Thioredoxin (Trx) and thioredoxin reductase (TrxR) along with an electron donor form a thioredoxin system. Such systems are widely distributed among the organisms belonging to the three domains of life. It is one of the major disulfide reducing systems, which provides electrons to several enzymes, such as ribonucleotide reductase, methionine sulfoxide reductase and glutathione peroxidase to name a few. It also plays an important role in combating oxidative stress and redox regulation of metabolism. Trx is a small redox protein, about 12 kDa in size, with an active site motif of Cys-X-X-Cys. The reduction of the disulfide in Trx is catalyzed by TrxR. Two types of thioredoxin reductases are known, namely NADPH thioredoxin reductase (NTR) with NADPH as the electron donor and ferredoxin thioredxoin reductase (FTR) which depends on reduced ferredoxin as electron donor. Although NTR is widely distributed in the three domains of life, it is absent in some archaea, whereas FTRs are mostly found in plants, photosynthetic eukaryotes, cyanobacteria, and some archaea. The thioredoxin system has been well studied in plants, mammals, and a few bacteria, but not much is known about the archaeal thioredoxin system. Our laboratory has been studying the thioredoxin systems of methanogenic archaea, and a major focus has been on Methanocaldococcus jannaschii, a deeply rooted archaeon that has two Trxs and one TrxR. My thesis research concerns the thioredoxin system of the late evolving members of the group which are exposed to oxygen more frequently than the deeply rooted members of the group, and have several Trxs and TrxRs. Methanosarcina mazei is one such organism, whose thioredoxin system is composed of one NTR, two FTRs, and five Trx homologs. Characterization of the components of a thioredoxin system sets the basis to further explore its function. I have expressed in Escherichia coli and purified the five Trxs and three TrxRs of M. mazei. I have shown the disulfide reductase activities in MM_Trx1 and MM_Trx5 by their ability to reduce insulin with DTT as the electron donor, and that in MM_Trx3 through the reduction of DTNB by this protein with NADPH as the electron donor, and in the presence of NTR as the enzyme. MM_Trx3 was found to be the only M. mazei thioredoxin to accept electrons through the NTR, and to form a complete Trx - NTR system. The Trx - FTR systems are well studied in plants, and such a system is yet to be defined in archaea. I have proposed a mechanism of action for one of the FTRs. FTR2 harbors a rubredoxin domain, and this unit is the only rubredoxin in this organism. Superoxide reductase, an enzyme that reduces superoxide radical to hydrogen peroxide without forming oxygen, utilizes rubredoxin as the direct electron source and this enzyme is found in certain anaerobes, including Methanosarcina species. Thus, it is possible that FTR2 provides electrons via a Trx to the superoxide reductase of M. mazei. This activity will define FTR2 as a tool in combating oxidative stress in M. mazei. In my thesis research I have laid a foundation to understand a complex thioredoxin system of M. mazei, to find the role of each Trx and TrxR, and to explore their involvement in oxidative stress and redox regulation. / Master of Science

Contamination of perchlorate and chlorate in nature is primarily the result of various industrial processes. The microbial respiration of these oxyanions of chlorine plays a major role in reducing the society’s impact on the environment. The focus with this thesis is to investigate the oxygen-dependent regulation of key components involved in the chlorate respiration in the gram‑negative bacterium Ideonella dechloratans. Chlorate metabolism is based on the action of the enzymes chlorate reductase and chlorite dismutase and results in the end products molecular oxygen and chloride ion. Up‑regulation of chlorite dismutase activity in the absence of oxygen is demonstrated to occur at the transcriptional level, with the participation of the transcriptional fumarate and nitrate reduction regulator (FNR). Also, the chlorate reductase enzyme was shown to be regulated at the transcriptional level with the possible involvement of additional regulating mechanisms as well. Interestingly, the corresponding chlorate reductase operon was found to be part of a polycistronic mRNA which also comprises the gene for a cytochrome c and a putative transcriptional regulator protein.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

