This thesis describes the structural studies of four different proteins from two organisms. Xyloglucan endotransglycosylases, XETs, are involved in plant cell wall expansion and remodeling by splitting and reconnecting xyloglucan-cellulose crosslinks. The first crystal structure of a XET enzyme has been determined to 1.8 Å. The structure provides insights into how XETs are able to bind a heavily branched xyloglucan sugar, as well as hints about the XET-transglycosylation mechanism. Mycobacterium tuberculosis (Mtb) is the cause of enormous human mortality each year. Despite the sequencing of the complete Mtb-genome, the biological function of a large fraction of the M. tuberculosis proteins is still unknown. We here report the crystal structures of three such proteins, Rv2740, Rv0216 and Rv0130. Rv2740 forms a Cystatin α+b fold with a deep active site pocket similar to a limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis. However, in contrast to the small limonene-based substrate of the Rhodococcus enzyme, Rv2740 is able to degrade large fatty acid and sterol epoxides, giving suggestions for the physiological substrates of this enzyme. The structure of M. tuberculosis Rv0216 exhibits a so-called double hotdog fold. Rv0216 shows similarity to a number of enzymes using thiol esters as substrates, including several R-enoyl hydratases and β-hydroxyacyl dehydratases. However, only parts of the hydratase / dehydratase catalytic site are conserved in Rv0216. Rv0130 in contrast, contains a highly conserved R-hydratase motif, housed in a dimer of two single hotdog folded molecules. This active site is situated in a long tunnel, formed by a sharp kink in the Rv0130 central helix. A number of previously predicted single / double hotdog folded proteins from M. tuberculosis seem to feature a similar substrate-binding tunnel, indicating that Rv0130 as well as some of these proteins, might act on long fatty enoyl chains.

<p>Gram-negative bacteria possess an asymmetric outer membrane in which the inner leaflet is composed primarily of phospholipids while the outer leaflet contains both phospholipids and lipopolysaccharide (LPS). LPS forms a structural barrier that protects Gram-negative bacteria from antibiotics and other environmental stressors. The lipid A anchor of LPS is a glucosamine-based saccharolipid that is further modified with core and O-antigen sugars. In addition to serving a structural role as the hydrophobic anchor of LPS, lipid A is recognized by the innate immune system in animal cells and macrophages. The enzymes of Lipid A biosynthesis are conserved in Gram-negative bacteria; in most species, a single copy of each bio-synthetic gene is present. The exception is lpxH, which is an essential gene encoding a membrane-associated UDP-2,3-diacylglucosamine hydrolase, which catalyzed the attack of water upon the alpha-phosphate of its substrate and the leaving of UMP, resulting in the formation of lipid X. Many Gram-negatives lack an lpxH orthologue, yet these species must possess an activity analogous to that of LpxH. We used bioinformatics approaches to identify a candidate gene, designated lpxI, encoding this activity in the model organism Caulobacter crescentus. We then demonstrated that lpxI can rescue Escherichia coli deficient in lpxH. Moreover, we have shown that LpxI possesses robust and specific UDP-2,3-diacylglucosamine hydrolase activity in vitro. We have developed high-yield purification schema for Caulobacter crescentus LpxI (CcLpxI) heterologously expressed in E. coli. We crystallized CcLpxI and determined its 2.6 Å x-ray crystal structure in complex with lipid X. CcLpxI, which has no known homologues, consists of two novel domains connected by a linker. Moreover, we have identified a point mutant of CcLpxI which co-purifies with its substrate in a 0.85:1 molar ratio. We have solved the x-ray crystal structure of this mutant to 3.0 Å; preliminary comparison with the product-complexed model reveals striking differences. The findings described herein set the stage for further mechanistic and structural characterization of this novel enzyme.</p> <p></p> <p>In this work, we also isolate and characterize LpxB, an essential lipid A biosynthetic gene which is conserved among all Gram-negative bacteria. We purify E. coli and Hemophilus influeznea LpxB to near-homogeneity on a 10 mg scale, and we determine that E. coli LpxB activity is dependent upon the bulk surface concentration of its substrates in a mixed micellar assay system, suggesting that catalysis occurs at the lipid interface. E. coli LpxB partitions with membranes, but this interaction is partially abolished in high-salt conditions, suggesting that a significant component of LpxB's membrane association is ionic in nature. E. coli LpxB (Mr ~ 43 kDa) is a peripheral membrane protein, and we demonstrate that it co-purifies with phospholipids. We estimate, by autoradiography and mass-spectrometry, molar ratios of phospholipids to purified enzyme of 1.6-3.5:1. Transmission electron microscopy reveals the accumulation of intra-cellular membranes when LpxB is massively over-expressed. Alanine-scanning mutagenesis of selected conserved LpxB residues identified two, D89A and R201A, for which no residual catalytic activity is detected. Our data support the hypothesis that LpxB performs catalysis at the cytoplasmic surface of the inner membrane, and provide a rational starting-point for structural studies. This work contributes to knowledge of the small but growing set of structurally and mechanistically characterized enzymes which perform chemistry upon lipids.</p> / Dissertation

