Converting the 3-quinuclidinone reductase from Agrobacterium tumefaciens into the ethyl 4-chloroacetoacetate reductase by site-directed mutagenesis

In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) was modified by site-directed mutagenesis. And we further obtained a saturation mutant library in which the residue 197 was mutated. A single-point mutation converted the wild enzyme that originally had no catalytic activity in reduction of ethyl 4-chloroacetoacetate (COBE) into an enzyme with catalytic activity. The results of enzyme activity assays showed that the seven variants could asymmetrically reduce COBE to ethyl (S)-4-chloro-3-hydroxybutyrate ((S)-CHBE) with NADH as coenzyme. In the library, the variant E197N showed higher catalytic efficiency than others. The E197N was optimally active at pH 6.0 and 40 degrees C, and the catalytic efficiency (k(cat)/K-m) for COBE was 51.36 s(-1)center dot mM(-1). This study showed that the substrate specificity of AtQR could be changed through site-directed mutagenesis at the residue 197.

Automated summary

In this study, the 3-quinuclidinone reductase from Agrobacterium tumefaciens was modified by site-directed mutagenesis to change its substrate specificity. A single-point mutation at residue 197 resulted in an enzyme with catalytic activity towards ethyl 4-chloroacetoacetate. The mutant E197N showed the highest catalytic efficiency for the substrate in enzyme activity assays.