Fumarate hydratases (FHs, fumarases) catalyze the reversible conversion of fumarate into l‐malate. FHs are distributed over all organisms and play important roles in energy production, DNA repair and as tumor suppressors. They are very important targets both in the study of human metabolic disorders and as potential therapeutic targets in neglected tropical diseases and tuberculosis. In this study, human FH (HsFH) was characterized by using enzyme kinetics, differential scanning fluorimetry and X‐ray crystallography. For the first time, the contribution of both substrates was analyzed simultaneously in a single kinetics assay allowing to quantify the contribution of the reversible reaction for kinetics. The protein was crystallized in the spacegroup C2221, with unit‐cell parameters a = 125.43, b = 148.01, c = 129.76. The structure was solved by molecular replacement and refined at 1.8 Å resolution. In our study, a HEPES molecule was found to interact with HsFH at the C‐terminal domain (Domain 3), previously described as involved in allosteric regulation, through a set of interactions that includes Lys 467. HsFH catalytic efficiency is higher when in the presence of HEPES. Mutations at residue 467 have already been implicated in genetic disorders caused by FH deficiency, suggesting that the HEPES‐binding site may be important for enzyme kinetics. This study contributes to the understanding of the HsFH structure and how it correlates with mutation, enzymatic deficiency and pathology.

Nifurtimox and benznidazole represent the only treatments options available targeting Chagas disease, the most important parasitic infection in the Americas. However, use of these is problematic as they are toxic and ineffective against the more severe stages of the disease. In this work, we used a multidisciplinary approach to characterise the fumarases from Trypanosoma cruzi, the causative agent of Chagas Disease. We showed this trypanosome expresses cytosolic and mitochondrial fumarases that via an iron-sulfur cluster mediate the reversible conversion of fumarate to S-malate. Based on sequence, biochemical properties and co-factor binding, both T. cruzi proteins share characteristics with class I fumarases, enzymes found in bacteria and some other protozoa but absent from humans, that possess class II isoforms instead. Gene disruption suggested that although the cytosolic or mitochondrial fumarase activities are individually dispensable their combined activity is essential for parasite viability. Finally, based on the mechanistic differences with the human (host) fumarase, we designed and validated a selective inhibitor targeting the parasite enzyme. This study showed that T. cruzi fumarases should be exploited as targets for the development of new chemotherapeutic interventions against Chagas disease.

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Human fumarase (HsFH) is a well-known citric acid cycle enzyme and is therefore a key component in energy metabolism. Genetic studies on human patients have shown that polymorphisms in the fumarase gene are responsible for diseases such as hereditary leiomyomatosis and renal cell cancer. As a first step in unravelling the molecular basis of the mechanism of fumarase deficiency in genetic disorders, the HsFH gene was cloned in pET-28a, heterologously expressed in Escherichia coli, purified by nickel-affinity chromatography and crystallized using the vapour-diffusion technique. X-ray diffraction experiments were performed at a synchrotron source and the structure was solved at 2.1 Å resolution by molecular replacement.

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PEREIRA DE PÁDUA, R. A. Structural and functional characterization of Trypanosoma cruzi fumarate hydratase isoforms. 119 f. Thesis (Doctorate). School of Pharmaceutical Sciences of Ribeirão Preto -University of São Paulo, Ribeirão Preto, 2014.Trypanosoma cruzi is a flagellate protozoan parasite that infects humans and causes Chagas disease, a tropical neglected disease that affects millions of people worldwide. Fumarate hydratases (FH), or fumarases, are enzymes responsible for the reversible stereo-specific hydration of fumarate into S-malate, and were recently considered to be essential to Trypanosoma brucei viability, suggesting, therefore, a potential role for FHs as macromolecular targets to the drug development against trypanosomatids. The present work focused on the functional, biochemical, biophysical and structural characterization of T. cruzi fumarases (TcFHs) and human fumarase (HsFH) to evaluate TcFHs role for T. cruzi, map the reaction mechanism and identify and exploit differences between the parasite and host enzymes in order to design selective inhibitors to the parasite enzyme. Sequence analysis revealed that TcFHs belong to class I fumarases (dimeric and iron-sulfur containing enzymes) and are not homologous to HsFH which belongs to class II fumarases (tetrameric iron independent enzymes). Cellular sub-localization studies confirmed the presence of a cytosolic and a mitochondrial fumarases in T. cruzi and gene knockout experiments suggested TcFHs are essential to the parasite. The kinetic characterization showed that TcFHs activity is highly sensitive to oxygen whereas HsFH activity remained stable in aerobic conditions. Electron paramagnetic experiments further revealed the presence of an iron-sulfur cluster highly sensitive to oxidation and involved in the catalytic mechanism in both TcFHm and TcFHc. TcFHs structural models, built by homology modeling using the Leishmania major fumarase crystal structure as template, were compared to the HsFH crystal structure and the differences were used to design a selective ligand to the parasite fumarases. The designed ligand showed to inhibit TcFHc with an IC 50 of 1 µM and showed no effect on the human fumarase activity. In vivo assays using T. cruzi epimastigotes demonstrated the trypanocidal effect of the designed inhibitor probably caused by stalling ATP production. The results obtained with the development of this project represent an innovative proposal on the development of new therapies against Chagas disease, the use of fumarase enzyme as a macromolecular target, as well as present a potent and selective inhibitor to the parasite enzyme to be further used as a prototype in the development of drugs against Chagas disease. The synthesis of inhibitor analogues with optimized pharmacological properties are currently in progress.

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