Two isozymes of triosephosphate isomerase have been isolated to homogeneity from human placenta. Triosephosphate isomerase A and triosephosphate isomerase B were compared in terms of their chemical, and biological properties.

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There are at least two models describing how the enzymes of metabolic pathways are arranged in living cells. The first is a stochastic model, where enzymes are freely-diffusing in the aqueous environment of the cell, and the second, the metabolon model, has pathway enzymes organized as enzyme complexes. Both are valid scientific hypotheses in that they make predictions that can be tested regarding pathway regulation, localization, and function. The goal of the work presented here was to test the metabolon model using the flavonoid biosynthetic pathway in Arabidopsis, which has been hypothesized to exist as a metabolic enzyme complex. Five novel mutants of the gene encoding the first enzyme of flavonoid biosynthesis, chalcone synthase (CHS), were characterized in an effort to develop tools for investigating the organization of flavonoid metabolism in Arabidopsis. A variety of mutant CHS genotypes were identified in this allelic series, including ones that displayed both null and temperature-sensitive phenotypes, based on endproduct analysis. Characterization of protein and RNA levels indicated that the stability of the CHS enzyme was reduced in some of the mutants as compared to wild type. In several of the alleles, homodimerization of CHS was also impaired. Effects of the mutations at the amino acid level were predicted from the three-dimensional crystal structure of the highly-homologous alfalfa CHS, which indicated substitutions at diverse sites on the enzyme, including ones that may disrupt folding and/or active site function. This allelic series should provide a useful genetic resource for ongoing studies of flavonoid enzyme structure, function, and subcellular organization. In an effort to determine the in planta location of the first two enzymes in flavonoid biosynthesis, CHS and chalcone isomerase (CHI), immunolocalization experiments were performed. Results indicate that CHS and CHI are abundant in epidermal and cortex cells of the root elongation zone and the root tip, consistent with the accumulation of flavonoid endproducts at these sites. At the subcellular level, both of these enzymes were found to localize to the endoplasmic reticulum (ER), consistent with the hypothesis that the enzymes of flavonoid biosynthesis are organized as a membrane-associated enzyme complex. Analysis of the tt7(88) mutant, which lacks the cytosolic domain of the putative 'anchor' P450 enzyme, flavonoid 3'-hydroxylase, showed an altered distribution of CHS and CHI as compared to wild type, however CHS and CHI were still found to be associated with ER. These results suggest that complex interactions occur within the flavonoid enzyme complex to mediate the subcellular distribution of its constituents. Also evident from these studies was the asymmetric distribution of CHS and CHI in cortex cells of the elongation zone, a finding that may provide clues about the physiological function of flavonoids in roots. Together, these immunolocalization data support the metabolon model for the organization of flavonoid biosynthesis in Arabidopsis. In an effort to develop tools to investigate the in vivo dynamics of flavonoid biosynthesis, fusion proteins between CHS or CHI and the reporter, green fluorescent protein (GFP), were produced. Transient transfection assays in epidermal cells from onion root bulbs and Arabidopsis seedlings indicated that the GFP component of the fusion constructs was functional, as determined via GFP fluorescence. To investigate the spatial and temporal dynamics of these fusion proteins in all cell types, Arabidopsis plants stably transformed with the CHI-GFP fusion constructs were generated. The analysis of these transgenic plants should provide information regarding the localization and dynamics of flavonoid biosynthesis in vivo, and thereby serve to offer new insights into the function and regulation of this important plant metabolic pathway. Overall, the research presented here represents a significant contribution toward understanding how subcellular organization may be important in regulating metabolism. / Ph. D.

Flavonoid biosynthesis is an important secondary metabolic pathway in higher plants with a range of vital functions in plants and animals. This pathway has been developed as a model system for the study of multi-enzyme complexes. The goal of the work presented here was to structurally characterize a series of loss-of-function chalcone synthase (CHS) alleles and to define the molecular basis of the interaction between CHS and the second enzyme of flavonoid biosynthesis, chalcone isomerase (CHI). CHS proteins encoded by five previously characterized alleles were characterized by homology modeling in an effort to explain the alterations in function, stability, and dimerization exhibited by these variants. Four of the encoded proteins have a single amino acid substitution and the fifth is a truncated protein resulting from a frameshift. Models for each of these proteins were generated in silico and analyzed after molecular dynamics simulations. This analysis suggested reasons for changes in catalytic ability and stability for three of the five CHS variants. To characterize the molecular basis of the CHS-CHI interaction, a model was developed using X-ray crystallography, small-angle neutron scattering (SANS), in silico docking, molecular dynamics simulations, and yeast 2-hybrid analyses. These enzymes appear to be interacting in a manner that could facilitate the flow of intermediates from one active site to another. These experiments also identified a series of amino acids that appear to be involved in the interaction, which are currently undergoing alteration and analysis using a yeast 2-hybrid assay to verify the authenticity of the model. The data presented herein could be used in future engineering experiments to alter pathway flux to control the levels or types of flavonoid endproducts, resulting in more nutritious plants or flowers with novel pigments. These experiments advance the study of the structure of multi-enzyme complexes, an area that currently contains little information. As well, this is the first known use of SANS for the investigation of the architecture of metabolons. The techniques described herein could easily be applied to other systems in an effort to better understand the organization of multi-enzyme complexes and the implications of these assemblies on metabolic regulation. / Ph. D.

