Trypanosoma cruzi cytochrome P450 reductases: structural and biological characterization.

PORTAL PATRICIO; DE VAS MATÍAS; SCHLESINGER MARIANA; FERNÁNDEZ VILLAMIL SILVIA; ALONSO GUILLERMO; TORRES HÉCTOR; FLAWIÁ MIRTHA; PAVETO CRISTINA.

Villa Carlos Paz. Cordoba, Argentina.

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).; 2008

Three different Trypanosoma cruzi genes codifying for cytochrome P450 reductases named TcCPR-A, TcCPR-B and TcCPR-C have been identified in our laboratory. The aminoacidic sequences present the characteristic binding domains to FMN, FAD and NADPH, with 11% identity, differing mainly in their amino-end. The recombinant enzymes expressed in bacteria demonstrated characteristic NADPH dependent cytochrome c reductase and dealkylkase activity. To produce high amounts of soluble recombinant proteins we coexpressed them in combination with chaperones. The soluble proteins were used to perform biochemical and structural characterization and to produce polyclonal sera against the TcCPRs. The Ni-NTA agarose purified proteins exhibited typical flavoprotein visible absorbance spectra. Subcellular localization was analyzed by Western blot and confocal microscopy using the specific antiserum. The expression vectors pTREX and pTEX-GFP, were used to generate the following constructions: pTREX-TcCPR-A, pTREX-TcCPR-B, pTREX-TcCPR-C and pTEX-TcCPR-C-GFP. Epimastigotes of the strain CL Brenner were transfected with these constructions and overexpression assessed by Southern, Northern and Western blot. TcCPR-B overexpression confers augmented resistance to the trypanocidal Nifurtimox and Benznidazole. We are currently establishing the redox state and survival in parasites challenged with these drugs.