The Aldo-Keto Reductases (AKR) are a group of oxidoreductase enzymes structurally and mechanistically distinct from the Alcohol Dehydrogenases (ADH). The AKRs are of importance for their ability to produce industrially useful compounds including chiral secondary alcohols. The ADH family have traditionally been exploited for chiral alcohol production; the AKR family have currently been underexploited for chiral alcohol production and present the opportunity to search for novel oxidoreductases with properties and substrate specificities distinct from the ADH enzymes. The AKR studied here, from the hyperthermophilic bacteria Thermotoga maritima has been characterised with respect to its biochemical and structural properties, and its potential as a biocatalyst evaluated. This enzyme is the second example of a thermophilic AKR to have its three dimensional structure solved, the other also being from Thermot. maritima. The AKR studied exhibits high stability with respect to temperature and moderate amounts of organic solvents. A large preference for the reduction reaction compared to the oxidation reaction was found, which has previously been observed in other AKRs. The X-ray crystal structure was solved to 2.6Å resolution in the apo form. The final structure has three loop sections which were not located due to disorder within the crystal, which are expected to become ordered upon cofactor and substrate binding. A section of one of these missing loops was found to bind at the active site of the enzyme, with a glutamate occupying the site of substrate carbonyl binding. The formation of a dimer, increased helix-dipole stabilisation and long distance ion pair interactions all act to increase thermostability of the AKR with respect to its mesophilic homologues. The X-ray crystal structure of Escherichia coli bacterioferritin has also been solved to 1.9Å resolution, which was co-purified along with the recombinant AKR enzyme. This structure shows the symmetrical binding of a heme molecule on the local two-fold axis between subunits and the binding of two metal atoms to each subunit at the ferroxidase centre. These metal atoms have been identified as zinc by the anaylsis of the structure and X-ray data and confirmed by microPIXE experiments. For the first time the heme has been shown to be linked to the internal and external environments via a cluster of waters positioned above the heme molecule. This information has provided a greater insight into the function and mechanism of bacterioferritin.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / In this study we examine the utility of using CD8+ T cell hybridomas to measure the ability of bone marrow dendritic cells (BMDCs) to internalize cancer proteins and display them to cytotoxic T cells, a process termed cross‐presentation. We test the ability of a newly generated T cell hybridoma called BUSA14 to detect cross‐presentation of the melanoma antigen gp100. BUSA14 produces a dose‐dependent response to human and mouse gp100 peptides. However, cross‐presentation of gp100 by BMDCs using SK‐MEL‐28 human melanoma cell lysates or direct MHC class I‐restricted presentation by B16 murine melanoma cells was not detected. Both SKMEL‐28 and B16 cells express gp100 protein by immunoblot, and gp100 as a membrane bound protein may be concentrated by cell fractionation techniques. We validated our crosspresentation assay with another T cell hybridoma B3Z to detect cross‐presentation of the model antigen ovalbumin. Lastly, we determined that although BUSA14 expresses the coreceptor CD8, BUSA14 lacks CD3 expression, which likely impairs the ability of this hybridoma to respond to engagement of the T cell receptor and contributes to the inability to detect presentation of native gp100 protein. To resolve these issues, we plan to use primary gp100‐specific T cells from pmel mice expressing the same T cell receptor as the BUSA14 hybridoma to detect presentation of gp100 protein. Ultimately, we plan to evaluate the requirements for cross‐presentation of gp100, including a role for gamma‐interferon‐inducible lysosomal thiol reductase (GILT), a disulfide bond reducing enzyme.

