Investigation of the potential active site of a cyanide dihydratase using site-directed mutagenesis

Abstract

Cyanide dihydratase has conserved residues in the amino acid sequence with nitrilase, and cyanide hydratase. The conserved amino acid residues in the cyanide dihydratase from Pseudomonas stutzeri AK61 were altered by site-directed mutagenesis. The enzyme completely lost its activity of the cyanide hydrolysis by the replacement of cysteine-163 to serine. The replacement of tyrosine-53 to phenylalanine caused an increase of the K m value of the enzyme for cyanide. Substitution of nine other residues seemed to affect the structure of the enzyme.