to precise changes in an amino acid (1% of the total of index patients).

3. The expression of GH1 in leukocytes of controls is neither constitutive nor constant as far as the observed variants of splicing. This methodology does not allow detecting without doubt the mutations in patients. However, it has allowed us to confirm the expression of mutants as in the case of a predicting substitution of change in amino acid, as well as discarding or confirming anomalies of splicing in carrying alleles of intronic changes.

4. The use of computer tools has allowed us to make a theoretical approach to the effect that two changes in amino acid (Phe25Tyr and Val173Leu) in protein GH have on their structure and interaction with GHR. The homology analysis of mutant proteins with those of the other genes of the cluster of the GH has allowed us to conclude that the Phe25Tyr substitution can have been generated by genic recombination with gene GH2, whereas it is not the case for the protein with the Val173Leu change.

5. The recombinant proteins obtained in the laboratory (WT and mutants) have been suitable for their study in vitro (inmunoreactivity, cellular proliferation and bioactivity).

6. The mutant GHs Phe25Tyr and Val173Leu show a diminished inmunoactivity and bioactivity. The cellular models of lines HepG2 and C-28/I2 have not allowed us to obtain results; in contrast, the model of primary culture of human foetal chondrocytes has allowed us to detect that the two mutant GHs stimulate with smaller intensity the IGF1 transcription and they cause an increase in the expression of GHR and IGF1R.

7. The change of Phe25Tyr amino acid incorporated in one allele of GH1 taken to heterozygosity by two non-related families is shown as growth delay during childhood accompanied by deficiency secretion answer of GH and diminution of IGF1. The response to the treatment with GH is suitable and allows us to recommend the treatment until the end of the growth. The Val173Leu change detected in a patient with short stature and RIUC, without apparent GH deficiency, allows us to suggest that precocious detection of mutations in gene GH1 when the secretion pretends to be normal, would allow a precocious treatment that would have improved its prognosis of final stature. The analysis of the complete haplotypes of GH1 allows us to interpret the effect of the combination of mutations in the protein with the genotypes for the SNPs.

Wood formation or xylogenesis is a fundamental process for so diverse issues as industry, shelter and a sustainable environment. Wood is comprised of secondary xylem, rigid large cells with thick cell walls that are lignified. The basis for the sturdy cells is an advanced composite made up of cellulose fibers cross-linked by hemicelluloses and finally embedded in lignin. This fiber-composite is the secondary cell walls of woody plants. Cell division and differentiation is regulated by switching on and off genes. Proteins encoded by these genes execute the major functions in the cells. They steer the entire machinery operating the structure and function of the cells, maintaining growth and synthesising essential products such as the cell wall carbohydrates.   Here we describe the investigation of genes and proteins involved in xylan formation as well as the development of a model system that will aid the functional analysis of wood formation. Xylan is the main hemicellulose or cross linking glycan in dicot wood and thereby one of the most abundant carbohydrates on earth. We demonstrate that hybrid aspen cell suspension cultures can be used as a model system for secondary cell wall formation. We have also examined glycosyltransferases from CAZy family 43 that play a part in secondary cell wall formation. We have focused on one of these, Pt×tGT43A, a likely ortholog of Arabidopsis IRX9, which plays a crucial role in xylan formation. The protein was transiently expressed in Nicotiana benthamiana and its function and localization is described. Also, we investigate a glycoside hydrolase, Pt×tXyn10A, involved in wood formation. Its role is not clear but it most likely modifies xylan as it gets incorporated into the secondary cell wall after secretion from the Golgi. This influences the interaction between cellulose, xylan and lignin in the finished wood cell. We have also cloned a transcription factor, Pt×tMYB021, a likely ortholog of Arabidopsis MYB46 and we show that it activates GT43A, GT43B and Xyn10A. By analysis of the promoter sequences we identify a CA-rich motif putatively important for xylem-specific genes.   By mastering proteins involved in xylogenesis we will acquire the tools to improve and develop the wood product market. Xylan is an immense unexploited source of renewable carbohydrate. New products envisioned include e.g. faster growing trees, changed fiber characteristics, optimised utilization of wood carbohydrates for biofuels and biomaterials as well as invention of intelligent materials by biomimetic engineering.
Vedbildning, eller xylogenes, är en grundläggande mekanism för så skilda områden som industri, boende och en hållbar miljö. Ved består av sekundärt xylem som är starka, stora celler med tjocka cellväggar som är lignifierade. Grunden för de starka cellerna är en avancerad komposit bestående av cellulosafibrer tvärbundna av hemicellulosa och slutligen ingjutet i lignin. Denna fiberkomposit är den sekundära cellväggen i vedartade växter. Celldelning och differentiering regleras genom att sätta igång och stänga av gener. Proteiner som kodas av dessa gener utför de viktigaste funktionerna i cellerna. De styr hela maskineriet som upprätthåller cellernas struktur och funktion, underhåller tillväxt samt tillverkar nödvändiga produkter såsom cellväggskolhydraterna. Här beskriver vi utforskningen av gener och proteiner som är inblandade i xylanbildning liksom utvecklandet av ett modellsystem som kommer vara en hjälp i den funktionella analysen av vedbildning. Xylan är den vanligaste hemicellulosan, eller korsbindande glykanen, i lövträd och därför en av de vanligaste kolhydraterna på jorden. Vi demonstrerar att hybridaspcellkulturer i suspension kan användas som ett modellsystem för sekundär cellväggsbildning. Vi har också undersökt glykosyltransferaser från CAZy-familj 43 som tycks spela en viktig roll i bildandet av sekundär cellvägg. Vi har fokuserat på en av dessa, Pt×tGT43A, en trolig ortolog till Arabidopsis IRX9 som spelar en viktig roll i xylanbildning. Proteinet har uttryckts övergående i Nicotiana benthamiana och dess funktion och lokalisering beskrivs. Dessutom undersöker vi ett glykosidhydrolas, Pt×tXyn10A, involverad i vedbildning. Dess roll är oklar men högst sannolikt modifierar det xylan medan det inkorporeras i sekundära cellväggen efter sekretion från Golgi. Detta influerar interaktionen mellan cellulosa, hemicellulosa och lignin i den slutliga vedcellen. Vi har också klonat en transkriptionsfaktor, Pt×tMYB021, en trolig ortolog till Arabidopsis MYB46 och vi visar att den aktiverar GT43A, GT43B och Xyn10A. Genom analys av promotorsekvenserna har vi identifierat ett CA-rikt motiv förmodat viktigt för xylemspecifika gener.Genom att bemästra proteinerna som är ansvariga för vedbildning får vi verktyg att utveckla skogsproduktsmarknaden. Xylan är en ofantligt stor outnyttjad källa till förnyelsebara kolhydrater. En vision är nya produkter som till exempel snabbväxande träd, ändrade fiberegenskaper, optimerat användande av vedkolhydrater för biobränsle och biomaterial såväl som utvecklandet av intelligenta material genom biomimetisk ingenjörskonst.
