Shaping the active site from a distance: Unravelling the regulatory role of the conserved N-terminal β-flap of thiol peroxidases through structure-function characterization of the Staphylococcal ortholog

Bacterial thiol peroxidases (Tpxs) are members of atypical two-cysteine peroxiredoxin, which detoxify hydroperoxides during host-pathogen interactions. Despite extensive research on bacterial Tpxs, the precise structure-based functional characterization of Staphylococcal Tpx (SaTpx) remains elusive. Herein, we cloned and purified SaTpx along with its plausible physiological redox partners to reconstitute its electron relay system. We solved the high-resolution crystal structure of SaTpx, revealing the essential active-site catalytic triad (Cys 60, Thr 57, Arg 128). The crystal structure of SaTpx reveals the presence of the highly conserved N-terminal {beta}-flap, absent in its mammalian orthologs (Peroxiredoxin 5). To investigate the plausible role of this highly conserved {beta}-flap, we cloned and characterized a deletion mutant, N{Delta}15-SaTpx, lacking this region. N{Delta}15-SaTpx showed reduced ability to mitigate organic hydroperoxides in vitro; partially impaired to prevent ROS-mediated ds-DNA nicking and failed to protect host E. coli cells from exogenous ROS stress. The ultra-high-resolution crystal structures of N{Delta}15-SaTpx and N{Delta}15-SaTpx with substrate mimic revealed a distorted catalytic triad (with flipped Arg 128), explaining its partially impaired catalytic activity. This study, for the first time, elucidates the critical regulatory role of the N-terminal {beta}-flap in stabilizing the catalytic triad of bacterial Tpx, providing insights for future inhibitor designing. HighlightsO_LIUltra high-resolution crystal structures of SaTpx and N{Delta}15-SaTpx C_LIO_LIUltra-high resolution crystal structure of hydroperoxide substrate mimic bound N{Delta}15-SaTpx C_LIO_LI{beta}-flap deletion mediated distortion of catalytic triad in N{Delta}15-SaTpx C_LIO_LIThe novel regulatory role of the conserved N-terminal {beta}-flap of Thiol peroxidases. C_LI