Pseudomonas fluorescens SBW25 is a gram-negative soil bacterium capable of growing on histidine as the sole source of carbon and nitrogen. Expression of histidine utilization (hut) genes is controlled by the HutC repressor with urocanate, the first intermediate of the histidine degradation pathway, as the direct inducer. Recent genome sequencing of P. fluorescens SBW25 revealed the presence of hutD in the hut locus, which encodes a highly conserved hypothetical protein. Previous genetic analysis showed that hutD is involved in hut regulation, in such a way that it prevents overproduction of the hut enzymes. Deletion of hutD resulted in a slow growth phenotype in minimal medium with histidine as the sole carbon and nitrogen source. While the genetic evidence supporting a role of hutD in hut regulation is strong, nothing is known of the mechanism of HutD action. Here I have cloned and expressed the P. fluorescens SBW25 hutD in E. coli. Purified HutD was subjected to chemical and structural analysis. Analytic size-exclusion chromatography indicated that HutD forms a dimer in the elution buffer. The crystal structure of HutD was solved at 1.80 Å (R = 19.3% and Rfree = 22.3%) by using molecular replacement based on HutD from P. aeruginosa PAO1. P. fluorescens SBW25 HutD has two molecules in an asymmetric unit and each monomer consists of one subdomain and two ß-barrel domains. Comparative structural analysis revealed a conserved binding pocket. The interaction of formate with a highly conserved residue Arg61 via salt-bridges in the pocket suggests HutD binds to small molecules with carboxylic group(s) such as histidine, urocanate or formyl-glutamate. The hypothesis that HutD functions via binding to urocanate, the hut inducer, was tested. Experiments using a thermal shift assay and isothermal titration calorimetry (ITC) analysis suggested that HutD binds to urocanate but not to histidine. However, the signal of HutD-urocanate binding was very weak and detected only at high urocanate concentration (53.23 mM), which is not physiologically relevant. The current data thus does not support the hypothesis of HutD-urocanate binding in vivo. Although the HutD-urocanate binding was not confirmed, this work has laid a solid foundation for further testing of the many alternative hypotheses regarding HutD function.

Made available in DSpace on 2017-07-24T19:38:13Z (GMT). No. of bitstreams: 1 SHEILA BOREIKO.pdf: 2469578 bytes, checksum: d94618ec9a9eb25acc74ab5a7a6ca5d3 (MD5) Previous issue date: 2014-03-26 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / Chagas' disease, caused by the protozoan Trypanosoma cruzi, is one of the seventeen neglected diseases according to World Health Organization. In the last two decades, this parasite specific metabolic pathways have been evaluated as therapeutic targets, making the prospect for the development of more specific and less toxic drugs. To achieve this goal, there is the need for studies to get knowledge on the pathway protein three dimensional structures.Protein structures can be studied experimentally by the X ray diffraction technique and computationally by homology modeling, however, other structural information can also be obtained by spectroscopic techniques. Thus, in this work, structural studies of the enzyme Urocanate Hydratase from Trypanosoma cruzi (TcUH), which participates in the histidine metabolic pathway, were carried out. The enzyme was expressed functionally in E. coli and,by affinity chromatography, effectively purified and crystallized, however, no minimum quality for X-ray diffraction was observed. Thus, we carried out the structural study by circular dichroism (CD), small angle X-ray scattering (SAXS) and homology modeling. The TcUH is mainly composed of α-helices and its denaturation process by temperature starts near 50 ° C, being irreversible after completed. The SAXS study indicated that the protein in solution was not monomeric. With the homology produced model, docking studies indicated that some promising molecules to be carefully studied for possible inhibition tests. / A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, é uma das dezessete doenças negligenciadas de acordo com a Organização Mundial de Saúde. Nas últimas duas décadas, vias metabólicas específicas deste parasita têm sido avaliadas como alvos terapêuticos, o que abre perspectivas para o desenvolvimento de medicamentos mais específicos e menos tóxicos. Para alcançar este objetivo, há a necessidade de estudos para conhecimento da estrutura tridimensional de proteínas que fazem parte destas vias. As estruturas das proteínas podem ser estudadas experimentalmente pela técnica de difração de raios X e computacionalmente pela modelagem por homologia, porém, outras informações estruturais também podem ser obtidas por técnicas espectroscópicas. Sendo assim, realizaramse, neste trabalho, estudos estruturais com a enzima Urocanato Hidratase de Trypanosoma cruzi (TcUH), que participa da via metabólica da histina. A enzima foi expressa em E. coli de forma funcional e, por meio de cromatografia de afinidade, purificada efetivamente e cristalizada, porém, não apresentou qualidade mínima para análise por difração de raios X. Assim, realizou-se o estudo estrutural por meio de dicroísmo circular (CD), espalhamento de raios X a baixo ângulo (SAXS) e modelagem por homologia. A TcUH é constituída majoritariamente por hélices-α e seu processo de desnaturação térmica inicia-se próximo a 50 °C, sendo irreversível após completa. O estudo de SAXS indicou que em solução a enzima não se apresenta monomérica. Com o modelo produzido por homologia, que apresentou razoáveis índices de qualidade, os estudos de docagem indicaram algumas moléculas promissoras que deverão ser estudadas criteriosamente para possíveis testes de inibição.