Thioredoxin-fold (TRX) oxidoreductases are universal catalysts that mediate oxidative protein folding and maintain redox balance across all domains of life. These enzymes act on a vast range of structurally and functionally diverse protein substrates yet perform their catalysis with remarkable precision, and how they achieve this has remained unresolved. Using Escherichia coli DsbA (EcDsbA) as a model TRX-fold enzyme, we captured high-resolution structures of EcDsbA in complex with a peptide derived from its physiological substrate LptD, an essential outer-membrane protein involved in lipopolysaccharide transport. These structures represent rare snapshots of transient enzyme–substrate intermediates, the structures revealed the LptD peptide engages EcDsbA within the hydrophobic groove near the catalytic C-P-H-C motif, diverging from the previously understood binding modes of EcDsbA substrates, the binding of LptD also prompted a induced subtle movement within the structure to accommodate bulky residues, suggesting a dynamic mechanism in which local flexibility and hydrophobic work to diversify EcDsbAs substrate range.
Despite this flexibility, the structure showed a conserved cis-proline “lock” adjacent to the catalytic motif remained rigid in all complexes. This element anchors substrate cysteines through backbone hydrogen bonds, locking them into the precise geometry required for disulphide bond formation. This simple yet elegant structural solution reconciles catalytic precision with substrate diversity. Interestingly, this same arrangement was found to be is conserved across thioredoxins, protein disulphide isomerases, peroxiredoxins, and other bacterial Dsb proteins, suggesting universal catalytic principle preserved throughout evolution. By uncovering this mechanism, our work clarifies why the TRX fold is one of nature’s most successful enzyme architectures. This provides a framework for engineering redox catalysts or designing drugs that target TRX-like enzymes involved in infection, cancer, and neurodegenerative disease.