QC20100730

Human growth hormone (GH) participates in human longitudinal growth, lipid and carbohydrate metabolism, and comprises a remarkably number of proteins with similar sequences, generated either genetically or post-translationally, that in some cases show clearly differentiated biological activities. Current methods employed for its quantification are mainly based on a specific immunodetection of the most concentrated GH variants in blood circulation. However, it is not clear neither which variants are recognised in each case, nor which is the real concentration of some of these variants. Probably related, present immunoassays show a disparity of results between them that difficults the comparison of data from different assays, with direct consequences in the clinical field. Within a doping context, the illegal administration of recombinant GH constitutes a complex challenge, given the fact that the pharmaceutical variant and the native 22 kDa GH variant do not show any structural difference that allows a direct detection. However, the administration of the pharmaceutical inhibits the natural production of the hormone, resulting in modifications between the relative concentration of some of these variants. In this case, which variants are detected is of utmost importance, since these constitute the base of this anti-GH doping method. Here, the relevance of some GH variants is addressed, including their generation, characterisation, analysis through specific antibodies and their detection on biological samples.
L'hormona de creixement humana (GH) participa en el creixement post-longitudinal i en el metabolisme de lípids i carbohidrats, i comprèn un extraordinari nombre de proteïnes de seqüències similars, generades tant genèticament com posttranslacional, que en alguns casos mostren activitats biològiques clarament diferenciades. Els mètodes actuals emprats per la seva quantificació es basen principalment en una immunodetecció específica de les variants de GH més concentrades en circulació sanguínia. Tanmateix, no resta clar quines variants es reconeixen en cada cas, ni quina és la concentració real d'algunes d'aquestes variants. Possiblement relacionat, els immunoassaigs actualment utilitzats mostren una disparitat de resultats que dificulten la comparació de dades d'assaigs diferents, amb conseqüències directes en el camp clínic. Dins d'un context de dopatge, l'administració il·legal de GH recombinant constitueix un desafiament complex, donat el fet que la variant farmacèutica i la variant de GH nativa de 22 kDa no mostren cap diferència que permeti una detecció directa. No obstant, l'administració del medicament farmacèutic inhibeix la producció natural de la hormona, derivant en canvis entre la concentració relativa d'algunes d'aquestes variants. En aquest cas, és de màxima importància quines variants són detectades, ja que això constitueix la base d'aquest mètode d'antidopatge de GH. Aquí, s'estudia la rellevància d'algunes variants de GH, incloent-hi la seva generació, caracterització, anàlisi via anticossos específics i la seva detecció en mostres biològiques.

Η διαδικασία της αύξησης ελέγχεται από έναν πολύπλοκο συνδυασμό πολλών παραγόντων σε διάφορα επίπεδα, που περιλαμβάνουν ενδογενείς παράγοντες, όπως είναι ο γονότυπος, οι ορμόνες, οι παράγοντες αύξησης και εξωγενείς παράγοντες, όπως είναι η διατροφή και η επίδραση του περιβάλλοντος. Οι ορμονικοί παράγοντες, που επηρεάζουν την αύξηση είναι κυρίως η αυξητική ορμόνη (GH) και οι ινσουλινόμορφοι αυξητικοί παράγοντες (IGFs). Στην διαδικασία της αύξησης συμμετέχουν, όμως, και άλλες ορμόνες, όπως η θυροξίνη, τα επινεφριδιακά ανδρογόνα, τα στεροειδή του φύλου, τα γλυκοκορτικοειδή, η βιταμίνη D, η λεπτίνη και η ινσουλίνη, που αλληλεπιδρούν με τον άξονα GH-IGF. Η αυξητική ορμόνη εκκρίνεται στην κυκλοφορία από τα σωματότροπα κύτταρα του πρόσθιου λοβού της υπόφυσης, υπό την επίδραση δύο υποθαλαμικών ορμονών του εκλυτικού παράγοντα της αυξητικής ορμόνης (GHRH), που διεγείρει την έκκριση της GH και της σωματοστατίνης (SS), που αναστέλλει την έκκρισή της. Μέχρι σήμερα στην διεθνή βιβλιογραφία έχουν περιγραφεί πολλές μεταλλάξεις του γονιδίου της GH ως αιτία κοντού αναστήματος στα παιδιά. Η παρούσα μελέτη εξέτασε ομάδα 11 παιδιών με κοντό ανάστημα, ρυθμό αύξησης κάτω από την 2η εκατοστιαία θέση και καθυστερημένη οστική ηλικία. Όλοι οι ασθενείς υπεβλήθησαν σε λεπτομερή κλινική εξέταση και πλήρη εργαστηριακό έλεγχο. Από την κλινική εξέταση και τον εργαστηριακό έλεγχο αποκλείστηκε η παρουσία κάποιας συστηματικής πάθησης. Στην συνέχεια υπεβλήθησαν σε προκλητές δοκιμασίες έκκρισης της GH, με κλονιδίνη και L-Dopa, σε έλεγχο της 24ωρης έκκρισης της GH και τη δοκιμασία γένεσης του IGF-I. Με βάση τα εργαστηριακά αποτελέσματα της έκκρισης της GH η ομάδα των ασθενών διαχωρίστηκε σε αυτούς με ιδιοπαθές κοντό ανάστημα (10 περιπτώσεις) και ένα ασθενή με νευροεκκριτική δυσλειτουργία της GH (GHND), ο οποίος είχε μειωμένη 24ωρη έκκριση GH. Από τους ασθενείς αυτούς ελήφθησαν βιοψίες ούλων, στους καλλιεργημένους ινοβλάστες των οποίων έγιναν οι μελέτες αύξησης των ινοβλαστών και περιφερικό αίμα, από το οποίο έγινε εξαγωγή γονιδιωματικού DNA. Έγινε πολλαπλασιασμός των γονιδίων του υποδοχέα της GH (GHR) και του γονιδίου της GH (GH1) με την αλυσιδωτή αντίδραση πολυμεράσης (PCR) και προσδιορισμός της αλληλουχίας τους. Ανιχνεύτηκαν μεταλλαγές στους 6 από τους 11 ασθενείς, που μελετήθηκαν, οι οποίες εντοπίζονταν στο ιντρόνιο 4 του γονιδίου GH1 και ένας ακόμη ασθενής που έφερε μεταλλάξεις στα ιντρόνια 1 και 2. Οι μεταλλάξεις αυτές δεν επηρέαζαν την διαδικασία του ματίσματος και τον σχηματισμό του mRNA και απομακρύνονταν με το μάτισμα. Στην βιβλιογραφία αναφέρονται περισσότεροι από 10 πολυμορφισμοί του γονιδίου GH1 που εντοπίζονται κυρίως στα ιντρόνια του γονιδίου και κάποιοι από αυτούς έχουν συσχετιστεί με ελαττωμένη έκφραση του γονιδίου GH1. Στον ασθενή με την GHND περιγράφηκε μια μεταλλαγή στη θέση +7 του ιντρονίου 4 του γονιδίου GH1. RT-PCR του GH1 cDNA έδειξε ότι η μετάλλαξη αυτή είναι υπεύθυνη για το εσφαλμένο μάτισμα του mRNA, με αποτέλεσμα την απαλοιφή του εξονίου 5 από το ώριμο μετάγραφο. Ο ασθενής με τη μεταλλαγή είναι ετεροζυγώτης και η ίδια μεταλλαγή σε ετερόζυγη κατάσταση, βρέθηκε και στους δύο γονείς του ασθενούς, οι οποίοι έχουν επίσης κοντό ανάστημα. Η μεταλλαγή αυτή οδηγεί στην παραγωγή μικρότερου μορίου GH. Η βιοδραστικότητα του παραγόμενου ανώμαλου μορίου της GH εκτιμήθηκε με την προσθήκη ορού του ασθενούς σε καλλιέργειες φυσιολογικών ινοβλαστών, με τη μέθοδο ενσωμάτωσης στο DNA της βρώμο-δεοξυουριδίνης (BrDU), η οποία έδειξε μειωμένη σύνθεση DNA συγκρινόμενη με την σύνθεση DNA παρουσία ορού φυσιολογικών ατόμων. Δηλαδή η περίπτωση αυτή οικογενούς κοντού αναστήματος, το οποίο κληρονομείται κατά τον επικρατούντα χαρακτήρα, οφείλεται σε μεταλλαγή στο ιντρόνιο 4 του γονιδίου GH1.
Growth can be defined as an increase in size by accretion of tissue. The control of the growth process is affected by many complex interacting factors including internal cues such as the genotype, external factors such as nutrition and environment, and internal signaling systems such as hormones and growth factors. The principal hormones influencing growth are Growth Hormone (GH) and the Insulin-like Growth Factors (IGFs), but many other hormones contribute, such as thyroxine, adrenal androgens, sex steroids, glucocorticoids, vitamin D, leptin and insulin, often channeled through interaction with the GH-IGF axis. GH is secreted from the anterior pituitary into the circulation. The pattern of GH secretion is determined primarily by the interaction between the hypothalamic peptides Growth Hormone Releasing Hormone (GHRH) and somatostatin (SS). Many mutations of the GH1 gene have been described as the cause of short stature in children. The present study examined 11 children with severe short stature, growth velocity below the 2nd centile and delayed bone age. All patients underwent thorough clinical examination and laboratory investigation in order to exclude an underlying chronic disease. Also GH secretion provocative studies, 24 hr endogenous secretion studies and IGF-I generation test were carried out. According to the results of these tests the patients we studied were divided in two groups: 10 of the patients had idiopathic short stature (ISS) and 1 patient had GH neurosecretory dysfunction (GHND). Fibroblast cultures were established from gingival biopsies obtained from the patients and genomic DNA was extracted from peripheral blood leukocytes. GH1 and GH receptor (GHR) genes were amplified by PCR and sequenced. Hot spot mutations were detected in GH1 intron 4 in 6 patients and mutations in introns 1 and 2 were detected in 1 patient. These mutations did not affect the splicing of the primary RNA transcript. A novel deletion of thymine 7 bp downstream from the 3' splice site of intron 4 was found in the patient who had GHND. RT-PCR of GH1 cDNA showed that this mutation causes aberrant GH mRNA splicing, changes the read frame, creates a new stop codon and results in the deletion of exon 5. This was also confirmed by restriction enzyme analysis of the mutant cDNA. Both short parents and the patient are heterozygotes for this mutation. BrDU incorporation in the DNA of normal fibroblast cultures in the presence of the patient’s blood serum showed reduced DNA synthesis compared to fibroblasts cultured in medium with normal human serum. Addition of high concentrations of GH (4 μg/ml) to the culture medium containing the patient’s serum led to a near normal DNA synthesis. This is a new case of familial short stature inherited as a dominant trait, due to a mutation in intron 4 of the GH1 gene.

