title: Exploring residues crucial for nitrilase function by site directed mutagenesis to gain better insight into sequence-function relationships.
abstract: Nitrilases represent a very important class of enzymes having an array of applications. In the present scenario, where the indepth information about nitrilases is limited, the present work is an attempt to shed light on the residues crucial for the nitrilase activity. The nitrilase sequences demonstrating varying degree of identity with P. putida nitrilase were explored. A stretch of residues, fairly conserved throughout the range of higher (96%) to lower (27%) sequence identity among different nitrilases was selected and investigated for the possible functional role in nitrilase enzyme system. Subsequently, the alanine substitution mutants (T48A, W49A, L50A, P51A, G52A, Y53A and P54A) were generated. Substitution of the rationally selected conserved residues altered the substrate recognition ability, catalysis and affected the substrate specificity but had very little impact on enantioselectivity and pattern of nitrile hydrolysis.
authors.affiliation: Biocatalysis and Protein Engineering Group, Department of Pharmaceutical Technology, National Institute of Pharmaceutical Education and Research Sector 67, S.A.S. Nagar-160 062, Punjab, India.
authors.forename: Shubhangi
authors.initials: S
authors.lastname: Kaushik
authors.forename: Utpal
authors.initials: U
authors.lastname: Mohan
authors.forename: Uc
authors.initials: U
authors.lastname: Banerjee
keywords: Nitrilase; catalysis; conserved residues; mutants; sequence identity; site directed mutagenesis