Study of the properties of the frataxin from Nannochloropsis gaditana

MARCHETTI-ACOSTA, NOELIA S.; BARCHIESI, JULIETA; GOMEZ-CASATI, DIEGO F.; BUSI, MARIA VICTORIA

Congreso; 3er. Reunión Red Argentina de Tecnología Enzimática (RedTez); 2021

Fe-S clusters are among the oldest and most versatile cofactors used by most living organisms. They are formed by atoms of iron and inorganic sulphide. Although they can be synthesized non-enzymatically in vitro, their biosynthesis is catalyzed by specific enzymes. Three complete systems responsible for the biosynthesis of groups [Fe-S] were discovered in bacteria: NIF (nitrogen fixation), SUF (sulphur mobilization) and ISC (iron and sulphur cluster) and in eukaryotes the presence of a ISC and SUF homologous system in mitochondria and chloroplasts, respectively. In this work we address the initial characterization of this pathway in algae. First, we performed the identification in silico of gene and protein sequences possibly related to the biogenesis of Fe-S clusters in green and brown algae (Nannochloropsis gaditana, Chlorella vulgaris and Ectocarpus siliculosus) using the Phytozome database (https://phytozome.jgi.doe.gov). After performing sequence alignments and considering the high conservation found between the amino acid sequences, the frataxin from N. gaditana (NangaFH) was selected to perform its functional characterization. The sequence of NangaFH contains 204 amino acids, a mitochondrial targeting transit peptide of 82 residues and a frataxin domain comprised between residues 94 to 199. Within this domain the iron-binding sites are conserved (E95, D103, E104, D107, A111, D114, A115, D122 and E124). The recombinant protein was expressed in E. coli cells and purified to homogeneity. We evaluated their ability of NangaFH to attenuate the Fenton reaction by measuring the inhibition of malondialdehyde production after the addition of thiobarbituric acid. As previously suggested, frataxin could function as iron chaperone and in this way, its presence could attenuate oxidative damage by metals. Results showed that NangaFH did not attenuated the Fenton reaction in contrast to the frataxins from C. vulgaris and A.thaliana (both proteins attenuated the Fenton reaction about 23%). To determine whether NangaFH overexpression decreases the sensitivity of E. coli cells to oxidative and metal stress, bacterial cells overexpressing this protein were incubated in the presence of hydrogen peroxide, chromium and zinc. Our results showed that the expression of NangaFH allowed a better growth of E. coli cells under oxidative conditions. These results suggest that algae frataxin would have a protective role against oxidative stress in algae