Xylanases (EC 3.2.1.8) are glycosyl hydrolases that catalyze the hydrolysis of internal β-1,4 glycosidic bonds of xylan backbones, and have potential economical and environment friendly applications in the paper pulp, food, animal feed, detergent industries, bio-ethanol and bio-energy production systems. A xylanase from Bacillus sp. NG-27 (BSX), which is an extracellular endoxylanase, belonging to glycosyl hydrolase family 10 (GH10), shows optimum activity at a temperature of 70 °C and at a pH 8.5. It has a (β/α)8-triosephosphate isomerase (TIM) barrel fold, which has been studied concerning its function, structural properties, design and evolution. BSX, apart from thermo-alkalophilic features, shows resistance to SDS denaturation and protease K degradation. Hence, BSX serves as an important model system for fundamental understanding of the structure-stability-evolution relations of the ubiquitous TIM barrel fold. While the factors responsible for the thermal stability of GH10 xylanases have been analyzed, the improvement of thermostability of already thermostable enzymes is an important challenge. In general, there are large differences in optimal temperature (Tm) between hyperthermostable proteins with respect to their mesophilic homologs, indicating considerable scope available for introducing novel protein engineering approaches to improve protein stability. Thermostability and thermotolerance are of particular importance for industrial enzymes, because higher operating temperatures allow higher reactivity, higher bioavailability, higher process yield, lower viscosity, and reducing the risk of contamination. Thus, finding enzymes that can function at high temperatures has immense industrial importance and constitutes an active area of research. Earlier studies on enzymatic activity and thermostability of a recombinant BSX (RBSX) with different extreme N-terminus mutants by biochemical/biophysical methods showed that a single amino acid substitution (Val1→Leu) markedly enhanced the thermostability of recombinant xylanase from 70 °C to 75 °C without compromising its catalytic activity and showed higher cooperativity in the thermal unfolding transition. Conversely, substitution of Val1→Ala (V1A) at the same position decreased the stability of the protein from 70 °C to 68 °C. Furthermore, it was observed that substitution of Phe4 by Ala decreased the stability by ~4 °C whereas substitution of Trp6→Ala and Tyr343→Ala decreased the stability by ~10 °C with respect to RBSX. On the other hand, substitution of Phe4 by another aromatic residue Trp (F4W) did not change the stability and activity of RBSX. However, structural details were not available at that time, precluding any structure-based rationalization of stability changes resulting from a single amino acid substitution. The thesis reports the crystal structures of a recombinant xylanase from Bacillus sp. NG-27 (RBSX) and its various N-terminal and C-terminal mutants namely V1A, V1L, F4A, F4W, W6A, and Y343A. The crystal structure of RBSX (PDB ID: 4QCE) was solved at a resolution of 2.35 Å whereas those of V1A mutant (PDB ID: 4QCF) and V1L mutant (PDB ID: 4QDM) were solved at a resolution of 2.26 Å and 1.99 Å respectively. On the other hand, the crystal structure of F4A was solved at a resolution of 2.23 Å whereas F4W, W6A, and Y343A mutants were solved at a resolution of 2.22 Å, 1.67 Å, and 2.30 Å respectively. The availability of experimentally determined RBSX structure and its various mutant structures has enabled a critical examination including from a network perspective, of factors influencing thermal stability. The crystal structures in combination with computational analysis have provided valuable insights into the structural features that govern protein thermostability. The thesis candidate established a link between N-terminal to C-terminal contacts and RBSX thermostability. The study reveals that augmenting N-terminal to C-terminal noncovalent interactions is associated with enhancement of the stability of the enzyme. Perhaps, for the first time, the study provides a network perspective of N-terminal to C-terminal interactions and shows that the stabilizing interactions are not restricted to terminal regions but propagate to different parts of the protein structure. Furthermore, analysis of structures of different aromatic mutants of RBSX and structural bioinformatics studies were combined to understand the role of long-range aromatic cluster in the form of 'aromatic-clique' in the thermal stabilization of proteins. The results highlight an additional source of stability in thermophilic proteins, which could arise due to the prevalence of aromatic-cliques. In addition, the work exemplifies the experimental evidence specifically through long-range aromatic clique, in reiterating the role of interactions between N- and C-termini in protein stabilization. The thesis candidate demonstrated the experimental evidence depicting the role of partially solvent exposed tryptophan residues in shielding a surface pocket, which influenced the solvation of backbone atoms and stability of the RBSX enzyme. The candidate carried out a comprehensive database analysis of available crystal structures to look into the possible role of partially exposed tryptophan in hyperthermophilic proteins. The study provides strong evidence that partially exposed tryptophan side-chain is recruited in hyperthermophilic proteins for occluding potential surface pockets, to provide backbone solvent shielding and local stabilization. The overall structure of this thesis is further explained through a chapter wise description below: Chapter 1 | An introduction and outline of the thesis This chapter starts with a general introduction about the diversity of microorganisms and their ability to thrive in extreme environments such as high temperature. The research on these enterprising organisms offers not just the insights into the resilience of life on earth or possibilities of life elsewhere in the universe but also can provide exciting opportunities for a variety of industrial, environmental, biomedical, and pharmaceutical applications. While the adaptation of the cell inventory is important, it is a