Only a few hyperthermophilic archaea are found to have a complete nonoxidative stage of pentose phosphate pathway (NOPPP), which indicates that most archaeal hyperthermophiles are partially missing pentose metabolizing enzymes. However, very limited research has been done in this interesting field. Although few hyperthermophilic enzymes in PPP have been studied in detail, the enzymatic activities reported previously were underestimated because assays were performed at temperatures much lower than 80??C. The commercially available auxiliary enzymes used in those assays were not thermostable, which limited assay temperature as well. The substrates used in those assays are extremely expensive, which is another factor limiting the study in this area. In this project, an inexpensive and accurate assay system was developed to overcome these limitations. Genes encoding several auxiliary enzymes for PPP enzyme assays were cloned from selected hyperthermophiles and overexpressed in E.coli Rossetta2 TM (DE3). These enzymes were glyceraldehydes-3-phosphate dehydrogenase from Thermotoga maritima (TmGAPDH), triosephosphate isomerase from Pyrococcus furiosus (PfTIM), glycerophosphate dehydrogenase from Pyrococcus furiosus (PfG3PDH) and Aeropyrum pernixK1 (ApG1PDH), transketolase from T. maritima (TmTK), xylose isomerase from T. maritima (TmXI) and Thermotoga neapolitana (TpXI), and xylulose kinase from T. maritima (TmXuK). Their activities were determined under anaerobic conditions at 80?? iv C in both cell free extracts and for purified enzymes. The assay system contained the following parts: A) TmGAPDH, TmXI or TpXI, TmXuK (TmTK), PfTIM, and PfG3PDH or ApG1PDH as auxiliary enzymes for TK (XuK) assay; B) TmGAPDH, PfTIM, and PfG3PDH or ApG1PDH for transaldolase (TAL) assay. D-xylose, instead of the traditional assay substrate xylulose-5-phosphate (xylulose), was used as the substrate in this assay system for the determination of TK (XuK) activity. The activities of XuK, TK, and TAL were also determined in several hyperthermophilic bacteria and archaea. All enzymes served as paradigms to prove the feasibility of using the new assay system for other hyperthermophilic PPP enzymes. The species of hyperthermophiles used in this study were T. maritima, P. furiosus, Thermococcus guaymasensis, T. neapolitana, Thermotoga hypogea and T. petrophila. Two different methods were tested for the TAL assay (part B), with either TmGAPDH or PfTIM plus TmG3PDH as the auxiliary enzymes. Similar characteristics were obtained using both methods. The existence of TAL in all selected hyperthermophiles might indicate that the existence of the PPP is functioning among them. The XuK assays in selected hyperthermophiles were successfully conducted using the new assay system (part A). T. petrophila showed the highest XuK activity (0.3 U/mg), indicating the feasibility of the assay system???s application in the determination of hyperthermophilic PPP enzymes at high temperatures (80??C). TmTK activity may also be determined using the new assay system if the auxiliary enzyme XuK activity would be higher. Therefore, the new assay system developed in this project demonstrates dual functions for both XuK and TK assays in hyperthermophiles.

