Estudo sobre os mecanismos de catálise, controle e reconhecimento molecular de enzimas da via de degradação do naftaleno

• Ano de defesa: 2022
• Autor(a) principal: Mozart Silvio Pereira
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; Brasil; ICX - DEPARTAMENTO DE QUÍMICA; Programa de Pós-Graduação em Química; UFMG
• Palavras-chave em Português: Biorremediação; Enzima; Inibição; Salicilato; Truncagem; Bioremediation; Enzyme; Inhibition; Salicylate; Truncation; Química orgânica; Hidrocarbonetos policíclicos aromáticos; Naftaleno; Pseudomonas; Enzimas; Catálise; Salicilatos; Inibidores enzimáticos; Cinética de enzimas; Cromatografia de afinidade
• Link de acesso: http://hdl.handle.net/1843/50328

Naphthalene is one of the most common polycyclic aromatic hydrocarbons in the environment. The degradation of naphthalene by Pseudomonas putida G7 is due to the presence of the NAH7 plasmid encoding the higher pathway enzymes involved in the conversion of naphthalene to salicylate and the lower pathway enzymes involved in the conversion of salicylate to pyruvate and acetate. This work aimed to understand the structural and reactional characteristics of NahC, NahF, NahI, and NahG of this pathway. In relation to NahC, this work focused on studies on the NahC active site to evaluate the interaction of the iron center with the 1,2-dihydroxynaphthalene and dioxygen substrates. The spin states of the structures that best described the systems were the singlet and triplet state for the NahC-1,2-dihydroxynaphthalene and NahC-1,2-dihydroxynaphthalene-dioxygen complex, respectively. ONIOM calculations showed that residues H200, H246 and Y256 in the second coordination sphere of the metal are potentially important for the catalysis mechanism of NahC. Salicylate inhibition studies on the NahF and NahI enzymes were performed for a better understanding of the role of these two aldehyde dehydrogenases in the metabolic pathway of Pseudomonas putida G7. The inhibition profile showed a mixed type of inhibition wherein the inhibitor binds to both the substrate binding site and the cofactor, for which NahI exhibited a greater sensitivity to inhibition regard NahF. In relation to NahG, the work aimed to understand the protein dynamics and interactions with FAD affecting the enzymatic catalysis. The studies with truncated cofactors showed changes in Kd and kcat values consistent with changes in the interaction energy of the FAD cofactor. Site-directed mutation experiments in the FAD binding loop were performed to evaluate the role of important residues in both FAD binding and NahG enzyme catalysis.