Enhancing the catalytic efficiency of nitrilase for sterically hindered substrates by distal sites engineering

Nitrilases are enzymes capable of converting nitriles into carboxylic acids under mild conditions. However, wild type nitrilases typically exhibit poor catalytic efficiency towards sterically hindered substrates. This study investigates the role of distal amino acid residues in enhancing the catalytic efficiency of nitrilase for hydrolyzing sterically hindered substrates. Initially, consensus sequence analysis and structure-guided loop scanning strategies were employed to identify key distal residues. Subsequently, site-directed saturation mutagenesis and combinatorial mutations led to the generation of a mutant T171P/T173E/H181F/T225A/N146D, which exhibited a 9.5-fold increase in enzyme activity compared to the wild type when p -methoxybenzonitrile was used as a substrate. Moreover, the mutant also showed excellent catalytic performance with various sterically hindered substrates and in scaled-up reactions. Molecular dynamics simulations revealed that distal mutations can induce conformational changes through residue interaction networks, thereby optimizing the enzyme's substrate-binding pocket and enhancing its catalytic efficiency. This study offers a comprehensive investigation into the role of distal amino acid residues in modulating the catalytic efficiency of nitrilase towards sterically hindered substrates and provides insights into enzyme function modification via distal site mutations. A quintuple mutant of nitrilase was constructed through distal sites engineering, resulting in a significant enhancement of catalytic activity for various sterically hindered nitrile substrates.