Structural and biophysical studies of three folate pathway enzymes of pathogenic microorganisms.

Author(s)

João Henrique Pimenta Giudice

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2018-02-05

Examining board members

Marcio Vinícius Bertacine Dias; Ana Marcia de Sá Guimarães; Gustavo Fernando Mercaldi; Manoel de Arcisio Miranda Filho

• Advisor: Marcio Vinícius Bertacine Dias

Abstract

In 2015, according to the World Health Organization, the total number of deaths in the human population was 52.5 million people, in which infectious diseases - specifically, respiratory infections - appear third in the ranking of causes of death. In total, 3.5 million deaths or 6.7% of the total occurred, showing a significant increase when compared to the previous survey conducted in 2011, in which infectious diseases accounted for 3.2 million or 5.9% of the total total. Of the infectious diseases, we can highlight Malaria, HIV / AIDS, Hepatitis, Tuberculosis - which appears in 8th place in this same ranking, and Leprosy - which is an important concern in Brazil. Tuberculosis, caused by Mycobacterium tuberculosis is one of the main causes of death due to infectious diseases in the human population, and already presents resistant strains to the treatment - denominated multiresistant strains (MDR) and extremely resistant strains (XDR). While leprosy, caused by Mycobacterium leprae, despite not lethal, causes the invalidity of patients and Brazil has the second largest number of cases for this disease. Another pathogen of medical importance is Pseudomonas aeruginosa, which causes serious illness in patients with low immunity, especially those in hospitals - especially patients with burns. The Folate pathway has brought the attention of the pharmaceutical industries to the development of new drugs, since this pathway offers selectivity for these new inhibitors. This is since Tetrahydrofolate is an essential component for prokaryote organisms, and only they, need to perform the new production of this cofactor, while Eukaryotes acquire it through food. However, the emergence of resistant strains, not only reinforces the need for the development of new drugs, but also the understanding of the mechanisms of resistance of these strains. The aim of the present work was to obtain biophysical and structural information of three folate pathway enzymes, 7,8 dihydroneopterin aldolase, Dihydropteroate synthase and Dihydrofolate reductase. In addition, this work aimed to understand the mechanism of resistance through the induction of mutations at the codons 53 and 55 in the enzyme dihydropteroate synthase (DHPS), preliminary structurally characterize the DHNA enzyme and structurally study the enzyme DHFR in complex with cycloguanil. Thus, the cloning of the coding region of the gene for DHNA and the expression of the recombinant protein, purification and biophysical characterization were performed in this work. In addition, site-directed mutagenesis was performed aiming of producing mutant DHPSs (T70S and P72R), which were used in ITC, fluorescence, and crystallization tests to demonstrate that M. lepreae resistance to sulfas may be due to the exchange of a proline to an arginine at position 72. Finally, for DHFR, it was possible to obtain its structure in complex with cycloguanil which made it possible to evaluate the structural differences between the open and closed conformation of this enzyme. In addition, we have confirmed that the nicotidamide group has a crucial importance for interaction with the ligands. (AU)

• FAPESP's process: 14/24486-2 - Structural basis of sensibility of Mycobacterium tuberculosis and M. leprae dihydropteroate synthase to the sulfonamide drugs
• Grantee: João Henrique Pimenta Giudice
• Support Opportunities: Scholarships in Brazil - Master