challenge for proteins to overcome high temperature in order to remain folded in the correct three-dimensional structure while maintaining adequate flexibility for their desired function. Hence, elucidation of the molecular basis of protein stability at extreme temperature continues to attract researcher over a board range of disciplines. The various structural features responsible for protein stability are outlined and the basic structural and molecular strategies for the adaptation to high temperatures revealed by structure analysis are delineated. Of all potentially deactivating factors of protein stability, temperature is the best studied. A brief outline of the strategies and approaches for the design of proteins to meet the desirable properties such as increased thermal stability are presented whereas the structural features responsible for stability of triosephosphate isomerase (TIM)-barrel fold is outlined under a separate section. Subsequently a short introduction of family 10 (GH10) xylanases, which has the ubiquitous TIM-barrel fold and their classifications are presented. A section is dedicated to describe various thermostable GH10 xylanases, their structural features responsible for stability, and current and potential biotechnological applications. At the end, the scope of the present work is detailed. Chapter 2| Crystallization, Data Collection and Data processing of recombinant BSX (RBSX) and its different variants: Chapter 2 presents the purification of recombinant xylanase from Bacillus sp. NG-27 (RBSX), its N-terminal variants (V1A, V1L), and aromatic variants (F4W, F4A, W6A, and Y343A). The expression and purification of RBSX and its variants were carried out at the laboratory of our collaborator Prof. V. S. Reddy, Plant Transformation Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, India. Initial crystallization trials were screened by hanging drop vapor diffusion method and micro-batch diffusion method using crystallization-screening kits (Crystal Screen and Crystal Screen 2) from Hampton Research, USA and a laboratory made screen, which was based on reported crystallization condition of native BSX. After a few rounds of trials and optimization of the crystallization condition, diffraction quality rod shaped crystals of recombinant BSX (RBSX) were obtained within ten days, when 2-µl protein solution (10 g/ml) was mixed with 2-µl reservoir solution composed of 0.12 M MgCl2, 0.1 M NaCl, 0.1M Tris-HCl pH 8.5 and 15% PEG 8000. Subsequently, crystal was used for X-ray data collection and it diffracted X-rays to better than 2.2 Å at the home source at cryo-temperature (100 K). RBSX crystals belong to orthorhombic space group P212121 with unit cell parameters a = 54.77 Å, b = 75.65 Å, c = 179.91 Å and α = β = γ =90°. A three-dimensional screening grid was prepared based on crystallization condition of RBSX by carefully varying salt concentration (NaCl and MgCl2 from 10mM to 300mM in the interval of 10mM), different PEG variants (PEG 1000, PEG 3350, PEG 4000, PEG 8000, and PEG 10000) in the range of 5% to 20%. Tris-HCl buffer of pH 8.0 and of pH 8.5 was used in the concentration range of 0.05M and 0.1M respectively. Rod shaped crystals were obtained using hanging drop vapor diffusion method from the condition of 0.1M NaCl, 80mM MgCl2, 0.05M Tris-HCl pH 8.5 and 18 % PEG 8000 and 0.1M NaCl, 60mM MgCl2, 0.1M Tris-HCl pH 8.5 and 16 % PEG 8000 for V1L mutant and V1A mutant respectively. The diffracting crystals of F4A mutant were obtained from the condition of 0.1M NaCl, 140mM MgCl2, 0.05M Tris-HCl pH 8.5 and 15% PEG 8000 by using hanging drop vapor diffusion method. On the other hand, F4W and Y343A, crystals were grown by micro-batch diffusion method containing 1.1-μ1 ratio of protein and crystallization solution of 0.1M NaCl, 120mM MgCl2, 0.1M Tris-HCl pH 8.5 and 18% PEG 8000 and 0.1M NaCl, 150mM MgCl2, 0.1M Tris-HCl pH 8.5 and 15 % PEG 6000 respectively . W6A mutant crystals were grown by hanging drop vapor diffusion method of 0.1M NaCl, 160mM MgCl2, 0.05M Tris-HCl pH 8.5 and 20% PEG 8000. All the crystals were obtained at 20 °C-22 °C in 5-10 days, and were used for diffraction experiments (details in the table below). Table 1 Protein Space a b c α β γ X-ray source PDB group (Å) (Å) (Å) (°) (°) (°) ID RBSX P212121 54.77 75.65 176.91 90 90 90 Home-source 4QCE V1A C2 73.57 80.12 69.90 90 110.81 90 Home-source 4QCF V1L P212121 54.88 76.58 176.73 90 90 90 Synchrotron 4QDM F4W P212121 55.27 77.32 176.75 90 90 90 Home- source 5EB8 F4A P212121 52.62 67.71 181.54 90 90 90 Home- source 5EFF W6A P212121 54.99 76.60 181.54 90 90 90 Synchrotron 5EFD Y343A C2 73.86 80.11 69.21 90 111.19 90 Home- source 5EBA The quality of all dataset was assessed by SFCHECK. The data sets were found appropriate and useful for structure determination as discussed in Chapter 3. Chapter 3 | Molecular Replacement, Model Building, Refinement, validation of recombinant xylanase (RBSX), and different mutant structures: Chapter 3 details the application of molecular replacement method to the structure solution of RBSX structure, N-terminal and aromatic mutants of RBSX, the course of iterative model building and the refinement carried out and the quality of the final protein structure models. The structure solution for all the structures was obtained by the molecular replacement (MR) method with the program PHASER-MR in the PHENIX package using a search model of native-enzyme (2F8Q). The asymmetric unit of RBSX, V1L, F4A, F4W, W6A crystals was expected to contain two molecules whereas V1A and Y343A crystal was expected to contain one molecule as indicated by Matthews’s coefficient calculation. The final round of refinement was carried out with restrained refinement with TLS parameters for all the structures. The most essential refinement statistics of the final model of RBSX, V1A, and V1L mutant structures are given in Table 2 whereas the same for aromatic mutant structures, F4A, F4W, W6A, and Y343A are given in Table 3. Table 2 Refinement Statistics RBSX V1A V1L Resolution (Å) 27.7-2.32 26.8-2.26 40.2-1.96 Rwork / Rfree (%) 17.9/22.7 17.4/22.5 15.2/19.0 Average B-factors (Å2) Protein 21.6 26.3 13.9 Ligand/ion 15.6 26.4 18.74 Water 20.6 27.2 23.2 