ESTUDOS ESTRUTURAIS E FUNCIONAIS DAS PROTEÍNAS ALANINA RACEMASE ISOFORMA LONGA DE Trypanosoma cruzi E GLICERALDEÍDO-3-FOSFATO DESIDROGENASE DE Naegleria gruberi

• Ano de defesa: 2017
• Autor(a) principal: Machado, Agnes Thiane Pereira
• Orientador(a): Iulek, Jorge
• Banca de defesa: Thiemann, Otávio Henrique , Silber, Ariel Mariano , Galvão, Carolina Weigert , Pereira, Romaiana Picada
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: UNIVERSIDADE ESTADUAL DE PONTA GROSSA
• Programa de Pós-Graduação: Programa Associado de Pós-Graduação em Química - Doutorado
• Departamento: Físico Química, Química Analitica, Química Organica, Química Inorgânica
• País: BR
• Palavras-chave em Português: Alanina racemase isoforma longa; Trypanosoma cruzi; Gliceraldeído-3-fosfato desidrogenase; Naegleria gruberi; análise estrutural; caracterização cinética
• Palavras-chave em Inglês: Alanine racemase long isoform; Trypanosoma cruzi; Glyceraldehyde-3-phosphate dehydrogenase; Naegleria gruberi; structural analysis; kinetic characterization
• Área do conhecimento CNPq: CNPQ::CIENCIAS EXATAS E DA TERRA::QUIMICA
• Link de acesso: http://tede2.uepg.br/jspui/handle/prefix/13

Study of protein three-dimensional structures allow us to investigate the relations between amino acid sequence, structure and function, what is important chiefly for proteins from pathogenic organisms or ones that belong to the same genus of these, such that they can be used as a structural model. In this context, this work aims at the structural characterization of the enzymes alanine racemase long isoform from Trypanosoma cruzi and glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi. The long isoform of alanine racemase catalyzes the conversion between L and D-alanine which, in turn, is part of one of the metabolic pathways in Trypanosoma cruzi, the etiologic agent of Chagas disease. The heterologous expression of this enzyme in Escherichia coli BL21 (DE3) GroEL was analyzed by SDS-PAGE, which revealed that the protein is in higher proportion in the insoluble fraction, thus it was necessary to establish a recovery protocol followed by an in vitro refolding. Data from enzymatic assays and circular dichroism revealed the success of the recovery/refolding protocol, which may in the future contribute to the search for specific inhibitors. Glyceraldehyde-3-phosphate dehydrogenase from Naegleria gruberi catalyzes the sixth step of the organism’s glycolytic pathway. NgGAPDH enzyme was expressed in E. coli (DE3) using the pET-15b vector, and then purified by tree chromatographic steps, two of nickel affinity and one of size exclusion. The enzymatic characterization was investigated with the enzyme without the his-tag; NgGAPDH presented higher activity at pH 8.0, 25 °C and 10 mM of arsenate, and positive cooperativity for substrates G3P and NAD+. His-tag depleted NgGAPDH crystals appeared in 3 days after drop settings, the best crystal diffracted to 1.94 A resolution and belongs to space group P21 with cell parameters a = 83.74 A, b = 94.55 A, c = 90.93 A, = 99.96 °. The final refined structure presents R = 0.1652 and Rfree = 0.2029. The catalytic domain formed by residues 134 to 313 is highly conserved, as expected, with the exception of Asn145, present only in NgGAPDH, while the other GAPDHs present either Ser or Thr on the corresponding position. Molecular dynamics analysis revealed that Asn145 has correlated motion with residues Ala123, Thr125 and Pro126 that belong to what was called "bonded loop". It should be emphasized that this is the first GAPDH from the phylum Percolozoa that has its three dimensional structure determined and kinetic parameters established, such that we expect to have contributed to the understanding of the evolution of this class of proteins.