Construction of a subunit-fusion nitrile hydratase and discovery of an innovative metal ion transfer pattern

Metallochaperones are metal-binding proteins designed to deliver the appropriate metal to a target protein. The metal is usually transferred between different proteins. In this study, we discovered that metal was transferred between the same subunit of a mutant nitrile hydratase (NHase). Various "activator proteins" mediate the trafficking of metal ions into NHases. We constructed fusion NHases by fusing the β- and α-subunits and/or the "activator proteins" of the NHase from Pseudomonas putida. The fusion NHases exhibited higher thermostability and tolerance to high concentrations of the product amide. The mechanism of the cobalt incorporation changed from a self-subunit swapping pattern to an apoprotein-specific molecular chaperone pattern in vivo and a metallochaperone pattern in vitro. Notably, the cobalt transfer occurred between the same α-subunit in the metallochaperone pattern. These results not only demonstrated the superiority of fusion-type NHases, but also revealed an innovative metal ion transfer pattern in metalloprotein biosynthesis.