Deciphering the structure and mechanism of SaGpx: A noncanonical glutathione peroxidase from Staphylococcus aureus

Glutathione peroxidases (Gpx) are well-characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. Nevertheless, bacterial Gpx homologs play pivotal role in pathogenesis. Staphylococcus aureus encodes two conserved Gpx homologs but lacks glutathione (GSH) and the glutathione reductase enzyme required for canonical Gpx function. Herein, we aimed to elucidate the structure and function of one of the S. aureus Gpx homologs (SaGpx, EC:1.11.1.9, Uniprot Id: Q2FYZ0). We solved the high-resolution (1.55 Å) crystal structure of the SaGpx C36S mutant, which demonstrates SaGpx adopts a canonical Gpx fold with a conserved catalytic tetrad composed of C36, Q70, W124 and N125. The crystal structure of SaGpx C36S, demonstrates a profound structural similarity with the mammalian Gpx4. The structure of SaGpx resembles a fully folded, reduced conformational state of the enzyme's active site with an intensive hydrogen-bonding network among the catalytic tetrad, required for the generation of cysteine thiolate nucleophile and for substrate binding. This analysis of the wild-type SaGpx and its active site amino acid mutants unravels the mechanistic details of its plausible catalytic mechanism. Instead of GSH, SaGpx prefers to utilize the reduced Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism involved the formation of a cysteine sulfenic acid intermediate, followed by the formation of an intramolecular disulfide bond (between C36 and C82), which is subsequently resolved by thioredoxin1. This work provides the first structure-based biochemical characterization of a bacterial glutathione peroxidase homolog, establishing the novel structural insights of SaGpx as a noncanonical thioredoxin-dependent glutathione peroxidase.