Enantioselective reactions are one of the ways to produce pure chiral compounds. Understanding the basis of this selectivity makes it possible to guide enzyme design towards more efficient catalysts. One approach to study enzymes involved in chiral chemistry is through the use of computational models that are able to simulate the chemical reaction taking place. The potato epoxide hydrolase is one enzyme that is known to be both highly enantioselective, while still being robust upon mutation of residues to change substrate scope. The enzyme was used to investigate the epoxide hydrolysis mechanism for a number of different substrates, using the EVB approach to the reaction both in solution and in several enzyme variants. In addition to this, work has been performed on new ways of performing simulations of divalent transition metals, as well as development of new simulation software.

Orientador: Luciana Gonzaga de Oliveira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T05:28:22Z (GMT). No. of bitstreams: 1 Rampasio_RaquelRodrigues_M.pdf: 2052024 bytes, checksum: 07f4cd2fcba87af264e6efceab06d527 (MD5) Previous issue date: 2012 / Resumo: Epóxidos enantiopuros e dióis vicinais têm sido utilizados na síntese de inúeras moléculas bioativas. Dessa forma, as epóxido-hidrolases microbianas capazes de hidrolisar enantioseletivamente epóxidos racêmicos emergiram como uma alternativa promissora na obtenção destes compostos. Recentemente, a linhagem P. stipitis CCT 2617 foi selecionada por apresentar atividade hidrolítica frente a epóxidos terminais e teve seu genoma completo publicado. Assim, esta levedura foi selecionada para o trabalho de clonagem e expressão de sua epóxido hidrolase. Neste trabalho, a clonagem do gene SEH1, que codifica para a epóxido hidrolase de P. stipitis, foi efetuada com sucesso em P. pastoris, tanto no vetor pPICZa A, quanto no vetor pPICZ B. A clonagem da proteína com a cauda de histidina deve auxiliar na detecção da expressão. A detecção de uma banda, referente a uma proteína de 46 kDa, no gel de eletroforese foi um indício de que a expressão da enzima SEH (contendo o fator a) ocorreu, porém, não conseguimos reproduzir este resultado posteriormente. Além disso, buscamos melhores alternativas para a detecção da atividade enzimática, como o teste de adrenalina e o ensaio baseado em substrato fluorogênico, que devem ser aperfeiçoados para a utilização com células íntegras. A modelagem computacional da estrutura tridimensional da PSEH resultou em um modelo contendo 40% de hélices a e 12% de folhas b. Determinamos que os resíduos que devem fazer parte do sítio ativo são Tyr319, Asp209, Asp352 e His383 e, tendo em vista que a PSEH deve se apresentar na forma de um homodímero com sítio ativo similar ao das EHs de P. aeruginosa, A. radiobacter e A. niger, nossa hipótese é que esta enzima deve hidrolisar epóxidos pouco volumosos e aromáticos com algum nível de enantiosseletividade / Abstract: Enantiopure epoxides and vicinal diols have been used to prepare a number of bioactive molecules. Thus, the microbial epoxide hydrolases able to enantioselectivity hydrolyze racemic epoxides emerged as a promising alternative in the synthesis of these compounds. Recently, the P. stipitis CCT2617 strain was selected due to the presence of hydrolytic activity against terminal epoxides and had its genome completely described. Therefore, this yeast was selected for cloning and expression of the gene SEH1, which was annoted as epoxide hydrolase. In this work, the cloning of SEH1 gene, codifying for the epoxide hydrolase of P. stipitis, was done with success in P. pastoris, both in pPICZa A and pPICZ B vectors. The cloning of the protein with a histidine tag should help in the detection of expression. The detection of a protein with 46 kDa evidenced that the expression of SEH enzyme (containing the a factor) is occurring, however, this result was not reproducible due to the sample degradation. Furthermore, better alternatives for the detection of enzyme activity were performed, as adrenaline test and fluorogenic assay, which must be optimized for application with whole cells. The 3D structure computational modeling of PSEH resulted in a model that contains 40% of a helices and 12% of b sheets. Our hypothesis is that the residues that make part of the active site are Tyr319, Asp209, Asp352 and His 383. And, considering that the PSEH should be in the homodimeric form with an active site similar to that of the EHs of P. aeruginosa, A. radiobacter and A. niger, this enzyme should hydrolyze small and aromatic epoxides probably with some enantioselectivity / Mestrado / Quimica Organica / Mestre em Química