<p>The aim of this work was to develop a production process for the enzyme xyloglucan <i>endo</i>-transglycosylase from <i>Populus tremula x tremuloides</i> (<i>Ptt</i>XET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale.</p><p>This work also shows how the wildtype <i>Ptt</i>XET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction.</p><p>A heterologous production system for <i>Ptt</i>XET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of <i>Ptt</i>XET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB).</p><p>For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF).</p>

La voie endo-lysosomale dirige les récepteurs membranaires vers le processus de dégradation lysosomale. En bref, les récepteurs sont marqués par l'ubiquitine, envoyés vers les endosomes précoces puis, pris en charge pas le système ESCRT (Endosomal Sorting Complexes Required for Transport) et intégrés dans des vésicules intraluminales. Ce système est composé des complexes ESCRT-0, I, II, II et VPS4. Certaines protéines ESCRT sont aussi recrutées lors de processus topologiquement similaires comme la cytokinèse ou le bourgeonnment viral de certains virus enveloppés. AMSH (Associated Molecule of the SH3 domain of STAM) est une ubiquitine-hydrolase associée au système ESCRT qui hydrolyse les chaînes d'ubiquitine liées par leur lysine K63. Elle interagit directement avec ESCRT-0 via la sous-unité STAM et avec les membres CHMP1A, 1B et 3 d'ESCRT-III. Bien qu'AMSH pourait recruter ces protéines ESCRT ou être elle-même recrutée par celles-ci, le mécanisme d'activation de son activité d'hydrolase est encore méconnu. Afin de mieux comprendre les bases structurales de l'activation d'AMSH, j'ai essayé danalyser des formes recombinantes de cette protéine par cristallographie aux rayons X et par diffusion des rayons X aux petits angles (SAXS) ce qui m'a permis d'obtenir deux modèles à basse résolution. De plus, j'ai caractérisé par SPR (Surface Plasmon Resonance) les interactions entre AMSH et CHMP1A, 1B et 3 et déterminé les résidus clefs du dernier complexe. Cela a montré que les surfaces d'interaction employées par le domaine MIT d'AMSH ne sont pas les mêmes pour CHMP3 et CHMP1A/1B. J'ai aussi découvert que l'activité enzymatique d'AMSH seule est très faible ce qui impliquerait une auto-inhibition en solution. L'hydrolyse des chaînes d'ubiquitine liées par leur lysine K63 pourrait alors être activée par une construction de STAM comprenant le domaine SH3 ainsi que les domaines liant l'ubiquitine VHS et/ou UIM.

Thesis (Ph.D. (Pharmaceutical Chemistry)--North-West University, Potchefstroom Campus, 2009.

Thesis (Ph.D. (Pharmaceutical Chemistry)--North-West University, Potchefstroom Campus, 2009.