Submitted by ANA KARLA MIRANDA PRADO null (karlinhamprado@hotmail.com) on 2017-04-24T20:41:26Z No. of bitstreams: 1 DissertaçãoAnaCorrecao final.pdf: 2950511 bytes, checksum: 267c66c96725c6306041b2f73554ba8d (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-04-26T13:59:46Z (GMT) No. of bitstreams: 1 prado_ak_me_sjrp.pdf: 2950511 bytes, checksum: 267c66c96725c6306041b2f73554ba8d (MD5) / Made available in DSpace on 2017-04-26T13:59:46Z (GMT). No. of bitstreams: 1 prado_ak_me_sjrp.pdf: 2950511 bytes, checksum: 267c66c96725c6306041b2f73554ba8d (MD5) Previous issue date: 2017-03-31 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Corynebacterium pseudotuberculosis é uma bactéria gram-positiva causadora da Linfadenite Caseosa (LC) em caprinos e ovinos. A LC é uma doença infectocontagiosa crônica que prejudica a produção de carne, leite e lã em vários países, incluindo o Brasil. Uma vez que o tratamento da doença é muitas vezes inviável e ineficaz, a eliminação dos animais infectados no rebanho tem sido uma das principais medidas de contenção da enfermidade. Vários grupos de pesquisa vem se dedicando ao estudo de C. pseudotuberculosis visando a identificação de fatores moleculares envolvidos na virulência e patogenicidade durante a infecção. Embora alguns destes componentes já tenham sido descritos, como a fosfolipase D, novos estudos são necessários para que seja possível compreender as diversas interações regulatórias que são intrínsecas a esse microrganismo, assim muitos grupos de pesquisas produzem de forma recombinante a enzima transaldolase de C. pseudotuberculosis, envolvida na glicólise e no metabolismo das pentoses-fosfato a fim de buscar inibidores que possam controlar sua ação enzimática. Nesse contexto, os flavonoides são compostos polifenólicos encontrados nas plantas e, em alguns casos, atuam como inibidores de infecções por microrganismos. Assim, a motivação deste trabalho consistiu em identificar e quantificar uma possível interação do flavonoide morina com a enzima transaldolase, a fim de bloquear a atividade de replicação e infecção de LC. Deste modo os objetivos desse estudo foram realizar a expressão, purificação, a caracterização da estrutura secundária e estabilidade térmica por dicroísmo circular e verificação de interação entre transaldolase e morina por espectroscopia de fluorescência da proteína transaldolase. Os resultados da expressão mostram que a proteína transaldolase com 40kDa foi purificada em cromatografia de afinidade seguida de cromatografia de exclusão molecular. A sua estrutura secundária apresentou 74% de alfa hélice, 0% de folha beta, 7,9% de alças e 18,1 % de estruturas aleatórias. A análise da desnaturação térmica mostrou que a temperatura de melting foi de 48°C, indicando que a proteína é estável. A interação entre Morina e Transaldolase apresentou mecanismo de supressão estáticodinâmico, com uma constante de associação moderada e um sítio de interação. A análise termodinâmica mostrou que o processo de interação é espontâneo ∆G<0, endotérmico ∆H>0 e entrópico ∆S<0. Assim, sabendo-se que a proteína Transaldolase é a proteína chave no processo das infecções por Corynebacterium pseudotuberculosis e considerando as propriedades antibacterianas e antiproliferativas do flavonoide morina, sugere-se que este composto possa ser investigado para os seus usos específicos. Sugere-se que a interação da transaldolase com a Morina possa exercer um papel de carreador ou seja, uma forma pela qual a proteína leva a molécula para o local que ela atua, e, assim, a Morina consiga realizar sua função antiproliferativa e bloquear as infecções. / Corynebacterium pseudotuberculosis is a gram-positive bacterium which causes Caseous Lymphadenitis (CL) in small ruminants. CL is a chronic infectious disease which impairs meat, whool and milk production in many countries including Brazil. Once the treatment for CL is not efficient, removing affected animals from herds represents one of the major strategies to prevent the disease from spreading. Many research groups have been looking for molecular components in C. pseudotuberculosis that are involved in virulence and infection, among which phospholipase D stands out as the major one described so far. However, new studies are necessary for the understanding of the microorganism’s biology, including its intrinsic regulation mechanisms. The object of study of this work was the enzyme transaldolase involved in glycolysis and in the metabolism of pentoses-phosphate, necessary for the formation of nucleic acids that the bacterium uses to replicate in order to find inhibitors that can control its enzymatic action. In this context, flavonoids are polyphenolic compounds found in plants and, in some cases, act as inhibitors of infections by microorganisms. Thus, the purpose of this work is to identify and quantify a possible interaction of the flavonoid morin with the enzyme transaldolase, in order to block LC´s replication activity and infection. The objectives of this study included expression and purification of C. pseudotuberculosis´s transaldolase protein, the characterization of the secondary structure and thermal stability by circular dichroism and the study of the interaction between transaldolase and morin by fluorescence spectroscopy. The transaldolase protein, with approximately 40kDa, was purified on affinity chromatography followed by molecular exclusion chromatography. Its secondary structure had 74% of alpha helix, 0% of beta sheet, 7.9% of loops (turn) and 18.1% of random structures. The thermal denaturation analysis showed that the melting temperature is 48 °C, indicating that the protein is stable. The interaction between Morina and Transaldolase presented a static-dynamic suppression mechanism, with a moderate association constant and one interaction site. The thermodynamic analysis showed that the interaction process is spontaneous ΔG <0, endothermic ΔH> 0 and entropic ΔS <0. Thus, with the knowledge that Transaldolase protein is the key protein in the process of Corynebacterium pseudotuberculosis infections and considering the antibacterial and antiproliferative properties of the flavonoid morine, it is suggested that this compound can be investigated for its specific uses. It is suggested that the interaction of transaldolase with Morina may play a role of carrier, that is, a way in which the protein takes the molecule to the place it acts, and thus Morina can perform its antiproliferative function and block the Infections.