A method for purification and radiolabelling phosphoglucose isomerase was devised in order to develop a sensitive quantitative radioimmunoassay for the detection of the enzyme irrespective of its catalytic activity. For four genetic variants of PGI no difference in the molecular specific activity was observed. In one variant (PGI-Denton), liver and heart tissue extracts, and in mature erythrocytes (as compared to normal erythrocytes), a decreased molecular specific activity was observed which initially may imply that these samples contain cross-reactive material which is not catalytically active.

Triosephosphate isomerase (EC 5.3.1.1., TPI) undergoes specific posttranslational modifications (deamidation and oxidation) which are believed to initiate protein turnover by destabilization of the dimer. The crystal structures, amino acid sequences, and aging related changes of TPI from various species have been independently characterized by several laboratories. TPI has thus become the prototype enzyme for examining the initial steps in protein turnover. The binding of substrate enhances the specific deamidation of the mammalian enzyme, and a general mechanism of 'molecular wear and tear' [Gracy, R. W., Yiiksel, K. 0., Chapman, M. L., and Dimitrijevich, S. D. (1990) in Isozymes-Structure, Function and Use in Biology and Medicine (Ogita, Z-I., and Markert, C. L., Eds) pp. 787-817, Wiley-Liss, New York] has been proposed to explain how enzymes may wear out.

As doenças tropicais têm um grande impacto sobre a saúde em países de baixa renda, estando relacionadas com condições de pobreza e desigualdade. A tripanossomíase africana é uma infecção parasitaria negligenciada incluída na agenda da Organização Mundial de Saúde. Esta enfermidade é causada pelo Trypanosoma brucei gambiense e Trypanosoma brucei rhodesiense, sendo transmitida pela mosca tsé-tsé (Glossina sp.) e geralmente fatal se não tratada. Os fármacos usados no seu tratamento são ineficazes, difíceis de administrar e causam severas reações adversas. Portanto, existe a necessidade do desenvolvimento de alternativas quimioterápicas eficazes e seguras. Assim, a enzima fosfoglicerato mutase (PGAM) surge como um importante alvo molecular. Esta enzima esta envolvida no metabolismo de glicose, sendo necessária para a viabilidade do parasito. Somado a isso, ela difere da enzima dos hospedeiros permitindo a identificação de inibidores específicos. Não obstante, esforços têm sido realizados para identificar inibidores da PGAM, bem como para elucidar sua estrutura e mecanismo de reação. Nosso propósito é obter o modelo de alta resolução desta macromolécula sem ligantes e conseqüentemente analisar a mudança de conformação que esta sofre ao se ligar ao seu substrato natural. A PGAM de Trypanosoma brucei obtida na expressão e purificação mostrou-se cataliticamente ativa nos ensaios cinéticos. Por experimentos de cromatografia de exclusão molecular observamos que a amostra purificada se comportava na forma de monômero. Dados de difração de raios-X foram coletados para cristais da macromolécula obtidos na ausência de ligantes. A estrutura cristalográfica foi resolvida a 2.3 Å, apresentando um dímero na unidade assimétrica. Ambas as moléculas do dímero estavam na forma livre e apresentava grande diferença conformacional se comparadas com as PGAMs de estruturas conhecidas que estão ligadas ao substrato ou produto natural. Por espalhamento de raios-X a baixos ângulos confirmamos que a enzima é monomérica em condições que mimetizam a fisiológica. A mudança conformacional induzida pelo ligante não afeta a topologia dos dois domínios da PGAM. Entretanto, há mudanças nos ângulos torcionais da cadeia principal dos laços que conectam os domínios da proteína. Além disso, o metal cobalto parece estar envolvido na estabilização da estrutura terciária da PGAM na conformação livre. Finalmente, este novo modelo estrutural pode contribuir para o esforço internacional de desenvolver fármacos tripanocidas / Tropical diseases represent a major burden on population health in low-incoming countries, being related to poverty and social disadvantage. African trypanosomiasis is a neglected parasitic infection on the agenda of World Health Organization. This disorder is caused by Trypanosoma brucei gambiensis and Trypanosoma brucei rhodesiensis, transmitted by the tsetse fly (Glossina sp.), and usually fatal if untreaded. The drugs used in the treatment are ineffective, difficult to administer, and cause severe adverse reactions. Therefore, there is a need to develop effective and safe chemotherapies. Thus, the enzyme phosphoglycerate mutase (PGAM) emerges as an important molecular target. This enzyme is involved in glucose metabolism, and is necessary for viability of the parasite. Moreover, it differs from the host enzyme allowing the identification of specific inhibitors. Nevertheless, efforts have been made in identifying PGAM inhibitors and to elucidate their structure and mechanism of reaction. Our purpose is to obtain the high resolution model of the macromolecule free from ligands and consequently to analyze the change in conformation that undergoes upon binding to its natural substrate. Trypanosoma brucei PGAM obtained in the expression and purification was shown to be catalytically active in the kinetics assays. In the size exclusion chromatography we observed that the purified sample behaves as a monomer. X-ray diffraction data were collected for crystals of the macromolecules obtained in the absence of ligands. The crystal structure was solved to 2.3 Å, showing a dimmer in the asymmetric unit. Both molecules of the dimmer were in free form, and had a large conformational difference compared with those of know PGAM structures that are connected to the natural substrate or product. Small angle X-ray scattering confirm that the enzyme is monomeric under conditions that mimic the physiological. Ligand-induced conformational change does not affect the topology of the two domains of the PGAM. However, there are changes in torsional angles of the main chain of the loops that connect the protein domains. Additionally, the metal cobalt seems to be involved in stabilizing the tertiary structure of PGAM in the free conformation. Finally, this new structural model may contribute to the international effort to develop trypanocidal drugs.

