N-terminal loops at the tetramer interface of nitrile hydratase act as "hooks" determining resistance to high amide concentrations.

Nitrile hydratase (NHase) has been extensively utilized in industrial acrylamide production. However, the vulnerability to high concentrations of acrylamide limits its further application. Herein, we redesigned the N-terminal loop at the tetramer interface of a thermophilic NHase from Pseudonocardia thermophila JCM3095 (PtNHase), and its catalytic activity, resistance to high acrylamide concentrations, and thermostability were improved. Amino acid residues located in the N-terminal loop of the tetramer interface that are responsible for enhancing the resistance to high acrylamide concentrations were identified via static structural analysis and molecular dynamics simulations. A variant library was used to fine-tune the tetramer interface. Variant αL6T exhibited 3.5-fold greater resistance to 50% (v/v) acrylamide, whereas its activity was 1.2-fold higher than that of the wild-type (WT) enzyme, revealing no activity-stability trade-off. Compared to the use of Escherichia coli harboring the WT enzyme, the use of E. coli harboring αL6T increased the acrylamide concentration from 398.1 g/L to 500 g/L. Crystal structure-guided analysis of αL6T and molecular dynamics simulations revealed that increased enzyme surface hydration and the introduction of positive cross-correlation into the N-terminal loop of the tetramer interface caused the two loop regions to hook to each other, thus improving the resistance to high acrylamide concentrations.