Cloning and expression of the isocitrate lyase gene from a nitrogen-fixing bacterium, Azotobacter vinelandii, and functional analysis of the enzyme by site-directed mutagenesis.
The gene encoding isocitrate lyase (ICL) from a nitrogen-fixing mesophilic bacterium, Azotobacter vinelandii strain IAM1078, was cloned, and the gene expression was examined. When sodium acetate or glucose was used as carbon source, similar growth was observed in this bacterium, but the ICL activity of cells grown with the former source was 43-hold higher than those with the latter. In addition, northern blot analysis revealed that expression of the ICL gene was induced by acetate. Based on a comparison of the amino acid sequences of the ICLs of various organisms, the ICL of this bacterium was found to be classifiable into subfamily 3, one of two phylogenetic groups of eubacteial ICLs. Replacement of the Ile504 in the ICL by Met, which is conserved in the corresponding position of cold-adapted ICLs of psychrophlic bacteria, resulted in decreased thermostability of activity, indicating that this amino acid residue is involved in thermal properties of this enzyme.
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Kita-ku, Sapporo, Japan.
Yumi
Y
Tanaka
Tomofumi
T
Hayashi
Naoto
N
Yamaoka
Yasuhiro
Y
Takada
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D016948:Azotobacter vinelandii / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development / Q000502:physiology; D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007522:Isocitrate Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009586:Nitrogen Fixation
D007522:Isocitrate Lyase