Site-directed mutagenesis for cysteine residues of cobalt-containing nitrile hydratase.
作者： Yoshiteru Hashimoto , Satoshi Sasaki , Sachio Herai , Ken-Ichi Oinuma , Sakayu Shimizu
DOI: 10.1016/S0162-0134(02)00373-2
关键词:
摘要: Abstract Three cysteine residues, which are completely conserved among α-subunits in all nitrile hydratases, thought to be the ligands of a metal ion catalytic center this enzyme. These residues (i.e. αC102, αC105 and αC107) high-molecular-mass hydratase (H-NHase) Rhodococcus rhodochrous J1 were replaced with alanine by site-directed mutagenesis using R. ATCC12674 host–vector system, resultant transformants investigated. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) for cell-free extracts each mutant transformant revealed that four αC105A, αC107A, αC102A/C105A αC105A/C107A) showed predominant α- β-subunit protein bands mobility identical those native H-NHase, while three αC102A, αC102A/C107A αC102A/C105A/C107A) did not produce corresponding proteins. The purified former enzymes neither enzymatic activity nor maximum absorption at 410 nm was detected wild type H-NHase. They also contain cobalt ions. Based upon these findings, found essential active expression
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