Fazemos parte de um cenário mundial em que o esgotamento das fontes de energias fósseis atrelado à poluição gerada por esse uso, preocupam os diferentes setores do comércio, da indústria, do governo e das instituições em defesa do meio ambiente. Nesse sentido, a busca por novas fontes energéticas renováveis tem dirigido diversas pesquisas, além de drenar bilhões de dólares em investimentos. Uma das linhas de pesquisa mais importantes é a da produção do etanol de segunda geração (2G), um etanol produzido a partir dos resíduos gerados na produção do etanol de primeira geração. No caso do Brasil, esses resíduos compreendem principalmente a palha e o bagaço de cana-de-açúcar; essa biomassa é formada majoritariamente por celulose (∼45%), hemicelulose (∼25%) e lignina (∼20), e sua hidrólise envolve pré-tratamentos adequados e uso de enzimas que agem especificamente em seus alvos. Dessa forma, a produtividade de etanol aumenta, sem necessariamente ampliar áreas de cultivo. Essa vertente é muito promissora, porém os custos ainda são relativamente altos e a aplicabilidade depende bastante de adaptações do setor industrial e aprimoramentos na produção em si (atividade específica das enzimas e sua ação sinérgica). O objetivo principal deste projeto é reconhecer e mapear as bases moleculares que comandam a atividade da enzima xilose isomerase (XI), que converte xilose (presença majoritária na hemicelulose) em xilulose, possibilitando a utilização desta por Saccharomyces cerevisiae (já que a xilose não é fermentescível), para obtenção do etanol de segunda geração como produto final. Para isso, foi realizada uma busca extensiva de genes de diversos microrganismos, que codifiquem para XI, e que essas ainda não possuam estruturas resolvidas publicadas. A maioria das ORFs (Open Reading Frame, do inglês), ou regiões codificadoras, foram amplificadas, clonadas em vetores específicos e transformadas em bactérias Escherichia coli Rosetta (DE3). Parte dessas cepas transformadas resultaram na produção da XI de interesse. Com isso, foi possível obter cristais e iniciar a resolução de estruturas cristalográficas. Esses resultados foram cruzados e correlacionados com os de atividade enzimática, cinética química e estabilidade térmica, fornecendo boa perspectiva para o entendimento das bases moleculares que regem a atividade xilose isomerásica. / We are part of a world scenario in which the depletion of fossil energy sources linked to the pollution generated by this use, concern the different sectors of commerce, industry, government and institutions in defense of the environment. In this regard, the search for new renewable energy sources has headed many researches, besides generating billions of dollars in investments. One of the most important research lines is the production of second generation ethanol (2G), an ethanol produced from the waste generated in the production of the first generation one. In the case of Brazil, these residues mainly include sugar cane straw and bagasse. This biomass is mostly composed of cellulose (∼45%), hemicellulose (∼25%) and lignin (∼20), and its hydrolysis involves adequate pre-treatments and the use of enzymes that specifically act on their targets. In this way, ethanol productivity increases without necessarily expanding growing areas. This aspect is very promising, but the costs are still relatively high and the applicability badly depends on adaptations of the industrial sector and improvements in the production itself (specific activity of the enzymes and their synergistic action with others). The main goal of this project is to recognize and map the molecular bases that control the activity of the enzyme xylose isomerase (XI), which converts xylose (the mostly present carbohydrate in hemicellulose) into xylulose, allowing its use by Saccharomyces cerevisiae (since xylose is not fermentable), to obtain the second generation ethanol as final product. To reach this, an extensive search of genes of several microorganisms, that code for XI, and still do not have solved high resolution structures published are carried out. Most ORFs (Open Reading Frames) were amplified, cloned into specific vectors and transformed into Escherichia coli Rosetta (DE3) bacteria. Some of these transformed strains leaded to the production of XI of interest. Furthermore, it was possible to obtain protein crystals and to start trying to solve crystallographic structures. These results were cross - checked and correlated with those of enzymatic activity, chemical kinetics and thermal stability, providing a good perspective for understanding the molecular bases which govern isomerase activity.

