Substrate access tunnel engineering for improving the catalytic activity of a thermophilic nitrile hydratase toward pyridine and pyrazine nitriles

Nitrile hydratase (NHase) is able to bio-transform nitriles into amides. As nitrile hydration being an exothermic reaction, a NHase with high activity and stability is needed for amide production. However, the widespread use of NHase for amide bio-production is limited by an activity-stability trade-off. In this study, through the combination of substrate access tunnel calculation, residue conservative analysis and site-saturation mutagenesis, a residue located at the substrate access tunnel entrance of the thermophilic NHase from extremophile Caldalkalibacillus thermarum TA2. A1, beta Leu48, was semi-rationally identified as a potential gating residue that directs the enzymatic activity toward various pyridine and pyrazine nitriles. The specific activity of the corresponding mutant beta L48H towards 3-cyanopyridine, 2-cyanopyridine and cyanopyrazine were 2.4-fold, 2.8-fold and 3.1-fold higher than that of its parent enzyme, showing a great potential in the industrial production of high-value pyridine and pyrazine carboxamides. Further structural analysis demonstrated that the beta His48 could form a long-lasting hydrogen bond with alpha Glu166, which contributes to the expansion of the entrance of substrate access tunnel and accelerate substrate migration. (C) 2021 Published by Elsevier Inc.

Automated summary

This study identified a potential gating residue, beta Leu48, in a thermophilic NHase from an extremophile, which can enhance the enzymatic activity towards various pyridine and pyrazine nitriles. The mutation showed a significant increase in specific activity, suggesting great potential for industrial production of high-value compounds. Structural analysis revealed that the mutant residue could form a hydrogen bond that expands the substrate access tunnel entrance and accelerates substrate migration.