CYTOCHROME P450 REDUCTASES IN Trypanosoma cruzi. CLONING AND CHARACTERIZATION.

PORTAL, PATRICIO; VILLAMIL, SILVIA F.; ALONSO, GUILLERMO D.; FLAWIÁ, MIRTHA M.; TORRES, HECTOR N.; PAVETO, CRISTINA.

Pinamar, Argentina.

Congreso; X Congress of the Pan-American Association for Biochemistry and Molecular Biology (PABMB), XLI Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology (SAIB) and the XX Annual Meeting of the Argentine Society for Neurochemistry (SAN; 2005

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

Recombinants genomic sequences of Trypanosoma cruzi, ORF 639 and ORF 819 encoding for homologues of FAD and FMN containing NADPH dependent Cyt P-450 reductases have been cloned and expressed in BL 21(DE3) pLys E cells by using pRSET-A-Tc-639 and pRSET-A-Tc-819 recombinant vectors.  Their structural similarities to Cyt P-450 reductases include a NADPH, FAD, FMN, and BH4 domains. Single gene copies for both sequences have been demonstrated by Southern blot analysis. The transcription of both genes was demonstrated by RT-PCR. Northern blot analysis showed a single transcript of 1.8 kb of ORF 639.  Predicted protein molecular masses of 67 kDa and 81 kDa were corroborated by Western blot using respective polyclonal antibodies raised against the purified expressed proteins as well as against the His-tag. Both enzymes reduce Cyt C using only NADPH as cofactor. Km for NADPH was 18 mM and 17mM for TcR-639 and TcR-819 respectively. The ability for both enzymes to reduce alternatives substrates and participate in Cyt P-450 dependent dealkylation process was demonstrated in a heterologous system. Tc-639 and Tc-819 reductase activities were inhibited by the flavoprotein specific inhibitor diphenyleneiodonioum (DPI).