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Previous issue date: 2011-04-15 === Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES === In 2009, tuberculosis (TB) was responsible for 1.3 million deaths worldwide. The incidence rates reached 9.4 millions and the World Health Organization (WHO) estimative indicates that one third of the world population is infected by Mycobacterium tuberculosis, the main agent responsible for the disease. The lack of new drugs released on market, the long period treatment presenting side effects (causing the abandon by the patients) and the cases with HIV co-infection contributed to the appearance of multi drug resistant strains (MDR-TB) and
extensively drug resistant strains (XDR-TB). It's clear, thus, that the development of new drugs to fight TB is necessary and fundamental to the success in eradicating this disease. The histidine biosynthesis pathway emerge in this context offering attractive targets, given that its present in prokaryotes, lower eukaryotes and plants, but absent in animals. The last enzyme in the route is called Histidinol Dehydrogenase and is responsible for the conversion of L-Histidinol into LHistidine. Its essentiality to the bacilli was confirmed by gene knockout, confirming its potential for the development of inhibitory compounds. In this work, a
purification protocol was developed, producing the enzyme in the homogeneous form in quantities sufficient to carry its biochemical characterization. The enzyme needs a divalent metal ion in the active site to catalyze the reaction. The kinetic constants were determined, as well as the mechanism, the pH rate profiles and the interaction of its substrates and products by isothermal titration calorimetry. A tridimensional model for its structure was constructed by sequence homology, allowing the analysis of the interaction of the substrates and metal in the active site. The results obtained will allow the rational design of molecules that act as inhibitors. === Em 2009 a tuberculose (TB) foi respons?vel por 1,3 milh?es de mortes no mundo inteiro. A incid?ncia de casos chegou ao patamar de 9,4 milh?es e as estimativas da Organiza??o Mundial da Sa?de (OMS) indicam que aproximadamente 1/3 da popula??o mundial est? infectada pelo Mycobacterium tuberculosis, principal agente causador da doen?a. A falta de novas drogas no mercado, o tratamento longo e com efeitos colaterais (levando ao abandono por parte dos pacientes) e os quadros de co-infec??o com HIV tem colaborado para o
surgimento de novas cepas resistentes as drogas atualmente em uso (MDR-TB e XDR-TB). Fica claro, portanto, que o desenvolvimento de novas drogas para o combate da TB ? necess?rio e fundamental para que se tenha sucesso na erradica??o desta doen?a. A via de bioss?ntese de histidina aparece nesse contexto oferecendo alvos atrativos, visto que est? presente em organismos procari?ticos, em organismos eucari?ticos inferiores e em plantas, mas ausente em animais. A ?ltima enzima pertencente ? via ? chamada de Histidinol
Desidrogenase e ? respons?vel pela convers?o de L-Histidinol em L-Histidina. Sua essencialidade para a viabilidade do bacilo foi comprovada atrav?s de nocaute g?nico, confirmando sua potencialidade para o desenvolvimento de compostos inibidores de sua atividade. Neste trabalho, um protocolo depurifica??o foi desenvolvido, produzindo a enzima na forma homog?nea em quantidades suficientes para realizar a caracteriza??o bioqu?mica da mesma. A enzima necessita de um ?on met?lico divalente no s?tio para catalisar a rea??o. Suas constantes cin?ticas foram determinadas, assim como o mecanismo, os perfis de pH, e a intera??o com os substratos e produtos atrav?s de calorimetria de titula??o isot?rmica. Um modelo tridimensional da sua estrutura foi constru?do por homologia de sequ?ncia, permitindo uma an?lise da intera??o dos substratos e do metal no s?tio ativo da enzima. Os resultados obtidos permitir?o o desenho
racional de mol?culas que atuem como inibidores.
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