Orientador: Anita Jocelyne Marsaioli / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-27T18:00:04Z (GMT). No. of bitstreams: 1 Costa_BrunaZucolotoda_D.pdf: 6212345 bytes, checksum: ec8b84a214cbc377e0f9f13d33ddc561 (MD5) Previous issue date: 2011 / Resumo: Os processos biocatalíticos foram abordados nesta tese de doutorado deste a triagem de micro-organismos para a seleção de biocatalisadores adequados até o uso de enzimas isoladas em reações de interesse biotecnológico. O primeiro capítulo apresenta o isolamento, identificação e a triagem enzimática de bactérias heterotróficas isoladas de rejeitos de mineração de cobre. A partir destas amostras foi possível isolar 189 bactérias, as quais apresentaram uma diversificada atuação catalítica. As bactérias isoladas foram identificadas por MALDI-TOF e por sequenciamento do gene RNAr 16S sendo encontrados diversos gêneros como Bacillus, Acinetobacter, Pseudomonas, Delftia, Stenotrophomonas, Hydrogenophaga, Rhodococcus, entre outros. O segundo capítulo apresenta o estudo do potencial catalítico de uma nova epóxido hidrolase de Aspergillus brasiliensis CCT1435, recombinante e expressa em E. coli. Esta EH é ativa em uma ampla faixa de pH e temperatura, apresentando um desempenho ótimo em pH 6,0 e 30 °C. Este trabalho ainda permitiu uma avaliação detalhada da aplicação biocatalítica desta EH na hidrólise do óxido de estireno em meio aquoso e bifásico. Por fim, o terceiro capítulo apresenta resultados preliminares envolvendo um processo quimio-enzimático para a produção de alcalóides pirrolidínicos a partir das respectivas 1,4-dicetonas. Esta metodologia é promissora uma vez que diversas 1,4-dicetonas foram convertidas nas suas respectivas iminas cíclicas, sendo que a subsequente etapa de redução com NaBH3CN promoveu a formação das pirrolidinas de interesse. Portanto, esta tese de doutorado apresenta um estudo amplo e diversificado envolvendo processos biocatalíticos aplicados em uma série de reações de interesse biotecnológico / Abstract: Biocatalytic processes have been addressed in this thesis from the microorganism screening for the selection of suitable biocatalysts to the use of isolated enzymes in biotechnological reactions. The first chapter presents the isolation, identification and enzymatic screening of heterotrophic bacteria isolated from copper mine drainages. From these samples, 189 bacteria were isolated, which showed diverse catalytic activities. The isolated bacteria were identified by MALDI-TOF and 16S rRNA gene sequencing and several genera were found: Bacillus, Acinetobacter, Pseudomonas, Delftia, Stenotrophomonas, Hydrogenophaga, Rhodococcus, among others. The second chapter presents the catalytic potential of a new epoxide hydrolase from Aspergillus brasiliensis CCT1435 (AbEH), recombinant and expressed in E. coli. This EH is active in a wide pH and temperature range, with a great performance at pH 6.0 and 30 °C. This work also presents the AbEH biocatalytic application for the styrene oxide hydrolysis in aqueous and biphasic media. Finally, the third chapter presents preliminary results involving a chemo-enzymatic method for the production of pyrrolidine alkaloids from the corresponding 1,4-diketones. This approach is promising since several 1,4-diketones were converted to their respective cyclic imines, and the subsequent reduction with NaBH3CN promoted the pyrrolidines formation. Therefore, this thesis presents a broad and diverse study of biocatalytic processes applied in a number of interesting biotechnological reactions / Doutorado / Quimica Organica / Doutora em Ciências

