Nitrilase from Rhodococcus rhodochrous J1. Sequencing and overexpression of the gene and identification of an essential cysteine residue.
The amino acid sequences of the NH2 terminus and internal peptide fragments of a Rhodococcus rhodochrous J1 nitrilase were determined to prepare synthetic oligonucleotides as primers for the polymerase chain reaction. A 750-base DNA fragment thus amplified was used as the probe to clone a 5.4-kilobase PstI fragment coding for the whole nitrilase. The nitrilase gene modified in the sequence upstream from the presumed ATG start codon was expressed to approximately 50% of the total soluble protein in Escherichia coli. The predicted amino acid sequence of the nitrilase gene showed similarity to that of the bromoxynil nitrilase from Klebsiella ozaenae. The 5,5'-dithiobis(2-nitrobenzoic acid) modification of the nitrilase from R. rhodochrous J1 resulted in inactivation with the loss of one sulfhydryl group/enzyme subunit. Of 4 cysteine residues in the Rhodococcus nitrilase, only Cys-165 is conserved in the Klebsiella nitrilase. Mutant enzymes containing Ala or Ser instead of Cys-165 did not exhibit nitrilase activity. These findings suggest that Cys-165 plays an essential role in the function of the active site.
Kobayashi
M
M
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Japan.
Komeda
H
H
Yanaka
N
N
Nagasawa
T
T
Yamada
H
H
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase; D003545:Cysteine