title: Characterization of chimeric and mutated isocitrate lyases of a mesophilic nitrogen-fixing bacterium, Azotobacter vinelandii, and a psychrophilic bacterium, Colwellia maris.
abstract: Chimeric enzymes between a cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia maris, (CmICL) and a mesophilic ICL of a nitrogen-fixing bacterium, Azotobacter vinelandii, (AvICL) were constructed by dividing the ICL genes into four regions of almost equal length and exchanging regions in various combinations. The chimeric ICL, which was replaced C-terminal region 4 of AvICL by the corresponding region of CmICL, showed much lower specific activity and lower optimum temperature and thermostability for activity than wild-type AvICL, indicating that region 4 is involved in its thermal properties. Furthermore, mutual substitution between the Met501 residue in region 4 of CmICL and the corresponding Ile504 residue of AvICL influenced the temperature dependence of their activities, suggesting that these amino acid residues are important to the respective mesophilic and cold-adapted properties of AvICL and CmICL.
authors.affiliation: a Biosystems Science Course, Graduate School of Life Science , Hokkaido University , Sapporo , Japan.
authors.forename: Tomofumi
authors.initials: T
authors.lastname: Hayashi
authors.forename: Wataru
authors.initials: W
authors.lastname: Matsuzaki
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takada
mesh_terms: D034241:Alteromonadaceae / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007522:Isocitrate Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D007522:Isocitrate Lyase
keywords: chimeric enzyme; cold-adapted enzyme; isocitrate lyase; mesophilic enzyme; site-directed mutagenesis