Investigation of the potential active site of a cyanide dihydratase using site-directed mutagenesis.
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.
Watanabe
A
A
Research Center for Advanced Science and Technology, University of Tokyo, Japan.
Yano
K
K
Ikebukuro
K
K
Karube
I
I
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D003545:Cysteine; D006867:Hydrolases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010649:Phenylalanine; D011549:Pseudomonas / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012694:Serine; D014443:Tyrosine
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014443:Tyrosine; D012694:Serine; D010649:Phenylalanine; D006867:Hydrolases; C083488:cyanide dihydratase; D003545:Cysteine