Female offspring of Sprague-Dawley rats fed a high-salt diet (HS) during pregnancy show an enhancement of mean arterial pressure (MAP) and heart rate (HR) response to acute stress in adulthood compared to offspring whose mothers were fed a normal-salt diet (NS) [1]. In the present study, we first examined the expression of soluble epoxide hydrolase (SEH) protein in brain tissue. Whole brains were collected and SEH gene (EPHX2) mRNA and SEH protein expression were analyzed using RT-PCR and Western blot, respectively. mRNA levels were relatively decreased in high-salt rats (1.0 ± 0.32 NS vs 0.39 ± 0.07 HS, n=6). However, the relative expression of SEH protein was significantly increased in HS rats (0.97 ± 0.06 NS vs. 1.72 ± 0.32 HS, n=10). SEH is an enzyme that inactivates epoxyeicosatrienoic acids (EETs), which can increase the level of oxygen free radical production and potentially produce an increase in blood pressure. Tempol, a free radical scavenger, was administered ntracerebroventricularly to HS (n=12) and NS (n=11) offspring to determine if the stressinduces cardiovascular hyperresponsiveness could be reversed. We were unable to conclusively show that this was the case. Hence, the expression of SEH protein in the brains of HS offspring was increased, but a role, if any, for this change in explaining the exaggerated response to acute stress remains elusive. Second, the expression of the glucocorticoid receptor (GR) gene was investigated. We focused on the methylation patterns of the exon 17 GR promoter and 17 CpG dinucleotide sites that include the NGFI-A transcription factor binding site. Female rats (HS n=8, NS n=8) were sacrificed and brains were immediately extracted. Tissue from the pituitary, hypothalamus, and hippocampus was removed and DNA was extracted from each of these areas. CT conversion was performed on the DNA samples followed by cloning and sequencing. Methylation patterns between HS and NS in the pituitary, hypothalamus, and hippocampus did not vary. RT-PCR and Western blot were performed to investigate differences in the levels of GR transcription and/or translation. There were no significant differences found. However, the trends found may suggest different levels of GR mRNA and protein between HS and NS female rats. DNA methylation may play a role in the regulation of GR in prenatal high-salt female offspring. Additional studies will be needed to pinpoint the mechanisms responsible for the exaggerated cardiovascular response to acute stress in HS offspring.

<p>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.</p><p><i>Mycobacterium tuberculosis</i> (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 <i>M. tuberculosis </i>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 <i>Rhodococcus erythropolis</i>. However, in contrast to the small limonene-based substrate of the <i>Rhodococcus</i> enzyme, Rv2740 is able to degrade large fatty acid and sterol epoxides, giving suggestions for the physiological substrates of this enzyme.</p><p>The structure of <i>M. tuberculosis</i> Rv0216 exhibits a so-called double hotdog fold. Rv0216 shows similarity to a number of enzymes using thiol esters as substrates, including several <i>R</i>-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 <i>R</i>-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 <i>M. tuberculosis</i> 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>

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.

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

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

L’époxyde hydrolase soluble (sEH) est une enzyme ubiquitaire, bifonctionnelle, codée par le gène EPHX2. La partie hydrolase (sEH-H) est responsable de la dégradation de facteurs endothéliaux vasodilatateurs, les acides époxyeicosatriénoïques (EETs), alors que la partie phosphatase (sEH-P) est impliquée dans le métabolisme des acides lysophosphatidiques (LPAs).L’objectif de ce travail a été de développer des outils méthodologiques permettant d'évaluer le rôle de la sEH dans la physiopathologie des maladies cardiovasculaires.Nous avons développé une méthode de quantification par CLHP-MS² des EETs et de leurs métabolites, les acides dihydroxyeicosatrienoic acids (DHETs). L'application de cette méthode montre que la dysfonction endothéliale des patients atteints d’hypertension artérielle et de diabète de type 2 est associée à une diminution de la libération locale des EETs lors de l'augmentation du débit sanguin, notamment liée à une augmentation d’activité de la sEH-H. L’inhibition pharmacologique de la sEH-H a permis de diminuer l’inflammation et l’atteinte glomérulaire dans un modèle murin d’insulino-résistance. De plus, l’étude des polymorphismes génétiques du gène EPHX2, codant la sEH, a permis de démontrer que la fonction sEH-H joue probablement un rôle important dans le contrôle de la fonction rénale et vasculaire des patients transplantés rénaux. Enfin, les résultats expérimentaux obtenus dans un modèle d’inactivation génétique de la sEH-P et l'étude des polymorphismes génétiques d'EPHX2 chez les patients insuffisants cardiaques suggèrent un rôle important de cette partie dans la régulation du métabolisme des lipides ainsi que dans le contrôle de l’homéostasie cardiovasculaire.Ainsi, les résultats obtenus au cours de ce travail soutiennent l’intérêt de développer des inhibiteurs pharmacologiques de la sEH-H pour traiter les maladies cardiovasculaires, rénales et métaboliques chez l’homme et suggèrent que la modulation de la sEH-P pourrait également constituer une nouvelle cible d'intérêt dans la prise en charge de ces pathologies. / Soluble epoxide hydrolase (sEH) is an ubiquitous bifunctional enzyme that is encoded by the EPHX2 gene. The hydrolase activity (sEH-H) is responsible for the conversion of the endothelial vasodilator epoxyeicosatrienoic acids whereas the phosphatase activity (sEH-P) is involved in the metabolism of lysophosphatidic acids (LPAs).The aim of this work was to develop chromatographic methods and molecular biology techniques to evaluate sEH activities in cardiovascular diseases.We developed a LC-MS/MS method to quantify EETs and their metabolites, the dihydroxyeicosatrienoic acids (DHETs). Using this method, we showed that the endothelial dysfunction of hypertensive and type 2 diabetic patients is associated with a decrease in the local production of EETs during flow increase notably due to increased sEH-H activity. In a murine model of insulin resistance, pharmacological inhibition of sEH-H improved renal function by decreasing inflammation, oxidative stress and glomerular lesions. Moreover, genetic investigations of EPHX2 revealed that sEH-H may play a substantial role in the control of renal and vascular function in kidney recipients. Finally, experimental results obtained in knock-in sEH-P deficient rats and genetics findings in patients with heart failure strongly suggest that sEH-P is involved in lipid metabolism and cardiovascular homeostasis.Taken together, these results strengthen the interest of developing pharmacological inhibitors of sEH-H to be tested in patients with cardiovascular, renal or metabolic diseases and suggest that the modulation of sEH-P represents a new therapeutic target to treat these pathologies.