No description available.

Enzymatic fluorination of natural products is extremely rare. Of the 4000 halogenated natural products identified, only 13 possess a fluorine atom. The C-F bond forming enzyme from the soil bacterium, Streptomyces cattleya, remains the only native enzyme to be identified that is capable of such biochemistry. It generates 5’-fluoro-5-deoxyadenosine (5‘-FDA) from S-adenosyl-L-methionine (SAM) and F-. The “fluorinase” is the first committed step toward the biosynthesis of the two fluorometabolites, 4-fluorothreonine and fluoroacetate, via the common intermediate, fluoroacetaldehyde (FAld). The enzymatic steps responsible for the conversion of 5’-FDA to the fluorometabolites remained to be fully characterised when this project began. Previously, a purine nucleoside phosphorylase was identified that was capable of generating 5-fluorodeoxyribose-1-phosphate (5-FDRP) from 5’-FDA. 5-FDRP is subsequently isomerised to 5-fluorodeoxyribulose-1-phosphate (5-FDRulP) by an aldose-ketose isomerase enzyme. Chapter 2 describes the identification of the isomerase gene from the genomic DNA of S. cattleya and the corresponding protein product was capable of generating 5-FDRulP from 5-FDRP. The next intermediate, FAld, is generated from 5-FDRulP by a fuculose aldolase. Attempts to identify the aldolase gene from S. cattleya were unsuccessful, however a putative fuculose aldolase from Streptomyces coelicolor was isolated that could generate FAld from 5-FDRulP, which is described in Chapter 3. Following the identification and over expression of a PLP-dependant transaldolase, which generates 4-fluorothreonine (4-FT) from FAld and L-threonine in S. cattleya, Chapter 4 details the successful in vitro reconstitution of fluorometabolite biosynthesis using five over- expressed enzymes. In Chapter 5, attempts to develop a novel assay for fluorinase activity was explored. The colorimetric detection of L-methionine produced by the fluorinase in a coupled L-amino acid oxidase and horseradish peroxidase assay, leading to the oxidation of a dye substance. This was carried out with interest in developing a high-throughput assay for fluorinase mutants, generated by random mutagenesis, in order to identify those with increased activity. In the event, it proved unsuccessful.