RMSD Bond distance (Å) 0.007 0.005 0.019 Bond angles (◦) 1.123 0.955 1.802 Luzzati coordinate 0.279 0.269 0.175 error (Å) Working set Table 3 Refinement Statistics F4A F4W W6A Y343A Resolution (Å) 18.15-2.23 18.97-2.22 32.1-1.67 34.03-2.30 Rwork / Rfree (%) 17.8/24.0 16.8/21.0 15.68/18.58 17.8/23.0 Average B-factors (Å2) Protein 13.1 19.5 16.7 26.8 Ligand/ion 14.2 20.0 21.4 26.1 Water 11.5 28.9 25.9 30.7 RMSD Bond distance (Å) 0.0144 0.0088 0.0139 0.0063 Bond angles (◦) 1.593 1.228 1.5995 1.0951 Luzzati coordinate 0.261 0.252 0.176 0.293 error (Å) Working set Chapter 4 | Mutations at the extreme N-terminus modulate thermostability of RBSX: Implications of interactions between termini for stability This chapter details the structural analysis of RBSX and its various extreme N-terminus mutations in relation to their different thermostability scale. Although several factors have been attributed to thermostability, the stabilization strategies used by proteins are still enigmatic. Studies on a RBSX, which has the ubiquitous (β/α)8-TIM (Triosephosphate isomerase) barrel fold showed that just a single mutation, Valine1→Leucine (V1L), though not part of any secondary structural element, markedly enhanced the stability from 70 °C to 75 °C without loss of catalytic activity. Conversely, substitution of Valine1→Alanine (V1A) at the same position decreased the stability of the enzyme from 70 °C to 68 °C. To gain structural insights as to how a single extreme N-terminus mutation can markedly influence the thermostability of the enzyme, the candidate has determined the crystal structure of RBSX and two mutants. Based on computational analysis of their crystal structures including residue interaction network, a link was established between N- to C-terminal contacts and RBSX thermostability. The study reveals that augmenting N- to C-terminal non-covalent interactions is associated with the enhancement of the stability of the enzyme. Perhaps, for the first time, the study provides a network perspective of N-terminal to C-terminal interactions and shows that the stabilizing interactions are not restricted to terminal regions but propagate to different parts of the protein structure. In addition, several lines of evidence were discussed that point to support the structural coupling between the chain termini and implications of stability changes in different proteins. It is proposed that the strategy of mutations at the termini could be exploited with a view to modulate stability without compromising on enzymatic activity, or in general, protein function, in diverse folds where N- and C-termini are in close proximity. Chapter 5 | Role of long-range aromatic cluster in the structural stability of RBSX Chapter 5 describes the different aromatic mutant crystal structures of RBSX namely F4W, F4A, W6A, and Y343A and the structural comparison with the RBSX crystal structure. Systematic studies of different alanine mutations (F4A, W6A, and Y343A) to disrupt this aromatic cluster showed that substitution of Phe4, Trp6, and Y343 by alanine drastically decreased the stability of recombinant BSX (RBSX). It was observed that substitution of Phe4 by Ala (F4A) decreased the RBSX stability by ~5 °C whereas substitutions of Trp6 by Ala (W6A) and Tyr343 by Ala (Y343A) markedly decreased the stability of the enzyme by ~10 °C. On the other hand, substitution of Phe4 by Trp (F4W) did not result any change in its thermal unfolding pattern of the enzyme. We observed that the mutated amino acid residues (Phe4, Trp6, and Tyr343) in the RBSX structure are part of an ‘aromatic-clique’. An aromatic-clique is defined as a cluster of aromatic residues in which each residue interacts with all other residues within the cluster through aromatic interactions. The study reveals that the decreased stability shown by F4A, W6A, and Y343A mutants resulted from cumulative effects in the loss of aromatic interactions and disruption of aromatic-clique, and reduced van der Waal interactions. In addition, the work exemplifies the importance of interactions between N-terminal and C-terminal through aromatic contacts or packing in folding and stability of the TIM-barrel fold protein. The structure based multiple sequence alignment of RBSX with other GH10 xylanase from Bacillus organisms revealed that aromatic-clique of interest is fully conserved in B. halodurans (BHX) and Bacillus firmus (BFX) xylanases, which are thermostable in nature, like RBSX. On the other hand, this aromatic-clique is not conserved in the GH10 xylanases from Bacillus N137, Bacillus alcalophilus, which are reported as thermo-labile in nature. Furthermore, analysis of available crystal structures of different thermostable xylanases from GH10 family showed the prevalence of aromatic-clique that may be playing a critical role in their structure-stability and folding. Lastly, a comprehensive analysis of homologous pairs of proteins from (hyper)thermophilic and mesophilic organisms was carried out and observed the high occurrence of aromatic-cliques in the thermophilic proteins in comparison to their mesophilic homologs. These results highlight an additional source of stability in thermophilic proteins, which can arise due to the prevalence of aromatic-cliques. The findings reported in the thesis provide important lessons for engineering xylanases for industrial applications. The strategy of mutations based on clustering of aromatic pairs in the form of ‘aromatic-clique’ may be effectively applied to other enzymes and provides new insights for engineers to design proteins for biotechnological applications. Chapter 6 | Tryptophan occludes surface pocket: Implications for protein stability Chapter 6 describes the structural feature of a partially exposed tryptophan residue, which effectively occludes a surface pocket and plays a critical role in RBSX thermo-stabilization. As a part our long-standing interest in the structural analysis of thermostable proteins, it was observed that just a single mutation, W6A of a recombinant xylanase (RBSX) from Bacillus sp. NG-27 decreased the stability from 70 °C to 60 °C. To gain structural insights into how a single mutation W6A can remarkably influence the thermostability of the