Ribonucleotide reductase (RNR) is a key enzyme in the de novo biosynthesis and homeostatic maintenance of all four DNA building blocks by being able to make deoxyribonucleotides from the corresponding ribonucleotides. It is important for the cell to control the production of a balanced supply of the dNTPs to minimize misincorporations in DNA. Because RNR is the rate-limiting enzyme in DNA synthesis, it is an important target for antimicrobial and antiproliferative molecules. The enzyme RNR has one of the most sophisticated allosteric regulations known in Nature with four allosteric effectors (ATP, dATP, dGTP, and dTTP) and two allosteric sites. One of the sites (s-site) controls the substrate specificity of the enzyme, whereas the other one (a-site) regulates the overall activity.  The a-site binds either dATP, which inhibits the enzyme or ATP that activates the enzyme. In eukaryotes, ATP activation is directly through the a-site and in E. coli it is a cross-talk effect between the a and s-sites. It is important to study and get more knowledge about the overall activity regulation of RNR, both because it has an important physiological function, but also because it may provide important clues to the design of antibacterial and antiproliferative drugs, which can target RNR. Previous studies of class I RNRs, the class found in nearly all eukaryotes and many prokaryotes have revealed that the overall activity regulation is dependent on the formation of oligomeric complexes. The class I RNR consists of two subunits, a large α subunit, and a small β subunit. The oligomeric complexes vary between different species with the mammalian and yeast enzymes cycle between structurally different active and inactive α6β2 complexes, and the E. coli enzyme cycles between active α2β2 and inactive α4β4 complexes. Because RNR equilibrates between many different oligomeric forms that are not resolved by most conventional methods, we have used a technique termed gas-phase electrophoretic macromolecule analysis (GEMMA). In the present studies, our focus is on characterizing both prokaryotic and mammalian class I RNRs. In one of our projects, we have studied the class I RNR from Pseudomonas aeruginosa and found that it represents a novel mechanism of overall activity allosteric regulation, which is different from the two known overall activity allosteric regulation found in E. coli and eukaryotic RNRs, respectively.  The structural differences between the bacterial and the eukaryote class I RNRs are interesting from a drug developmental viewpoint because they open up the possibility of finding inhibitors that selectively target the pathogens. The biochemical data that we have published in the above project was later supported by crystal structure and solution X-ray scattering data that we published together with Derek T. Logan`s research group. We have also studied the effect of a novel antiproliferative molecule, NSC73735, on the oligomerization of the human RNR large subunit. This collaborative research results showed that the molecule NSC73735 is the first reported non-nucleoside molecule which alters the oligomerization to inhibit human RNR and the molecule disrupts the cell cycle distribution in human leukemia cells.

The study herein described involved the detection of pyridine N-oxide reductase activity in cell-free extracts of E. coli 9723, the determination of co-factors necessary for the enzymatic process, a study of the optimum conditions for enzyme catalysis, and a general characterization of the enzyme.

The objectives of these investigations were (1) the purification of MG reductase from sheep liver and (2) studies of some of its characteristics. MG reductase was purified 40 fold and showed a single band on SDS-PAGE. Molecular weight estimations with SDS-PAGE showed a molecular weight of 44,000; although gel filtration with Sephadex G-150 gave a molecular weight of 87,000 indicating that the enzyme might be a dimer. The Km for MG is 1.42 mM and for NADH it is 0.04 mM. The pH optimum for the purified enzyme is pH 7.0. Isoelectric focusing experiments showed a pI of 9.3. In vivo experiments involving rats treated with 3,3',5-triiodothyronine (T_3) and 6-n-propyl-2-thiouracil (PTU) indicated that MG reductase was depressed by T_3 and elevated by PTU.

