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Elucidation of stability determinants of cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Colwellia maris, by construction of chimeric enzymes

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Title: Elucidation of stability determinants of cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Colwellia maris, by construction of chimeric enzymes
Authors: Watanabe, Seiya Browse this author
Yasutake, Yoshiaki Browse this author
Tanaka, Isao Browse this author →KAKEN DB
Takada, Yasuhiro4 Browse this author →KAKEN DB
Authors(alt): 高田, 泰弘4
Issue Date: 2005
Publisher: Society for General Microbiology
Journal Title: Microbiology
Volume: 151
Start Page: 1083
End Page: 1094
Publisher DOI: 10.1099/mic.0.27667-0
PMID: 15817777
Abstract: To elucidate determinants of differences in thermostability between mesophilic and psychrophilic monomeric isocitrate dehydrogenases (IDHs) from Azotobacter vinelandii (AvIDH) and Colwellia maris (CmIDH), respectively, chimeric enzymes derived from the two IDHs were constructed based on the recently resolved three-dimensional structure of AvIDH, and several characteristics of the two wild-type and six chimeric IDHs were examined. These characteristics were then compared with those of dimeric IDH from Escherichia coli (EcIDH). All recombinant enzymes with a (His)6-tag attached to the N-terminal were overexpressed in the E. coli cells and purified by Ni2+-affinity chromatography. The catalytic activity (kcat) and catalytic efficiency (kcat/Km) of the wild-type AvIDH and CmIDH were higher than those of EcIDH, implying that an improved catalytic rate more than compensates for the loss of a catalytic site in the former two IDHs due to monomerization. Analyses of the thermostability and kinetic parameters of the chimeric enzymes indicated that region 2, corresponding to domain II, and particularly region 3 located in the C-terminal part of domain I, are involved in the thermolability of CmIDH, and that the corresponding two regions of AvIDH are important for exhibiting higher catalytic activity and affinity for isocitrate than CmIDH. The relationships between the stability, catalytic activity and structural characteristics of AvIDH and CmIDH are discussed.
Type: article
URI: http://hdl.handle.net/2115/5545
Appears in Collections:理学院・理学研究院 (Graduate School of Science / Faculty of Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 高田 泰弘

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