Catechol-O-methyltransferase from Paracoccidioides spp.: advances in biochemical and structural characterization

Name: Marielly Moura MartinsType: MSc dissertationPublication date: 30/03/2022Advisor:

Namesort descending Role
Juliana Barbosa Coitinho Goncalves Advisor *

Examining board:

Namesort descending Role
Débora Maria Abrantes Costa External Examiner *
Juliana Barbosa Coitinho Goncalves Advisor *
Renato Graciano de Paula Internal Examiner *

Summary: Fungi of the genus Paracoccidioides are causative agents of systemic mycosis Paracoccidioidomycosis (PCM), which primarily affects the lungs and may affect other tissues. PCM is endemic in Latin America and Brazil is the country that accounts for 80% of reported cases, followed by Colombia and Venezuela. The treatment is carried out with antifungal agents and is prolonged, expensive and with side effects, which discourages patients from following it, increasing the recurrence of the disease, in addition to the already reported cases of resistance. Thus, the search for new pharmacological targets remains important. The protein catechol-O-methyltransferase (COMT) may be a possible pharmacological target for the treatment of PCM because in other fungi it seems to participate in its survival in the host, however, in Paracoccidioides it has never been studied and its three-dimensional structure has not been resolved. COMT has low genetic variability among Paracoccidioides species and low similarity to human COMT. For this, Escherichia coli Rosetta(DE3) and Arctic Express bacteria were transformed with the expression vector pET28a(TEV) containing the comt gene were used for recombinant expression of this protein in order to obtain it in large quantity and functional. Different expression, lysis and affinity purification conditions were evaluated. After evaluation of different protocols, COMT showed soluble expression, albeit in small amounts, both when expressed in E. coli Arctic Express bacteria and in Rosetta. In E. coli Arctic Express, expression was confirmed by Western blot using anti-His antibody. In addition, samples obtained from the two strains and purified by affinity chromatography showed catechol-O-methyl-transferase activity. In silico analyzes were performed and showed that the three-dimensional structure of the COMT of Paracoccidioides predicted by the I-TASSER server presents the typical folding of O-methyltransferases with little divergence between different MTs identified in the PDB (especially located in the loop regions). Furthermore, by structural alignments, amino acid residues that interact with the substrate and with the COMT cofactors were also predicted and can guide docking studies for the design of inhibitors for the protein. With the characterization of COMT, it is intended that it will be possible to better understand the biology of fungi of the genus Paracoccidioides and create solid knowledge that can be used for the development of new and more powerful drugs, contributing to diversify the available antifungal arsenal.

Keywords: catechol-O-methyltransferase, Paracoccidioides, heterologous expressionAccess to document

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