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Cloning, sequencing, and expression of a gene encoding the monomeric isocitrate dehydrogenase of the nitrogen-fixing

Takehiko Sahara1, Yasuhiro Takada, Yoji Takeuchi

  • 1Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan.

Bioscience, Biotechnology, and Biochemistry
|May 15, 2002
PubMed
Summary
This summary is machine-generated.

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Azotobacter vinelandii isocitrate dehydrogenase (IDH) gene regulation was studied. Nutrient availability controls IDH expression via two mRNA transcripts, impacting enzyme activity.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Isocitrate dehydrogenase (IDH) is a key enzyme in cellular metabolism.
  • Understanding IDH gene regulation is crucial for metabolic engineering and biotechnology.
  • The specific regulatory mechanisms of bacterial IDH, like that in Azotobacter vinelandii, are not fully elucidated.

Purpose of the Study:

  • To purify and characterize Azotobacter vinelandii isocitrate dehydrogenase (IDH).
  • To clone, sequence, and analyze the icd gene encoding IDH.
  • To investigate the transcriptional regulation of the icd gene in Escherichia coli.

Main Methods:

  • Enzyme purification and electrophoretic homogeneity assessment.
  • Gene cloning, DNA sequencing, and N-terminal amino acid sequencing.

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  • Gene expression analysis in E. coli using enzyme activity assays and mRNA quantification (primer extension).

    • Main Results:

    • The icd gene was cloned and sequenced, showing high homology to other bacterial IDHs.
    • Two distinct mRNA species (TS-1 and TS-2) with different 5'-untranslated regions were identified.
    • Promoter elements upstream of the TS-1 site were essential for IDH activity.
    • Nutrient availability significantly influenced the ratio of TS-1 to TS-2, correlating with IDH activity levels.

    Conclusions:

    • The icd gene of A. vinelandii is regulated at the transcriptional level.
    • Differential expression of two mRNA transcripts, controlled by nutrient conditions, modulates IDH activity.
    • This regulatory mechanism allows A. vinelandii to adapt its metabolic state to environmental nutrient levels.