A tripanossomose africana, ou doença do sono, como é popularmente conhecida, atingiu 25 mil pessoas só em 1995. Estima-se que a doença seja responsável pela morte de 10.000 pessoas a cada ano, segundo os dados da Organização Mundial da Saúde. Sua distribuição é exclusivamente africana, devido ao fato de sua transmissão depender da Tsé-Tsé, mosca do gênero Glossina que encontra lá condições favoráveis para sua proliferação. Atualmente os medicamentos existentes não são eficientes e devem ser ministrados em doses altas, provocando graves efeitos colaterais. O parasita Tripanosoma brucei, na forma encontrada no sangue dos mamíferos (tripomastigota), é o responsável pela doença e, assim como toda a família Trypanosomatidea, é altamente dependente de glicose. Esses parasitas consomem a quantidade de glicose equivalente à sua massa em aproximadamente sete horas. A via glicolítica torna-se, portanto, chave para o desenvolvimento de inibidores que possam ser utilizados no combate a estes parasitas. Nesse sentido, temos realizado estudos com a enzima glicose-6-fosfato isomerase (glucose-6-fosfato isomerase; Phosphoglucose Isomerase PGI; EC 5.3.1.9), responsável pela isomerização reversível da D-glicose-6-fosfato e D-frutose-6-fosfato e participa na glicólise como a segunda enzima da via. Os trabalhos realizados até aqui culminaram na sua expressão em forma recombinante, purificação através de coluna de afinidade, e caracterizações enzimáticas. Sua atividade específica foi determinada através de métodos já estabelecidos encontrados na literatura. O IC50 da enzima frente a quatro inibidores da reação foi determinado. O trabalho finaliza com a construção de um modelo estrutural da enzima determinado através de métodos de modelagem molecular por homologia. / The African trypanosomiasis, or sleeping sickness as is popularly known, affected 25 thousand people only in 1995. It is estimated that the disease is responsible for 10 thousand deaths per year, according to data provided from the World Health Organization (WHO). The distribution of the disease is exclusively African due to the transmission being dependent on the tsé-tsé vector. A fly, belonging to the Glossinia genus, finds in the African continent favorable conditions for its proliferation. Presently the existing drugs are not efficient and have to be applied in high dosage, resulting in severe side effects. The bloodstream form (tripomastigote) of the parasite Trypanosoma brucei is responsible for the disease and such as the whole Trypanosomatidae family is dependent on glucose. Those parasites consume a quantity of glucose equivalent to its mass in approximately seven hours. This characteristic results in the glycolitic pathway been a key target for drug development against those parasites. In this direction we are developing research with the enzyme glucose-6-phosphate isomerase (Phosphoglucose lsomerase PGI; EC 5.3.1.9) responsible for the reversible isomnerisation of D-glucose6-phosphate and D-fuctcose-6-phosphate. PGI participates as the second enzyme in the glycolytic pathway. The work developed so far resulted in the expression of the recombinant form of the parasite PGI, its affinity purification and enzimatic characterization. The specific activity was determined with established methods. The IC50 of four inhibitors was determined and a structural model of T brucei PGI was built by molecular modeling techniques.