enzyme, we determined the crystal structure of W6A mutant and compared the same with the crystal structure of RBSX. We serendipitously observed that substitution of Trp6 by alanine (W6A) in the protein results a small surface pocket, which was shielded by the bulky side-chain of Trp6 in the native structure. Inspection of the molecular structure of native protein structure revealed that side chain of Trp6 occludes the surface pocket, sterically impeding entry of solvent molecules including water. We demonstrated the experimental evidence depicting how a partially exposed tryptophan, which was shielding a surface pocket (tryptophan-shield), can directly influence the backbone solvation, and modulate the stability of the enzyme. Furthermore, computational analysis of high-resolution structures of hyperthermophilic proteins reveals that bulky and aromatic indole side-chain of tryptophan effectively occludes surface pockets in several hyperthermophilic proteins. The study provides a strong evidence that partially exposed tryptophan side-chain is recruited in hyperthermophilic proteins for occluding potential surface pockets to provide backbone solvent shielding and local stabilization. Chapter 7 | Summary and future direction Chapter 7 summaries the important findings of the present study from the crystal structure and computational analysis of a recombinant xylanase (RBSX) and its various N-terminal and C-terminal mutants and also outlines the future direction of the work. Appendix A details SFCHECK output for the processed data for all the structures reported in the thesis. Appendix B Reprints of the publications

Η αυξητική ορμόνη (GH) παίζει ιδιαίτερα σημαντικό ρόλο, καθώς προάγει κυρίως τη μεταγεννητική κατά μήκος αύξηση, και ελέγχει το μεταβολισμό των λιπιδίων και των υδατανθράκων, τη σύνθεση των πρωτεϊνών και τη διέγερση του ανοσοποιητικού συστήματος. Η ανεπάρκεια GH (GHD) διαγιγνώσκεται είτε με παθολογικές συγκεντρώσεις της GH στον ορό μετά από πρόκληση με φαρμακολογικούς παράγοντες που διεγείρουν την έκκριση της (κλασσική GHD), είτε με φυσιολογικές προκλητές δοκιμασίες αλλά παθολογικό 24ωρο εκκριτικό ρυθμό της GH (GH νευροεκκριτική δυσλειτουργία, GHND). Οι GHD και GHND ασθενείς έχουν σοβαρή καθυστέρηση αύξησης και ανταποκρίνονται καλά στην εξωγενή θεραπεία με hGH. Κοντό ανάστημα που σχετίζεται με ανεπάρκεια αυξητικής ορμόνης (GHD) μπορεί να είναι σποραδικού τύπου και ιδιοπαθής, αλλά στο 5-30% των περιπτώσεων υπάρχει προσβεβλημένος πρώτου βαθμού συγγενής, που υποδηλώνει γενετική αιτιολογία. Μεταλλάξεις του γονιδίου της GH (GH1) ευθύνονται για την εκδήλωση οικογενούς μεμονωμένης ανεπάρκειας GH (IGHD). Ο εγγύς υποκινητής του GH1 γονιδίου παρουσιάζει υψηλού βαθμού πολυμορφισμό, με τουλάχιστον 16 αναγνωρισμένους πολυμορφισμούς (SNPs) σε έκταση 535 βάσεων, που εκδηλώνονται σε σύνολο 40 απλότυπων, κάποιοι από τους οποίους επηρεάζουν την έκφραση της GH. Σκοπός της παρούσας εργασίας ήταν η ανεύρεση αλλαγών στην αλληλουχία του GH1 γονιδίου της αυξητικής ορμόνης (GH) και του εγγύς υποκινητή του σε ασθενείς με οικογενή μεμονωμένη ανεπάρκεια GH (IGHD), αλλά και η ανάλυση του τρόπου κληρονομικότητας αυτών των αλλαγών. Μελετήθηκαν 33 IGHD ασθενείς (29 GHD και 4 GHND), τα μέλη των οικογενειών τους (22 οικογένειες) και 31 μάρτυρες. Απομονώθηκε γονιδιωματικό DNA από λεμφοκύτταρα περιφερικού αίματος και πραγματοποιήθηκε πολλαπλασιασμός του GH1 γονιδίου και του υποκινητή του με την αλυσιδωτή αντίδραση πολυμεράσης (PCR). Τα δείγματα αλληλουχήθηκαν και προσδιορίστηκαν αλλαγές της αλληλουχίας με βάση την NCBI, blast- Μ28466.1-βάση δεδομένων. Στους ασθενείς μετρήθηκαν τα επίπεδα IGF-1, τα επίπεδα των άλλων ορμονών του πρόσθιου λοβού του αδένα της υπόφυσης, η μέγιστη τιμή GH μετά από προκλητές δοκιμασίες με κλονιδίνη και L-Dopa, και στους 4 GHND ασθενείς πραγματοποιήθηκε 24ωρη καταγραφή της αυθόρμητης έκκρισης της GH. Οι πολυμορφισμοί (SNPs) που ανιχνεύθηκαν στο GH1 γονίδιο και τον υποκινητή του σε ασθενείς και μάρτυρες, ελέγχθηκαν για τη συχνότητα των γονοτύπων τους, και συσχετίστηκαν με κλινικοεργαστηριακά χαρακτηριστικά, ενώ ο δυνητικός λειτουργικός ρόλος των μεταλλάξεων ελέγχθηκε με λογισμικά προγράμματα. Η αλληλούχιση του GH1 γονιδίου και του υποκινητή του ανέδειξε 18 γνωστούς από τη βιβλιογραφία SNPs στον υπό μελέτη πληθυσμό και τρεις νέες (novel) μεταλλάξεις στις 3 από τις 22 οικογένειες. Ανάλυση με το λογισμικό πρόγραμμα MatΙinspector των 2 ετερόζυγων σημειακών μεταλλάξεων που εντοπίστηκαν στον GH1 υποκινητή, -485G>C and -400G>A, αποκάλυψε θέση πρόσδεσης για τον μεταγραφικό παράγοντα E-twenty six-1 (ETS-1). Ανάλυση της τρίτης ετερόζυγης μετάλλαξης (G>A) στη θέση +300 του εσωνίου 1 του GH1 γονιδίου με τα λογισμικά ESEfinder3 και ASSP αποκάλυψε τη δημιουργία μιας κρυφής θέση ματίσματος και διάσπαση της περιοχής ματίσματος εξωνίου (ESE) ενός ψευδοεξωνίου, μειώνοντας τον αριθμό προσδενόμενων πρωτεϊνών πλούσιων σε σερίνη-αργινίνη (SR). Η στατιστική ανάλυση των συχνοτήτων των γονοτύπων στους πολυμορφισμούς υψηλής συχνότητας, και η συσχέτιση τους με παραμέτρους που σχετίζονται με την αύξηση, την έκκριση της GH, αλλά και την ανταπόκριση στην hGH αγωγή, έδειξε ότι οι GHD ασθενείς πιθανόν να εμφανίζουν διαφορετική μεταγραφική δραστηριότητα στο GH1 γονίδιο λόγω των θέσεων -278, -57 του υποκινητή, -6 της 5΄ UTR περιοχής και της θέσης +1169 του γονιδίου, που έρχεται σύμφωνο και με άλλες μελέτες, αλλά και της θέσης -31, που αναφέρεται για πρώτη φορά. Αυτοί οι πολυμορφισμοί συσχετίστηκαν με μειωμένα επίπεδα IGF-1. Από την άλλη, οι SNPs που αναγνωρίσθηκαν στους GHND ασθενείς, παρόλο που είναι παρόμοιοι με αυτούς των GHD ασθενών, συσχετίστηκαν με παραμέτρους αύξησης και όχι με τα επίπεδα IGF-1. Οι 18 πολυμορφισμοί και οι 3 μεταλλάξεις που εντοπίστηκαν στους GHD και GHND ασθενείς φαίνεται να συμβάλουν μερικώς και μεμονωμένα ή συνεργικά στη μεταγραφή του GH1 γονιδίου και συνεπώς στην έκκριση της GH, ενώ η διαφορετική συμμετοχή των SNPs στους GHD και GHND ασθενείς πιθανόν αντανακλά και τη διαφορετική εκδήλωση της νόσου. Η κατανόηση της φαινοτυπικής ποικιλότητας των ασθενών με IGHD και των γενετικών αιτιών μπορεί να βοηθήσει στη κατανόηση των μηχανισμών που ενέχονται στον έλεγχο της αύξησης, αλλά και στη βελτίωση της παρακολούθησης και της θεραπείας των ασθενών.