A Leishmaniose é uma infecção causada pelo protozoário do gênero Leishmania e transmitida pela picada de insetos. Os tratamentos atuais são caros e demorados, envolvendo compostos baseados em Sb(V), anfotericina B lipídica e miltefosina. Estudos recentes sugerem que a enzima tripanotiona redutase (TR) poderia ser um alvo específico no desenvolvimento de novos fármacos, pois ela é essencial e exclusiva dos tripanossomatídeos. Desta forma, este trabalho apresenta a síntese de novos compostos derivados da iminodibenzila com estrutura similar à clomipramina, um conhecido inibidor da TR. Tais compostos são conjugados de iminodibenzila com etilenodiamina, etanolamina e dietilenotriamina através de epicloridrina (dado-en, dado-ea e dado-dien , respectivamente). Foram obtidos com rendimentos superiores a 94% e caracterizados através de LC_MS e espectroscopia no infravermelho. Também se fez a avaliação da afinidade relativa dos novos ligantes por cobre através do teste de recuperação da fluorescência da calceína, concluindo-se que o ligante dado-dien possui uma afinidade maior com Cu (II) quando comparado com os outros ligantes sintetizados. Foram formados complexos metálicos através da adição de sulfato de cobre (II), acompanhando-se através de espectroscopia ultravioleta-visível para se obter uma estimativa da relação metal: ligante, concluindo-se que os complexos formados eram [Cu(dado-ea)]2+, [Cu(dado-en)2]2+ e [Cu(dado-dien)2]2+. A lipofolicidade foi estimada através de simulação computacional, constatando-se que os complexos formados são todos muito lipofílicos, sendo que o logP descreve na seguinte: [Cu(dado-en)2]2+ > [Cu(dado-dien)2]2+ > [Cu(dado-ea)]2+. Ensaios de atividade pró-oxidante através da oxidação da sonda fluorimétrica DHR revelaram que o complexo [Cu(dado-ea)]2+ apresentou a maior taxa de atividade pró-oxidante, sendo independente da concentração de peróxido de hidrogênio no meio reacional e alcançando seu limiar quando presente em concentração aproximada de 10 µM. Por fim, os compostos sintetizados foram testados quando à suas citotoxidades frente a promastigotas L. amazonenses e com macrófagos RAW 264,7. Quando o protozoário foi exposto a concentrações variadas dos compostos deste estudo, observou-se que os ligantes ea, en e dien tiveram os menores valores de IC50 (todos < 3µM), porém eles são muito tóxicos para serem utilizados como fármacos, Dentre os ligantes sintetizados, somente o dado-en apresentou um resultado numa faixa viável (IC50 19,9 ± 2,2 µM). Contra macrófagos RAW 264,7, dado-en apresentou IC50 > 50µM, ou seja, ele é mais tóxico para o protozoário do que para os macrófagos. A complexação com cobre não representou um aumento interessante na citotoxicidade dos compostos. O conjunto de resultados apresentados neste indica um potencial uso do composto dado-en no combate à leishmaniose. / Leishmaniasis is an infection caused by protozoa of the genus Leishmania. and transmitted by insects. Current treatments are expensive and time-consuming, involving Sb(V)-based compounds, lipossomal amphotericin B and miltefosine. Recent studies suggest that inhibition of trypanothione reductase (TR) could be a specific target in the development of new drugs, because it is essential and exclusive to trypanosomes. Thus, this work presents the synthesis of new compounds derived from iminodibenzyl with structures related to clomipramine, a known inhibitor of TR. These compounds were conjugates of iminodibenzyl with ethylenediamine, ethanolamine and diethylenetriamine, linked by epichlorohydrin. They were obtained with yields higher than 94% and their structures were confirmed by LC-MS and infrared spectroscopy. The relative affinity for copper of the new chelators was assessed by the recovery of calcein fluorescence, which showed that dado-dien has the highest affinity for Cu(II) among the new ligands. Metal complexes were synthesized by addition of copper(II) sulphate and studied by ultraviolet-visible spectroscopy, showing that the stoichiometries were [Cu(dadoea)]2+, [Cu(dado-en)2]2+ and [Cu(dado-dien)2]2+. Lipophilicity was assessed by computational methods, indicating that the complexes were all very lipophilic, with logP decreasing in the following order: [Cu(dado-en)2]2+ > [Cu(dado-dien)2]2+ > [Cu(dado-ea)]2+. Pro-oxidant activity assays by oxidation of DHR fluorimetric probe showed that [Cu(dado-ea)]2+ has the highest rate of oxidation, independent of hydrogen peroxide concentration, reaching its highest value when at ~ 10 &#181;M. Finally, the compounds were tested for citotoxicity to L. amazonensis promastigotes and RAW 264,7 macrophages. When the protozoa was exposed to varying concentrations of the compounds, it was observed that chelators ea, en and dien had the lowest values of IC50 (< 3 &#181;M). but they are too citotoxic to be used as drugs. Among the synthesized chelators, only dado-en presented a result in a pharmaceutically interesting range (IC50 19.9 ± 2.2 &#181;M). Against RAW 264,7 macrophages dado-en IC50 > 50 &#181;M. Complexation to copper did not enhance the citotoxicity of the compounds. Overall, the results in this study indicate a potential use of dado-en in combating leishmaniasis.

