Abstact

Thioredoxin-fold oxidoreductases drive oxidative protein folding and redox homeostasis across all domains of life. They catalyse thiol-disulfide exchange in diverse substrates, yet how they reconcile catalytic precision with substrate diversity remains unclear. Here we show, using high-resolution structures and functional analyses of the Escherichia coli oxidoreductase DsbA, that a conserved cis-proline loop adjacent to the catalytic Cys-Pro-His-Cys motif serves as a universal catalytic lock. The loop positions the substrate cysteine in a right-handed disulfide geometry optimal for exchange, while surrounding surfaces accommodate sequence variation. Substitution of the cis-proline abolishes turnover, whereas mutation of the preceding glycine preserves geometry but reduces efficiency. Comparative structural analyses demonstrate that this cis-proline-dependent hydrogen-bonding scaffold is conserved across thioredoxins, protein disulfide isomerases, peroxiredoxins and bacterial Dsb proteins. This conserved mechanism explains how catalytic fidelity is maintained while enabling substrate versatility and provides a foundation for enzyme engineering and therapeutic development.