Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue.
Peptides obtained by cleavage of a Rhodococcus rhodochrous K22 nitrilase, which acts on aliphatic nitriles such as acrylonitrile, crotonitrile, and glutaronitrile, have been sequenced. The data allowed the design of oligonucleotide probes which were used to clone a nitrilase encoding gene. Plasmid pNK21, in which 2.05-kb sequence covering the region encoding the nitrilase was was placed under the control of the lac promoter, directed overproduction of enzymatically active nitrilase in response to addition of isopropyl beta-D-thiogalactopyranoside in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the cell extract showed that the amount of nitrilase was about 40% of the total soluble proteins, leading to the establishment of a simple purification of the nitrilase. The nucleotide sequence of the nitrilase gene predicts a protein composed of 383 amino acids (M(r) = 42,275), including only one cysteine. The amino acid sequence homology between the Rhodococcus nitrilase and the Klebsiella ozaenae bromoxynil nitrilase [Stalker et al. (1988) J. Biol. Chem. 263, 6310-6314] was 38.3%, and a unique cysteinyl residue (Cys-170) in the former nitrilase was conserved at the corresponding position in the latter nitrilase. Cys-170 of the Rhodococcus nitrilase was replaced by Ala or Ser by site-directed mutagenesis. Both mutations resulted in the complete loss of nitrilase activity, clearly indicating that this cysteinyl residue is essential for the catalytic activity.
Kobayashi
M
M
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Yanaka
N
N
Nagasawa
T
T
Yamada
H
H
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D013329:Structure-Activity Relationship
D000619:Aminohydrolases; C076876:aliphatic nitrilase; C021867:nitrilase