Orientador: Fátima Pereira de Souza / Coorientador: Marcelo Andrés Fossey / Banca: Tatiana Elias Colombo / Banca: José Ramon Beltran Abrego / Resumo: Corynebacterium pseudotuberculosis é uma bactéria gram-positiva causadora da Linfadenite Caseosa (LC) em caprinos e ovinos. A LC é uma doença infectocontagiosa crônica que prejudica a produção de carne, leite e lã em vários países, incluindo o Brasil. Uma vez que o tratamento da doença é muitas vezes inviável e ineficaz, a eliminação dos animais infectados no rebanho tem sido uma das principais medidas de contenção da enfermidade. Vários grupos de pesquisa vem se dedicando ao estudo de C. pseudotuberculosis visando a identificação de fatores moleculares envolvidos na virulência e patogenicidade durante a infecção. Embora alguns destes componentes já tenham sido descritos, como a fosfolipase D, novos estudos são necessários para que seja possível compreender as diversas interações regulatórias que são intrínsecas a esse microrganismo, assim muitos grupos de pesquisas produzem de forma recombinante a enzima transaldolase de C. pseudotuberculosis, envolvida na glicólise e no metabolismo das pentoses-fosfato a fim de buscar inibidores que possam controlar sua ação enzimática. Nesse contexto, os flavonoides são compostos polifenólicos encontrados nas plantas e, em alguns casos, atuam como inibidores de infecções por microrganismos. Assim, a motivação deste trabalho consistiu em identificar e quantificar uma possível interação do flavonoide morina com a enzima transaldolase, a fim de bloquear a atividade de replicação e infecção de LC. Deste modo os objetivos desse estudo foram... / Abstract: Corynebacterium pseudotuberculosis is a gram-positive bacterium which causes Caseous Lymphadenitis (CL) in small ruminants. CL is a chronic infectious disease which impairs meat, whool and milk production in many countries including Brazil. Once the treatment for CL is not efficient, removing affected animals from herds represents one of the major strategies to prevent the disease from spreading. Many research groups have been looking for molecular components in C. pseudotuberculosis that are involved in virulence and infection, among which phospholipase D stands out as the major one described so far. However, new studies are necessary for the understanding of the microorganism's biology, including its intrinsic regulation mechanisms. The object of study of this work was the enzyme transaldolase involved in glycolysis and in the metabolism of pentoses-phosphate, necessary for the formation of nucleic acids that the bacterium uses to replicate in order to find inhibitors that can control its enzymatic action. In this context, flavonoids are polyphenolic compounds found in plants and, in some cases, act as inhibitors of infections by microorganisms. Thus, the purpose of this work is to identify and quantify a possible interaction of the flavonoid morin with the enzyme transaldolase, in order to block LC's replication activity and infection. The objectives of this study included expression and purification of C. pseudotuberculosis's transaldolase protein, the characterization of the secondary structure and thermal stability by circular dichroism and the study of the interaction between transaldolase and morin by fluorescence spectroscopy. The transaldolase protein, with approximately 40kDa, was purified on affinity chromatography followed by molecular exclusion chromatography. Its secondary structure had 74% of alpha helix, 0% of beta sheet, 7.9% of loops (turn) and 18.1% of ... / Mestre

Transaldolase-Mangel (TALDO) ist ein extrem seltener, angeborener Stoffwechseldefekt, von dem weltweit nur 34 Fälle bekannt sind. Der Defekt geht auf den Verlust des Enzyms Transaldolase 1 aus dem nicht-oxidativen Pentosephosphat-Weg (nicht-oxPPW) zurück und äußert sich in einem weiten Spektrum klinischer Symptome. Die schwerwiegendsten Folgen sind Leber- und Nierenmangelfunktionen, die zum sehr frühen Tod führen können. Desweiteren leiden 15 % der Patienten an wiederkehrenden Infektionen. Neutrophile Granulozyten (Neutrophile) sind die häufigsten weißen Blutkörperchen im Menschen und essentiell für die angeborene Immunantwort gegen Infektionserreger. Ich habe hier funktionale Aspekte von TALDO-Neutrophilen untersucht. Der oxidative Pentosephosphat-Weg (oxPPW) stellt das Reduktionsäquivalent NADPH bereit, welches indirekt für die Entstehung von reactive oxygen species (ROS)-abhängigen Neutrophil Extracellular Traps (NETs) verantwortlich ist. Der Beitrag des nicht-oxPPW zur ROS-abhängigen NET-Bildung ist bislang nicht bekannt. In dieser Arbeit konnte ich für Neutrophile aus