Name: Fabiano Torres Cruz
Type: MSc dissertation
Publication date: 28/03/2018
Advisor:

Namesort descending Role
Alexandre Martins Costa Santos Co-advisor *
Juliana Barbosa Coitinho Goncalves Advisor *

Examining board:

Namesort descending Role
Alexandre Martins Costa Santos Co advisor *
Fabiana Vasconcelos Campos Internal Examiner *
Juliana Barbosa Coitinho Goncalves Advisor *
Mariana Amalia Figueiredo Costa External Examiner *

Summary: Paracoccidioidomycosis (PCM), a systemic mycosis caused by fungi of the genus Paracoccidioides, is endemic in Latin America, with Brazil being the most affected country. Currently, treatment with traditional antifungals is usually long, costly, restricted to a few classes of drugs and with cases of resistance already reported. The catechol O-methyltransferase (COMT) protein, which has no structure solved in Paracoccidioides or other fungi, catalyzes the methylation of a catechol substrate using S-adenosylmethionine and Mg2+ ions as cofactors and appears to participate in important processes in pathogenic fungi. Thus, its biochemical and structural characterization could contribute to the development of new and more effective drugs for the treatment of PCM. To study COMT, its coding sequence was cloned into the pET-28aTEV vector, which adds an N-terminal histidine-tag (His-tag) to the expressed protein. The recombinant vector was then inserted into the strains of Escherichia coli BL21, ArcticExpress, Rosetta and Rosetta-gami. Recombinant COMT was expressed in the soluble fraction of ArcticExpress and it was subjected to nickel affinity chromatography followed by gel filtration (Ultrahydrogel and Superose 12 columns). Western blot with anti-Histag antibody showed the two major bands observed on SDS-PAGE (30 and 60 kDa) of the fraction obtained after affinity. The gel filtration profile showed three main peaks (fractions 1, 2 and 3) which, on SDS-PAGE, showed the presence of a prominent band near 60 kDa. Samples from the three fractions were used in an activity assay to evaluate the catechol consumption at 35 ºC and pH 7.5, and fraction 2 showed a higher activity/protein ratio. Theoretical calculations of the masses of gel filtration chromatography peaks indicate that COMT behaves as a dimer. Fraction 2, although not pure, was subjected to thermal denaturation test, WHERE a process of aggregation or denaturation was detected at temperatures above 320 K (~47 ° C). Circular dichroism of fraction 2 indicated an α-helix and β-sheet profile different from other COMT. An in silico model has also been generated and the structural data obtained can contribute as a guide for future biochemical characterization.

KEY WORDS: Paracoccidioides, Paracoccidioidomicose, PCM, catechol O-methyltransferase, COMT.

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