202 - Biochemical and Structural Characterization of 1-Cys Peroxiredoxin from the Human Opportunistic Pathogen Aspergillus Fumigatus

Abstract

Peroxiredoxin (Prx) are Cys-based, thiol-dependent peroxidases that contain one or two conserved Cys residues. To date, all characterized Prx enzymes display the universal motif PxxxT/SxxC. Here, we aim to structurally and biochemically characterize AfPrxA, a 1-Cys Prx (Prx6 subfamily) from the human opportunistic pathogen Aspergillus fumigatus . Remarkably, AfPrxA presents a unique motif among Prx enzymes (SxxxHxxC) and still displays an extraordinary reactivity towards H 2 O 2 . Indeed, the reactivity of AfPrxA towards H 2 O 2 was 3.7×10 7 M -1 s -1 as determined through a HRP competitive assay. Therefore, we are now attempting to obtain the crystal structure of AfPrxA. To date, the best diffraction pattern was obtained with AfPrxA pre-reduced, alkylated with iodoacetamide and treated with trypsin (resolution 3A); the refinement is in progress. Additionally, we are investigating the reducing system of AfPrxA, which varies for Prx6 type of enzymes in distinct organisms. Therefore, we produced recombinant enzymes of the thioredoxin (Trx) system from Aspergillus fumigatus and none of them could reduce AfPrxA. In contrast, ascorbate and dihydrolipoic acid supported the peroxidase activity of AfPrxA. Once ascorbate could reduce this protein in vitro (rate constant in the 10 3 M -1 s -1 range), we quantified the intra-cellular erytroascorbate concentration as approximately 200 µM. Therefore, erytroascorbate can act as a reducing agent of AfPrxA in vivo. To investigate the possible involvement of AfPrxA in the virulence of A. fumigatus , we constructed a knockout strain of A. fumigatus (ΔAfPrxA), and a strain expressing an AfPrxA allele without the catalytic cysteine (AfPrxAC28S). Currently, phagocytosis, CFU and TNF-α assays are under evaluation in macrophages (J774) cultures exposed to A. fumigatus strains. Acknowledgements: CEPID Redoxoma; FAPESP; Capes-CNPq.