Effects of the substituted amino acid residues on the thermal properties of monomeric isocitrate dehydrogenases from a psychrophilic bacterium, Psychromonas marina, and a mesophilic bacterium, Azotobacter vinelandii

Extremophiles : Life Under Extreme Conditions
Kango Tsubouchi, Yasuhiro Takada

Abstract

A cold-adapted monomeric isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina (PmIDH), showed a high degree of amino acid sequential identity (64%) to a mesophilic one from a mesophilic bacterium, Azotobacter vinelandii (AvIDH). In this study, eight corresponding amino acid residues were substituted between them by site-directed mutagenesis, and several thermal properties of the mutated IDHs were examined. In the PmIDH mutants, PmL735F, substituted Leu735 of PmIDH by the corresponding Phe of AvIDH, showed higher specific activity and thermostability of activity than wild-type PmIDH, while the H600Y and N741P mutations of PmIDH resulted in decreased specific activity and thermostability of activity. On the other hand, among the AvIDH mutants, AvP718T showed lower optimum temperature and thermostability of activity than wild-type AvIDH. In PmIDH variously combined the H600Y, L735F and N741P mutations, PmH600YL735F, including the H600Y and L735F mutations, showed higher specific activity than PmH600Y and similar optimum temperature and thermostability of activity to PmH600Y. Furthermore, PmL735FN741P exhibited higher specific activity and thermostability of activity than PmN741P. These results indicated tha...Continue Reading

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Methods Mentioned

BETA
column chromatography
PCR
electrophoresis
PCRs

Related Concepts

Isocitrate Dehydrogenase (NAD+)
Mutagenesis Process
Azotobacter vinelandii
Site
Genus Marina
Mutant Proteins
Study

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