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Domain structure and interaction within the pentafunctional arom polypeptide.

A R Hawkins1, M Smith

  • 1Dept. of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, England.

European Journal of Biochemistry
|March 28, 1991
PubMed
Summary
This summary is machine-generated.

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The Aspergillus nidulans AROM locus encodes a pentafunctional enzyme. Researchers delineated its functional domains, revealing two independent regions and domain interactions critical for enzyme activity.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • The shikimate pathway is essential for producing aromatic compounds in plants and microorganisms.
  • The AROM locus in Aspergillus nidulans encodes a multifunctional enzyme catalyzing five steps in this pathway.

Purpose of the Study:

  • To delineate the functional domains of the pentafunctional AROM polypeptide.
  • To investigate the folding and functional independence of these domains.
  • To explore potential interactions between domains.

Main Methods:

  • Oligonucleotide-mediated site-directed mutagenesis was used to create subfragments of the AROM locus.
  • Subfragments were expressed in Escherichia coli using the trc promoter.
  • Functional analysis was performed in appropriate E. coli mutants.

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Main Results:

  • The AROM polypeptide was divided into two independently folding regions: N-terminal (3-dehydroquinate synthase and EPSP synthase) and C-terminal (shikimate kinase, 3-dehydroquinase, and shikimate dehydrogenase).
  • Evidence suggests an interaction between DHQ synthase and EPSP synthase domains, stabilizing EPSP synthase activity.
  • An isoenzyme of 3-dehydroquinase (catabolic DHQase) was expressed, constituting up to 20% of soluble cell protein.

Conclusions:

  • The AROM polypeptide exhibits domain organization with independent folding and function.
  • Interactions between specific domains are crucial for enzyme stability and activity.
  • The study provides insights into the structure-function relationship of multifunctional enzymes in metabolic pathways.