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Related Experiment Videos

Soybean DapA mutations encoding lysine-insensitive dihydrodipicolinate synthase

G W Silk1, B F Matthews

  • 1United States Department of Agriculture, Agricultural Research Service, Plant Molecular Biology Laboratory, Beltsville, MD 20705, USA.

Plant Molecular Biology
|March 1, 1997
PubMed
Summary

Soybean dihydrodipicolinate synthase (DS) enzyme activity is inhibited by lysine. Mutating the DapA gene created soybean DS enzymes insensitive to lysine, offering potential for improved crop yields.

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Area of Science:

  • Plant biochemistry
  • Enzyme engineering
  • Molecular biology

Background:

  • Lysine synthesis in plants is crucial for growth and development.
  • The enzyme dihydrodipicolinate synthase (DS), encoded by the DapA gene, catalyzes a rate-limiting step in this pathway.
  • Wild-type soybean DS exhibits high sensitivity to feedback inhibition by lysine.

Purpose of the Study:

  • To engineer soybean dihydrodipicolinate synthase (DS) with reduced feedback inhibition by lysine.
  • To investigate the effect of specific amino acid substitutions on DS enzyme activity and regulation.
  • To develop a modified DS enzyme for potential applications in agriculture.

Main Methods:

  • Cloning and expression of the soybean (Glycine max cv. Century) DapA gene in Escherichia coli.

Related Experiment Videos

  • Site-directed mutagenesis using PCR to create three distinct DapA gene mutants.
  • Assessing dihydrodipicolinate synthase (DS) activity and feedback inhibition by lysine in E. coli.

    • Main Results:

    • The cloned wild-type soybean DapA gene expressed a functional DS enzyme sensitive to lysine inhibition.
    • Three DapA mutants were successfully constructed, with single or double amino acid substitutions.
    • Mutant DS enzymes showed complete insensitivity to lysine inhibition at concentrations up to 1 mM.

    Conclusions:

    • Specific mutations in the soybean DapA gene can confer resistance to lysine feedback inhibition.
    • Engineered DS enzymes offer a promising strategy for enhancing lysine biosynthesis in plants.
    • This research has implications for improving crop nutritional value and yield.