Peu d'enzymes de synthèse de composés odorants sont connues chez le genre Rosa. Ce travail de thèse a permis l'identification de quelques-unes de ces protéines grâce à la technologie des puces à ADN, à l'analyse de l'expression des gènes par RT-PCR quantitative en temps réel (qPCR) et à l'analyse des parfums par chromatographie en phase gazeuse (CPG). Une puce confrontant les ADNc d'une rose parfumée à ceux d'une rose non parfumée a permis de corréler l'expression d'un gène, codant pour une Nudix hydrolase, très fortement exprimé dans la rose parfumée, avec la présence des monoterpènes dans le parfum de nombreux cultivars de rosiers. La caractérisation d'un rosier dont l'expression de ce gène est fortement réduite par ARN interférants, a permis de confirmer le rôle de celui-ci dans la synthèse des monoterpènes. La phénylacétaldéhyde synthase (PAAS) est une autre enzyme participant à la synthèse du parfum. Trois allèles de cette protéine ont précédemment été mis en évidence. Les résultats de qPCR et de CPG dans une population hybride ont permis de montrer que l'allèle a1 est le seul à pouvoir induire la synthèse et l'émission de 2-phényléthanol. Les activités respectives des différentes isoformes ont été testées in vitro chez la levure et in planta dans des feuilles de tabac et des cals de rosier : ces expériences montrent que les trois isoformes ont des activités comparables. L'absence de synthèse de 2-phényméthanol chez les plantes présentant les isoformes a2 et a3 réside donc dans la très faible expression de leurs allèles, induisant probablement une faible concentration de l'isoforme dans les cellules

As mudanças climáticas estão causando prejuízos em vários setores da economia mundial. Na reunião da COP21, que teve como foco estas mudanças climáticas, participantes do mundo todo decidiram tomar atitudes urgentes para tentar conter aumento da temperatura média global. Dentro deste cenário, a produção e o consumo de energia têm uma importância central, onde fontes de energia renováveis vêm sendo preferidas às fontes de energias fósseis. O Brasil tem uma participação importante na geração de energia renovável mundial aportando um 40% do total de sua matriz energética. A degradação dos componentes da parede celular vegetal tem um vasto potencial na geração de biocombustíveis e outros compostos verdes a partir da celulose, hemicelulose e lignina. Para isto estudos das enzimas capazes de degradas estes componentes vem sendo realizados, com ênfase nas enzimas hidrolases de glicosídeos. Dentre as hidrolases, encontram-se as arabinanases, enzimas capazes de hidrolisar o arabinano, componente polissacídeo da hemicelulose, em L-arabinose. Neste trabalho, estudos envolvendo duas arabinanases de Bacillus licheniformis foram realizados, iniciando na etapa de clonagem dos genes. Os produtos foram transformados em Escherichia coli e expressos e purificados. A avaliação da estabilidade térmica indicou uma afinidade das enzimas por metais divalentes. Tentativas de cristalização resultaram na formação de um cristal, que possibilitou a determinação da estrutura uma das arabinanases. Através de ensaios bioquímicos, foi determinada a especificidade por substrato, temperatura e pH ótimos e a atividade frente a metais. Foi observado que as enzimas são seletivas para arabinano não ramificado, tem temperatura ótima em 45 e 40 graus, para BlAbn-1 e BlAbn-2, respectivamente, e pH ótimo em 8 e 7. Por último, foram realizados ensaios complementares de sinergismo e atividade oxidativa. Embora os ensaios de atividade oxidativa tenham sido inconclusivos, os ensaios de sinergismo mostraram que a enzima BlAbn-1 é capaz de aumentar em 30% a atividade do coquetel enzimático Accellerase 1500 sobre biomassa pré-tratada e sobre celulose pura. Este efeito é ainda maior na presença de sulfato de níquel. / Climate change is causing losses in different sectors of the world economy. At the meeting of COP21, focused on climate changes, participants from around the world decided to take urgent actions to try to halt the increase in global average temperature. Within this scenario, the production and consumption of energy are of central importance, where renewable energy sources have been preferred to fossil fuels. Brazil has an important role in the global renewable energy generation by contributing 40% of its total energy mix. The degradation of the components of plant cell wall has a vast potential in the generation of biofuels and other green chemical from cellulose, hemicellulose and lignin. Thus, studies of enzymes that degrade these components have been carried out, with emphasis on glycoside hydrolases. Among the hydrolases are the arabinanases, enzymes capable of hydrolyzing arabinan, a polysaccharide component of hemicellulose, in L-arabinose. In this work, studies involving two arabinanases from Bacillus licheniformis were carried out, starting in gene cloning step. The products were transformed into Escherichia coli, expressed and purified. The evaluation of the thermal stability of the enzymes showed an affinity for divalent metals. Crystallization attempts resulted in the formation of a single crystal, which made it possible to determine the crystal structure of one arabinanase. Through biochemical assays, it was determined the substrate specificity, optimum temperature and pH and activity against metals. It was observed that the enzymes are selective for non-branched arabinan, have optimum temperature at 45 and 40 degrees, to BlAbn-1 and BlAbn-2, respectively, and optimum pH of 8 and 7. Finally, additional tests were performed to evaluate the possible synergism and oxidative activity. Although the oxidative activity assays were inconclusive, the synergism tests showed that BlAbn-1 is able to increase by 30% the activity of the enzymatic cocktail Accellerase 1500 on pre-treated biomass and on pure cellulose. This effect is even greater in the presence of nickel sulfate.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-05-18T19:09:17Z No. of bitstreams: 2 Tese - Marcela Suriani Ribeiro - 2017.pdf: 1795199 bytes, checksum: fb63e9f789cfefb42f31cba029a29f4f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-05-19T10:43:53Z (GMT) No. of bitstreams: 2 Tese - Marcela Suriani Ribeiro - 2017.pdf: 1795199 bytes, checksum: fb63e9f789cfefb42f31cba029a29f4f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-05-19T10:43:53Z (GMT). No. of bitstreams: 2 Tese - Marcela Suriani Ribeiro - 2017.pdf: 1795199 bytes, checksum: fb63e9f789cfefb42f31cba029a29f4f (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-04-28 / 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.

