Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase.
Three cysteine residues, which are completely conserved among alpha-subunits in all nitrile hydratases, are thought to be the ligands of a metal ion in the catalytic center of this enzyme. These cysteine residues (i.e. alpha C102, alpha C105 and alpha C107) in the high-molecular-mass nitrile hydratase (H-NHase) of Rhodococcus rhodochrous J1 were replaced with alanine by site-directed mutagenesis using the R. rhodochrous ATCC12674 host-vector system, and the resultant transformants were investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the cell-free extracts of each mutant transformant revealed that four mutant transformants (i.e. alpha C105A, alpha C107A, alpha C102A/C105A and alpha C105A/C107A) showed predominant alpha- and beta-subunit protein bands with a mobility identical to those of the native H-NHase, while three mutant transformants (i.e. alpha C102A, alpha C102A/C107A and alpha C102A/C105A/C107A) did not produce the corresponding proteins. The purified former four mutant enzymes showed neither enzymatic activity nor the maximum absorption at 410 nm which was detected in the wild type H-NHase. They also did not contain cobalt ions. Based upon these findings, these three cysteine residues were found to be essential for the active expression of H-NHase.
Institute of Applied Biochemistry, The University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan.
Yoshiteru
Y
Hashimoto
Satoshi
S
Sasaki
Sachio
S
Herai
Ken-Ichi
K
Oinuma
Sakayu
S
Shimizu
Michihiko
M
Kobayashi
D000595:Amino Acid Sequence; D003035:Cobalt / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D006836:Hydro-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D021122:Protein Subunits; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase; D003545:Cysteine