Fuente: PubMed "industrial biotechnology" J Phys Chem B. 2026 Apr 1. doi: 10.1021/acs.jpcb.6c00014. Online ahead of print.ABSTRACTThe reaction mechanism of Bacillus safensis nitrilase (BsNIT) 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 (TS1) for PD and the ammonia release step (TS5) 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 BsNIT for industrial biocatalysis.PMID:41920775 | DOI:10.1021/acs.jpcb.6c00014