A nitrato redutase (NR) atua juntamente com a nitrito redutase (NiR) catalisando a primeira etapa da redução do nitrato. A NR, no citossol, é ativada, principalmente, pela luz e reduz o nitrato a nitrito. Em seguida, este é reduzido a amônio. Trabalhos anteriores demonstraram que a isoforma citossólica da NR está presente nas folhas e raízes do abacaxizeiro; já as associadas à membrana plasmática (NRMP) ainda não se tem registro. Sabe-se, no entanto, que a NRMP apresenta modos diferentes de ativação, como já constatados para outras espécies. Dentre os fatores que afetam sua atividade pode-se citar a temperatura. Pesquisas realizadas no Laboratório de Fisiologia Vegetal do IBUSP, acerca da influência do termoperíodo sobre os metabolismos nitrogenado e fotossintético de plantas de abacaxizeiro, aventaram a hipótese de que haveria uma nitrato redutase específica de membrana plasmática presente nas células radiculares, a qual seria regulada por termoperíodo, diferindo, portanto, da isoforma citossólica presente nas folhas. Assim, o presente trabalho teve como objetivo principal demonstrar a existência de uma isoforma da NR associada à membrana plasmática, a qual seria responsável pelo incremento da atividade dessa enzima registrada nas raízes de Ananas comosus, quando plantas cultivadas in vitro são submetidas ao termoperíodo (28°C dia/ 15°C noite). Para tanto, determinou-se o tempo mínimo de exposição das plantas de abacaxizeiro ao termoperíodo, necessário à indução da nitrato redutase radicular. Além disso, estudou-se a influência da idade das plantas na resposta ao tratamento com baixa temperatura noturna. Plantas cultivadas in vitro com 90 dias de idade ou com idades variadas foram transferidas para câmaras de crescimento com temperatura constante (28°C dia/noite controle experimental) ou com termoperíodo (28°C dia/ 15°C noite), fotoperíodo de 16 horas e intensidade luminosa de 55 moles m-2 s -1. Elas permaneceram nessas condições por 1, 3, 5, 7, 15, 30, 40, 50 ou 60 dias. Após cada período, a atividade in vivo da NR foi analisada durante a fase de ausência de luz. Para que fosse possível identificar uma possível NRMP nas células radiculares de abacaxizeiro, um método de ensaio in vitro foi padronizado e a melhor técnica de isolamento de frações de membrana plasmática foi selecionada. Após as plantas com 60 dias de idade serem submetidas por 30 dias ao tratamento termoperiódico ou à temperatura constante, as frações de membrana plasmática das células radiculares foram isoladas e o ensaio in vitro da NR foi realizado, utilizando-se NADH, NADPH ou succinato como doadores de elétrons. Os resultados indicaram que o tempo mínimo de exposição das plantas de abacaxizeiro ao termoperíodo foi de 30 dias. O método de ensaio enzimático in vitro foi padronizado para as plantas de abacaxizeiro e a técnica de isolamento de frações de membrana plasmática que se mostrou mais adequada para essa bromélia foi a de fracionamento por sistema de duas fases com Dextran T-500 e PEG 3350. O grau de pureza das frações, avaliado pela detecção da atividade da enzima citoplasmática malato desidrogenase (MDH), foi em média de 95%, evidenciando a eficácia da pradronização do método. Os resultados obtidos para as frações de membranas plasmáticas, extraídas das raízes das plantas que estiveram sob o tratamento termoperiódico, mostraram que a baixa temperatura noturna influenciou positivamente a atividade da nitrato redutase. O aumento da atividade foi observado quando NADH, NADPH ou succinato foram utilizados como doadores de elétrons. Isso significa que, provavelmente, mais de uma isoforma da NRMP está presente nas raízes de abacaxizeiro. Além disso, há indícios de que a isoforma que está ligada externamente à membrana por uma âncora glicosídica (que utiliza succinato como doador de elétrons) está presente nas células radiculares do abacaxizeiro e respondeu positivamente ao estímulo da baixa temperatura noturna. Em contrapartida, nenhuma diferença pôde ser observada quando as atividades da NR foram medidas nas frações de citoplasma das plantas controle e daquelas que foram tratadas com termoperíodo. Não foi detectada atividade nas frações citossólicas quando succinato foi oferecido como poder redutor da NR. Concluiu-se, portanto, que o incremento na atividade da NR, verificado nas plantas que foram tratadas com termoperíodo, deveu-se à indução