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Related Experiment Videos

Does Escherichia coli possess a second citrate synthase gene?

A J Patton1, D W Hough, P Towner

  • 1Department of Biochemistry, University of Bath, England.

European Journal of Biochemistry
|May 15, 1993
PubMed
Summary

A mutation in Escherichia coli citrate synthase (gltA) gene created an inactive hexameric enzyme. A revertant produced an active dimeric enzyme encoded by a distinct gene, revealing new insights into citrate synthase evolution.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Escherichia coli has a hexameric citrate synthase (gltA) with allosteric kinetics.
  • A citrate synthase-deficient strain (K114) was mutated to a revertant (K114r4) with altered enzyme properties.

Purpose of the Study:

  • To investigate the genetic basis of altered citrate synthase properties in E. coli.
  • To characterize the mutant citrate synthase enzyme and its gene.

Main Methods:

  • Gene cloning and sequencing of gltA from wild-type and mutant E. coli strains.
  • Enzyme purification and characterization of the mutant citrate synthase.
  • N-terminal amino acid sequencing of the purified mutant enzyme.

Main Results:

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  • A single mutation (Asp362Asn) was identified in the gltA gene of K114 and K114r4.
  • Expression of the mutated gltA gene resulted in an inactive hexameric enzyme.
  • The revertant K114r4 produced an active, immunologically distinct dimeric citrate synthase encoded by a different gene.

Conclusions:

  • The Asp362 residue is critical for the catalytic activity of hexameric E. coli citrate synthase.
  • The dimeric citrate synthase in K114r4 is a novel enzyme, distinct from the native E. coli enzyme.
  • Comparative N-terminal sequencing provides insights into citrate synthase diversity across different domains of life.