Development of High-Performance Nitrile Hydratase Whole-Cell Catalyst by Automated Structure- and Sequence-Based Design and Mechanism Insights

Nitrile hydratase (NHase) is a metalloenzyme that catalyzes the conversion of nitrile to amide and is widely used in the biocatalysis of bulk chemicals such as acrylamide and nicotinamide. Improving the thermostability, activity, and soluble expression of natural NHase is crucial for its industrial application. However, conventional engineering strategies are often based on the design and evaluation of single-point mutations, followed by multiple rounds of iterative combinations, which are inefficient and difficult to predict the evolutionary direction of the combinatorial mutations due to epistatic effects. In this study, we used PROSS, an automated design tool based on structural and sequence information, to design a thermophilic NHase from Pseudonocardia thermophila JCM3095 (PtNHase). By sequentially applying subunit-independent mutations, subunit-synergistic mutations, and single-point revertant mutations, we obtained the superior mutant A2B1–β221. This mutant exhibited 1.4-fold and 2.3-fold higher activity towards acrylonitrile and 3-cyanopyridine, respectively, compared to the wild type. Additionally, A2B1–β221 showed a significant enhancement in thermostability. Moreover, benefiting from the enhanced soluble expression, a high-performance whole-cell catalyst for NHase was obtained. Furthermore, conventional molecular dynamics simulations and metadynamics simulations were employed to resolve the molecular mechanisms underlying the high activity and thermostability of A2B1–β221. This study not only provided highly efficient whole-cell catalyst for NHase, but also demonstrated the efficacy of utilizing automated design tools and molecular dynamics simulations in the engineering of heterologous multimeric proteins, offering valuable insights into their applicability.