Unique biogenesis of carbon‐nitrogen‐cleaving enzyme harnessing a new mechanism

Nitrile hydratase (NHase) catalyzes hydration of nitrile (R‐CN) to amide. Rhodococcus rhodochrous J1 produces two NHases (H‐NHase and L‐NHase), depending on an inducer in the presence of cobalt (Co). Both enzymes have cobalt as an active center. H‐NHase is inducibly overexpressed ~50% of all soluble protein by the addition of urea to the media. It is used for the industrial production of acrylamide and nicotinamide. We discovered a novel post‐translational maturation in both NHases. The incorporation of Co into NHase depended on the α ‐subunit exchange between Co‐free NHase (apo‐NHase) and its Co‐containing mediator, this unique mechanism having been named “self‐subunit swapping” and the corresponding mediator (NhhAG [consisting of NhhG and H‐NHase α ‐subunit] and NhlAE [consisting of NhlE and L‐NHase α ‐subunit]) having been recognized as a subunit swapping chaperone. We also discovered that Co was inserted into both the Co‐free apo‐NhlAE (or apo‐NhhAG) and the Co‐free apo‐α‐subunit in an NhlE (or NhhG)‐dependent manner in the presence of Co and DTT in vitro . MALDI‐TOF MS analysis revealed that the non‐oxidized cysteine residues in apo‐NhlAE and apo‐NhhAG were post‐translationally oxidized after Co insertion. These findings suggested that both NhlE and NhhG show two activities, i.e., Co insertion and cysteine oxidation. They functioned not only as a self‐subunit swapping chaperone but also a metallochaperone that includes a redox function. Both NhlAE and NhhAG consisted of heterotrimers ( α e 2 and α g 2 ), and the resultant NhlE isolated from NhlAE exhibited a dimeric assembled form (e 2 ), indicating that the structures of α e 2 and α g 2 would be α ‐e 2 (not e‐a‐e) and α ‐g 2 (not g‐ α ‐g), respectively. Current works focus now on the elucidation of the swapping mechanism.