Quantum Molecular Dynamics Study on the Reaction Mechanism of Nitrilase toward an Aliphatic Dinitrile Substrate.

The reaction mechanism of nitrilase () toward the aliphatic dinitrile substrate pentanedinitrile (PD) was elucidated using quantum mechanical dynamics (QMD) simulations containing a cluster model having the catalytic triad (C164, K130, and E38). The elucidated reaction mechanism pathway revealed a sequential conversion of PD to 4-cyanobutanoic acid (CA) and pentanedioic acid (PA), involving two hydrolytic (2HO) steps mediated by the catalytic triad. Key intermediates and transition states were identified, highlighting water-mediated proton-transfer relays, thioimidate formation, and acyl-enzyme formation. The comparative analysis with the aromatic substrate benzonitrile (BN) showed a conserved reaction mechanism pathway but lower activation energy barriers for aliphatic substrates (PD and CA), highlighting a reduced π-electron delocalization and steric effects in aliphatic substrates. The identified rate-limiting step corresponded to the nucleophilic attack step () for PD and the ammonia release step () for CA, with relative energies of 14.2 kcal mol and 14.3 kcal mol, respectively. The elucidated reaction mechanism advances the insight and mechanistic understanding of nitrilase biocatalysis, substrate preferences, and rational understanding of the engineering of for industrial biocatalysis.