From past to present: resurrection and structural analysis of ancestral nodes of the peroxiredoxin protein family

ALDANA GOMEZ; BELEN CABRAL; RAMIRO LLOVERA; ALAN WOODLEY; AGUSTIN ORMAZABAL; PAMELA L. TOLEDO; YOSUKE HOSHINO; ERIC A. GAUCHER; DIEGO S. VAZQUEZ

Cordoba

Congreso; LI Reunión Anual de la Sociedad Argentina de Biofísica; 2023

Sociedad Argentina de Biofísica

Peroxiredoxins (Prxs) are a family of key enzymes for the reduction of reactive oxygen and nitrogen species generated by normal cellular metabolism. Their activity depends on at least one absolutely conserved cysteine (peroxidatic Cys or CP) located in the primary motif PXXX(T/S)XXCP, while some Prxs subfamilies possess a second cysteine, called resolving cysteine (CR), which forms an inter- or intramolecular disulfide bond with CP. Prxs exhibit a great versatility and adaptation to different chemical substrates, oligomeric states and functionality, raising the question of how these antioxidant enzymes evolved from the ancestral nodes. To this end, we resorted to the reconstruction and resurrection of some key ancestral sequences of the main nodes to structurally characterize them by in vitro and in silico assays.The three-dimensional structures were modeled with AlphaFold. The best models were tested through molecular dynamics simulations in different chemical states of CP. The results of these simulations, along with data from the ConSurf database, were used to identify conserved residues in the active site. These conserved residues were important for analyzing cavities and transport of two significant substrates of Prxs: hydrogen peroxide and peroxynitrite. Furthermore, recombinant expression and purification were optimized. The resulting high-purity proteins were subjected to a structural characterization including the oligomeric state at different pHs, fine secondary structure content by circular dichroism and fluorescence, thiol quantification and peroxidase activity.Our results suggest that the ancestral sequences exhibit properties of correctly folded proteins under the conditions tested. Bioinformatic analysis reveals that both the position of CP and Arg are highly conserved and the active site cavities are smaller in size compared to modern Prxs, suggesting that both substrate entry and exit are energetically favored in the ancestral nodes.