drei TALDO-Patienten eine jeweils komplett abwesende Entstehung ROS-abhängiger NETs und einen deutlich verringerten oxidativen Burst nach PMA-Stimulation zeigen. Um diese Beobachtungen in einem unabhängigen Modelsystem zu bestätigen, habe ich mit Hilfe des CRISPR-Cas9-Systems, ‚knock-out‘ Mutanten von Transaldolase 1 und dessen Partnerenzym Transketolase in der Neutrophil-ähnlichen Zelllinie PLB-985 hergestellt. Die dergestalt genetisch manipulierten Zellen waren nicht mehr zu PMA-induziertem Zelltod in der Lage. Dies ist somit der erste genetische Beweis für die Abhängigkeit des oxidativen Burst und der Bildung von NETs vom nicht-oxPPW. Diese Erkenntnis trägt zum einen zum mechanistischen Verständnis der NET-Entstehung bei und liefert zum anderen eine potentielle Erklärung für einige der bei TALDO beobachteten Symptome. Desweiteren wurden einige der metabolischen Erfordernisse für die Bildung von NETs mit Hilfe von Inhibitoren untersucht. Die erhaltenen Erkenntnisse zeigen, dass das initiale Maximum des oxidativen Bursts für NET-Bildung unerheblich ist und vielmehr die ROS-Generierung nach ca. 50 Minuten entscheidende Bedeutung für diese hat. / Transdaldolase 1-deficiency (TALDO) is a rare genetic disease with only 34 described cases globally. Transaldolase 1 is part of the non-oxidative pentose phosphate pathway (PPP) and its deficiency results in many clinical symptoms including kidney and liver failure, which can lead to early child-mortality. Some of these patients suffer from recurrent infections, for example in the respiratory tract. Neutrophils are the most abundant white blood cells and essential for the innate immune defence against bacterial and fungal pathogens. The PPP generates reduced NADPH that is crucial for the generation of superoxide by the NADPH oxidase NOX2. In turn, NOX2 is essential for neutrophil extracellular trap (NET) formation. NETs occur through the neutrophil-specific cell death netosis and consist of chromatin decorated with granular proteins. Here I report that neutrophils of three TALDO patients did not make NETs. Deletion of transaldolase 1, and its partner enzyme transketolase, in the neutrophil-like PLB-985 cell line reduced ROS generation and cell death. This confirms that transaldolase 1 is required for NET formation. We present, to the best of our knowledge, the first genetic evidence that the non-oxidative PPP is required for ROS generation and NET formation. Furthermore, some of the metabolic requirements for NET formation were assessed. The obtained data indicate that the initial peak of the oxidative burst is irrelevant for NET formation but the ROS generation after 50 minutes on the contrary has crucial significance.

The rapid increase in antibiotic resistance demands the identification of novel antibiotics with novel targets. One potential antibacterial target is the biosynthesis of peptidoglycan cell wall, which is both ubiquitous and necessary for bacterial survival. Both the caprazamycin-related compounds A-90289 and muraminomicin, as well as the capuramycin-related compounds A-503083 and A-102395 are potent inhibitors of the translocase I enzyme, one of the key enzymes required for cell wall biosynthesis. The caprazamycin-related compounds contain a core nonproteinogen b-hydroxy-a-amino acid referred to as 5’-C-glycyluridine (GlyU). Residing within the biosynthetic gene clusters of the aforementioned compounds is a shared open reading frame which encodes a putative serine hydroxymethyltransferase (SHMT). The revelation of this shared open reading frame resulted in the proposal that this putative SHMT catalyzes an aldol-type condensation reaction utilizing glycine and uridine-5’-aldehyde, resulting in the GlyU core. The enzyme LipK involved in A-90289 biosynthesis was used as a model to functionally assign this putative SHMT to reveal its functions as an l-threonine: uridine-5’-aldehyde transaldolases. Biochemical analysis indicates enzymatic activity is dependent upon pyridoxal-5’-phosphate, is non-reactive with alternative amino acids, and produces acetaldehyde as a co-product. Structural characterization of the enzymatic product is consistent with (5’S,6’S)-GlyU indicating that this enzyme orchestrates a C-C bond breaking and formation resulting in two new stereocenters to make a new l-a-amino acid. The same activity was demonstrated for the LipK homologues involved in the biosynthesis of muraminomicin, A-503083, and A-102395. This l-threonine: uridine-5’-aldehyde transaldolase was used with alternative aldehyde substrates to prepare unusual l-a-amino acids, suggesting the potential for exploiting this enzyme to make new compounds.