Abstract 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.
Cunliffe et al. (Tue,) studied this question.