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

The reaction mechanism of <i>Bacillus safensis</i> nitrilase (<i>BsNIT</i>) 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 (2H₂O) 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 (<b>TS1</b>) for PD and the ammonia release step (<b>TS5</b>) for CA, with relative energies of 14.2 kcal mol^-1 and 14.3 kcal mol^-1, respectively. The elucidated reaction mechanism advances the insight and mechanistic understanding of nitrilase biocatalysis, substrate preferences, and rational understanding of the engineering of <i>BsNIT</i> for industrial biocatalysis.