pela baixa temperatura noturna da NRMP. Esta pesquisa trouxe contribuições importantes acerca da existência de uma nitrato redutase associada à membrana plasmática em abacaxizeiro, nunca antes detectada em uma bromélia e muito pouco estudada nos demais vegetais. As padronizações realizadas serão essenciais para aplicação em outras pesquisas do Laboratório de Fisiologia Vegetal do IBUSP, abrindo oportunidades para se aprofundar ainda mais o tema sobre o controle da ativação da NR. / The nitrate reductase (NR) acts together with the nitrite reductase (NiR) to catalyze the first step of the nitrate reduction. The NR localized in the cytosol is activated mainly by light and reduces nitrate to nitrite, followed by its reduction to ammonium. Previous work demonstrated that the cytosolic isoform of NR is present in leaves and roots of Ananas comosus, although the isoform associated with the plasma membrane (PM-NR) has not yet been registered in this species. The PM-NR has different modes of activation in comparison to the cytosolic NR, as already demonstrated in other species. Among the factors that affect its activity can be mentioned the temperature. Experiments developed in the Laboratory of Plant Physiology of IBUSP hypothesized that exists a specific plasma membrane NR in plant roots regulated by thermoperiod, differing from the cytosolic isoform present in leaves. The present work aimed to demonstrate the existence of this isoform of NR present in the plasma membrane, which would be responsible for the increase of its activity in Ananas comosus roots when plants were cultivated in vitro under thermoperiod of 28°C day/ 15°C night. Initially, it was important to determine the minimum time of exposure to thermoperiod necessary for the induction of nitrate reductase in roots of pineapple plants. Furthermore, it was analyzed the influence of age in the response of plants to low night temperature treatment. For this purpose, plants with different ages cultivated in vitro were transferred to growth chambers either with constant temperature (28°C day/night experimental control) or with thermoperiod (28°C day/ 15°C night). The plants were cultivated in these conditions during 1, 3, 5, 7, 15, 30, 40, 50 or 60 days and then the in vivo NR activity was analyzed in the shoot and root tissues during the dark period. In order to identify a probable PM-NR in the pineapple root cells, it was also necessary to develop a NR in vitro assay protocol specific for Ananas comosus and the appropriate technique for plasma membrane isolation (Dextran T-500 and PEG 3350). The purity of the fractions, determined by the activity of cytoplasmic enzyme malate dehydrogenase (MDH), was on average 95%, indicating the effectiveness of the method. The next step was to evaluate the NR activity in citoplasmic and plasma membrane fractions of root tissues of Ananas comosus. Using 60 daysold plants exposed either to 30 days under the thermoperiodic treatment or to constant temperature, the plasma membrane fractions of roots were isolated and the in vitro NR assay was performed using NADH, NADPH or succinate as electron donators. The results indicated that the minimum thermoperiod exposure time necessary to induce NR activity was 30 days. Furthermore, it was demonstrated that low temperatures during the dark period positively influenced the activity of nitrate reductase in plasma membrane fractions and that the increase in its activity was observed when NADH, NADPH or succinate were used as electron donors. On the other hand, no difference in NR activity was observed in the cytoplasmic fraction of control plants and those which were treated with thermoperiod. Moreover, no NR activity was detected in cytosolic fractions when succinate was provided as electron donor. All together, this results showed that probably diferents isoforms are presents in pineapple roots. The extracellular isoform that is attached to the plasma membrane by a lipophilic anchor (using succinate as electron donor) can be present in root cells of pineapple and responded positively to the low night temperature stimulus. This study has made important contributions to the knowledge of the metabolism and physiology of Ananas comosus. This is the first time that the existence of a nitrate reductase associated with the plasma membrane, a very little studied enzyme, is documented in Bromeliaceae.