N-Acylethanolamines (NAEs) are bioactive acylamides that are present in a wide range of organisms. Because NAE levels in seeds decline during imbibition similar to ABA, a physiological role was predicted for these metabolites in Arabidopsis thaliana seed germination and seedling development. There is also a corresponding increase of AtFAAH (fatty acid amide hydrolase), transcript levels and activity, which metabolizes NAE to ethanolamine and free fatty acids. Based on whole genome microarray studies it was determined that a number of up-regulated genes that were responsive to NAE were also ABA responsive. NAE induced gene expression in these ABA responsive genes without elevating endogenous levels of ABA. It was also determined that many of these NAE/ABA responsive genes were associated with an ABA induced secondary growth arrest, including ABI3. ABI3 is a transcription factor that regulates the transition from embryo to seedling growth, the analysis of transcript levels in NAE treated seedlings revealed a dose dependent, inverse relationship between ABI3 transcript levels and growth, high ABI3 transcript levels were associated with growth inhibition. Similar to ABA, NAE negatively regulated seedling growth within a narrow window of early seedling establishment. When seedlings are exposed to NAE or ABA within the window of sensitivity, the induction of genes normally associated with the ungerminated desiccation tolerant state resumed. The NAE tolerant FAAH overexpressor and the NAE sensitive FAAH knockout both had a NAE/ABA sensitive window similar to the wild type A. thaliana. The abi3-1 ABA insensitive mutant does not undergo growth arrest upon exposure to ABA, but NAE did induce growth arrest when treated within the sensitivity window. This evidence showed that although NAE functions within an ABA dependent pathway, it also functions in an ABA independent signaling pathway. The FAAH overexpressor is tolerant to NAE through its ability to quickly metabolize NAE from the growth media, yet it is hypersensitive to ABA. The FAAH overexpressor also displayed hypersensitivity to GA, which improved its delayed germination in non-stratified seed, while the FAAH knock out showed GA insensitivity. Overall, these results showed that NAE functions as a negative regulator of germinating seed and seedling growth in ABA dependent and independent signaling pathways, and that altered NAE metabolism may interfere with ABA/GA perception in germinating seed.