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bioRxiv · 10.1101/2025.11.24.690144

Deciphering the Structure and Mechanism of SaGpx: A Non-Canonical Glutathione Peroxidase from Staphylococcus aureus

Maji, S.·Shukla, M.·Bhattacharyya, S.
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Abstract

Staphylococcus aureus encounters massive oxidative stress during infection. To counter this, the bacterium developed robust antioxidative defense mechanism. Glutathione peroxidases (Gpx) are well characterized antioxidative enzymes in eukaryotes; however, their bacterial counterparts remain poorly explored. S. aureus possesses two putative Gpx genes but lacks GSH biosynthetic machinery and glutathione reductase required for canonical Gpx function, suggesting alternate electron donor system(s) may be involved. This study aimed to elucidate structure-based biochemical characterization of one of the S. aureus glutathione peroxidases homologs (SaGpx, Uniprot Id: Q2FYZ0) and identify its plausible electron donor system. Herein, we cloned, purified and determined the high-resolution crystal structure of SaGpx (1.5 [A] resolution) using X-ray diffraction crystallography. In vitro biochemical characterization of the highly conserved active site amino acid point mutants, as well as their structural disposition suggests their precise roles in the enzymes catalysis. The crystal structure of SaGpx revealed that the enzyme adopts a canonical glutathione peroxidase fold with conserved catalytic tetrad composed of C36, Q70, W124 and N125. Also, SaGpx shows similarity with mammalian Gpx4, which was previously shown to exert phospholipid hydroperoxide peroxidase activity. Furthermore, biochemical assays suggest that SaGpx utilizes Staphylococcal thioredoxin1 as its cognate electron donor. The catalytic mechanism follows an atypical 2-cysteine peroxiredoxin-like pathway involving the formation of a sulfenic acid intermediate, followed by an intramolecular disulfide bond subsequently resolved by thioredoxin. 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.

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Maji, S., Shukla, M., Bhattacharyya, S.. 2025-11-24. Deciphering the Structure and Mechanism of SaGpx: A Non-Canonical Glutathione Peroxidase from Staphylococcus aureus. https://doi.org/10.1101/2025.11.24.690144

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