Gene cloning of cold-adapted isocitrate lyase from a psychrophilic bacterium, Colwellia psychrerythraea, and analysis of amino acid residues involved in cold adaptation of this enzyme.
The gene (icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme.
Sato
Yuhya
Y
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita 10-jo Nishi 8-chome, Kita-ku, Sapporo 060-0810, Japan.
Watanabe
Seiya
S
Yamaoka
Naoto
N
Takada
Yasuhiro
Y
D000085:Acetates / Q000494:pharmacology; D034241:Alteromonadaceae / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics* / Q000378:metabolism; D003080:Cold Temperature; D004790:Enzyme Induction / Q000187:drug effects / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D007522:Isocitrate Lyase / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*
D000085:Acetates; D018387:Codon, Initiator; D007522:Isocitrate Lyase