A metagenômica trouxe novas perspectivas ao estudo de comunidades microbianas no ambiente, permitindo explorar tanto a diversidade taxonômica de microrganismos ainda não-cultivados, como o acesso direto a genes e vias metabólicas. Neste trabalho, foram construídas bibliotecas metagenômicas a partir de amostras de sedimentos de mangue da Baía de Guanabara (RJ), impactadas com hidrocarbonetos de petróleo e metais pesados. Proteobacteria (33,3%), bactérias afiliadas a redutoras-de-sulfato (29,7%) e Firmicutes (20%) representaram os grupos principais nas amostras ambientais, baseado em análises filogenéticas de rDNA 16S, ao passo que isolamentos seletivos utilizando diesel e naftaleno permitiram a recuperação preferencial de delta-Proteobacteria e actinomicetos. Bibliotecas metagenômicas dos sedimentos enriquecidos com óleo diesel, com insertos entre 25 e 35 Kb clonados em fosmídeos, foram triadas para detecção de genes catabólicos de monoxigenases (alkB1) e expressão de epóxido-hidrolases, esterases, lipases e monoxigenases em ensaios de alto desempenho (HTS, high throughput screening). Clones reativos a alkB1 foram detectados, porém não foram funcionais nas condições de HTS testadas. Nas bibliotecas de fosmídeos triadas, vários clones apresentaram atividade enzimática, sendo que dois apresentaram atividade de lipase-esterase com alta seletividade, elevada taxa de conversão de substratos e excesso enantiomérico (ee >99%). Os resultados de HTS comprovaram a eficiência do uso da clonagem direta de DNA ambiental na expressão de vias metabólicas de interesse com potencial de aplicação biotecnológica. / Metagenomics brought a new perspective to the study of microbial communities in the environment, enabling access to the taxonomic diversity of uncultured microorganisms, as well as direct access to genes and metabolic pathways. In the current study, metagenomic libraries were constructed from mangrove sediment samples of the Guanabara Bay (RJ, Brazil), impacted with oil hydrocarbons and heavy metals. Proteobacteria (33.3%), sulfate-reducing affiliated bacteria (29.7%) and Firmicutes (20%) represented the main groups in the environmental samples based upon 16S rDNA phylogenetic analysis, whereas selective isolation using diesel and naphtalene yielded delta-Proteobacteria and actinomycetes. Metagenomic libraries of diesel-enriched sediment samples, with 25 to 35 Kb fosmid inserts, were screened for detecting monooxigenase genes (alkB1) and expression of epoxide hydrolases, esterases, lipases and monooxigenases in high throughput screening (HTS) assays. Clones reactive to the alkB1 probe were detected, but were not functional under the HTS conditions used. Several functional clones were detected in the clone library, and two showed lipase-esterase activity with high rates of substrate conversion and enantiomeric ratio (ee >99%). The results obtained on HTS showed the efficiency of the direct cloning of environmental DNA for the expression of metabolic pathways with potential biotechnological application.