Unraveling the Structure–Function Relationship and Mechanism of an Important Spiro‐Forming Nitrilase Using Metadynamics and Quantum Molecular Dynamics

The nitrilase from Bacillus safensis (BsNIT) is a spiro-forming enzyme with significant potential in the bioremediation of nitrile pollutants such as benzonitrile and glutaronitrile. Despite its environmental and industrial relevance, its structure-function relationships and mechanistic details remain poorly understood. This study employs metadynamics and quantum molecular dynamics (QMD) simulations to delineate BsNIT's structure-function relationships with relevant substrates. Metadynamics simulations identified distinct substrate association and dissociation pathways, with the T1 tunnel emerging as the primary diffusion route for substrates and products. Tyrosine-gated residues within the tunnel, alongside conserved active site residues, were crucial for orienting nitrile substrates and enabling efficient binding. Comparing BsNIT to Spirosoma linguale DSM 74 (SINIT) provides a clearer understanding of how variations in active site architecture and mechanisms, particularly the events revealed in our QM studies, favor certain nitrilases for amide formation while others preferentially catalyze hydrolysis. QMD simulations further revealed mechanistic insights, including Cys164's nucleophilic attack and Glu48's proton hopping via a water-mediated relay, which plays a critical role for nitrile hydrolysis. The critical transition state (TS1), corresponding to covalent substrate binding, exhibited an energy barrier of 14.8 kcal mol^-1, defining it as the rate-limiting step. Based on these studies' key mutations in the tunnel gating residues (Y276, Y278, and Y279) and mutations of salt-bridge residues (R67-D275, K75-E271, and K68-E229) are proposed to enhance BsNIT's substrate specificity for more bulky nitrile pollutants with increased efficiency. This computational analysis highlighted BsNIT's structural adaptations for catalytic efficiency, particularly in its interactions with benzonitrile and glutaronitrile. The study provides mechanistic insights into substrate binding, product release, and active site dynamics, and a comparative study with amide-forming nitrilase SlNIT for enhancing our understanding of how BsNIT's structure facilitates its function. These insights pave the way for the development of engineered BsNIT variants with enhanced activity and specificity toward specific nitrile pollutants, potentially leading to more effective bioremediation strategies.