Growth hormone (GH) plays a pivotal role in a number of physiological processes by promoting postnatal longitudinal body growth, lipid and carbohydrate metabolism, protein biosynthesis and activation of the immune system. GH deficiency (GHD) is diagnosed either by subnormal levels of serum GH during two hGH stimulation tests by pharmacological agents that physiologically stimulate GH secretion (classic form of GHD), or normal serum GH levels, but subnormal 24hr GH profile (Neurosecretory GH deficiency, GHND). Children with GHD and GHND have severe growth retardation and respond to exogenous human GH (hGH) therapy with significant catch-up growth. Short stature associated with isolated GH deficiency (GHD) is both sporadic and idiopathic, but between 5 and 30% have an affected first degree relative consistent with a genetic etiology. Mutations identified on the GH gene (GH1) associate with the manifestation of familial isolated GH deficiency (IGHD). The proximal promoter region of GH1 exerts a highly polymorphic region, with at least 16 single nucleotide polymorphisms (SNPs) identified over a region of 535bp, manifested by a total of 40 haplotypes, some of which affect the GH1 expression. The aim of this study was to identify possible changes in the sequence of the GH1 gene of growth hormone (GH) and its promoter region in patients with familial isolated GH deficiency (IGHD), together with the analysis of the inheritance pattern of such genetic changes. 33 IGHD patients (29 GHD and 4 GHND), their 1st degree relatives (22 families) and 31 controls were investigated. Genomic DNA was extracted from the lymphocytes of the subjects peripheral blood and the GH1 gene was amplified by the Polymerase Chain Reaction (PCR). The samples were sequenced and the changes were identified according to the sequence M28466.1 of the NCBI blast database. The levels of IGF-1 and other hormones of the pituitary were measured in the patients and the highest level of GH during the clonidine and L-Dopa hGH stimulation test were recorded, whereas in the 4 GHND patients a spontaneous 24hr GH profile was conducted. The sequencing results were analyzed for the frequencies of the genotypes of the identified SNPs and for any possible correlations with the clinical or biochemical characteristics of the patients and the controls. Additionally, the possible functional role of the found mutations was assessed using specific programs. The sequencing of GH1 and its promoter revealed 18 SNPs in the whole sample and 3 novel mutations in 3 of the 22 investigated families. Analysis with MatInspector of the 2 heterozygous nucleotide mutations located at the promoter regions -485GC and -400GA, respectively, revealed a binding sequence for the transcription factor E-twenty six-1 (ETS-1). Analysis of the third heterozygous mutation (GA) at the +300 region of intron 1 with ESEfinder3 και ASSP revealed a cryptic splicing position and disruption of the exon splicing enhancement (ESE) region by reducing the binding affinity of serine-arginine proteins (SRs). The frequency and correlation analysis showed that the GHD patients possible exert differential transcriptional activity at the GH1 gene via the SNPs at positions -278, -57 of the promoter, -6 of 5΄ UTR region, +1169 of intron 4, which is in accordance to previous studies, and also the newly reported SNP at -31 region. These SNPs associated also with decreased IGF-1 levels. On the other hand, the SNPs identified in the GHND patients, although similar to the GHD patients, correlated with several growth parameters, irrespective to the IGF-1 levels. The 18 SNPs and 3 mutations identified in the sample seem to contribute partially and individually or in synergy to the transcriptional regulation of GH1 in the GHD and GHND patients. The SNPs contribution is differentially exerted in the GHD patients, when compared to the GHND patients and this possibly reflects the different expression of the disease. The investigation of phenotypic variance in IGHD patients and their genetic predisposition can potentially improve our perception of the underlying mechanisms of growth and offer valuable information for the better therapeutic management of IGHD patients.

碩士
國立中興大學
動物科學系所
97
The study is divided into two parts. In trial 1, the polymorphisms of mtDNA D-loop and 13 protein codens in ostrich were analyzed. Investigating the polymorphisms in GH1 and MB gene, the aim of trial 2 was to combine these mutations with traits. Initially, SSCP analysis was performed to sieve out different patterns from samples. The individuals with different SSCP patterns were selected to detect and characterize gene polymorphisms by comparative sequencing. The results showed that 16 kinds of constitution from 15 SNPs in ostrich D-loop, definded into 3 clades in phylogenetic tree were drawn by MEGA 4. Sixtysix SNPs were found in all protein codons, incoulding 60 synonymous substitutions and 6 synonymous substitutions were presented at osthich mtDNA. The ATPase8 was considered as the highest conserved region with no polymorphism. The highest diversity in 13 proteins coding regions, NDVI, found 15 SNPs. In trial 2, there is no different patterns were found in GH1 gene, but two G to A mutations were found in MB gene. Geneotyping by minisequencing were showed frequencies of MB1 mutation in 85nt, GG, GA and AA, were evaluated as 0.273, 0.576, and 0.151 respectively. The allelic frequencies of G and A, were 0.561 and 0.439 between populations. The geneotype frequencies of MB2 mutation in 85nt, GG, GA and AA, were evaluated as 0.561, 0.515, and 0.1 respectively. The allelic frequencies of G and A, were 0.591 and 0.409 in MB1. The G85A at MB1 have siganificant difference（P＜0.05）on average daily gain（ADG）. We proposed GA genotype was better choice for ostrich breeding.