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Partial characterization and three-dimensional structural localization of eight mutations in exon 7 of the human phenylalanine hydroxylase gene associated with phenylketonuria

Academic Article
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Overview

authors

  • Bjorgo, E.
  • Knappskog, P. M.
  • Martinez, A.
  • Stevens, Raymond
  • Flatmark, T.

publication date

  • October 1998

journal

  • European Journal of Biochemistry  Journal

abstract

  • The molecular basis for the metabolic defect in patients with phenylketonuria has been characterized for seven missense point mutations (R252G/Q, L255V/S, A259V/T and R270S) and a termination mutation (G272X) in an evolutionarily conserved motif of exon 7 in the catalytic domain of the human phenylalanine hydroxylase (hPAH) gene. The mutations were expressed in three heterologous in vitro systems. When expressed as fusion proteins with maltose-binding protein in Escherichia coli five of the mutant proteins demonstrated a defect in the normal ability of hPAH to fold and assemble as homotetramer/dimer, and they were mostly recovered as inactive aggregated forms. Only for the R252Q and L255V mutants were catalytically active tetramer and dimer recovered and for R252G some dimer, i.e. 20% (R252Q, tetramer), 44% (L255V, tetramer) and 4.4% (R252G, dimer) of the activity for the respective wild-type (wt) forms. When expressed by a coupled in vitro transcription-translation system, all the mutant enzymes were recovered as a mixture of non-phosphorylated and phosphorylated forms with a low homospecific activity (i.e. maximum 11% of wt-hPAH for the L255V mutant). When transiently expressed in human embryonic kidney (A293) cells a very low level of immunoreactive PAH protein was recovered in spite of normal PAH mRNA levels. All these mutations resulted in variant hPAH proteins which revealed a defect in oligomerization, an increased sensitivity to limited proteolysis in vitro, reduced cellular stability and a variable reduction in their catalytic activity. All these effects seem to result from structural perturbations of the monomer, and based on the crystal structure of the catalytic domain of hPAH, an explanation is provided for the impact of the mutations on the folding and oligomerization of the monomers.

subject areas

  • Amino Acid Sequence
  • Base Sequence
  • Catalysis
  • Cell Line
  • DNA Primers
  • Exons
  • Humans
  • Kidney
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation
  • Phenylalanine Hydroxylase
  • Phenylketonurias
  • Protein Biosynthesis
  • Protein Conformation
  • Transcription, Genetic
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Research

keywords

  • mutation
  • phenylalanine hydroxylase
  • phenylketonuria expression
  • protein stability
  • three-dimensional structure
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Identity

International Standard Serial Number (ISSN)

  • 0014-2956

Digital Object Identifier (DOI)

  • 10.1046/j.1432-1327.1998.2570001.x

PubMed ID

  • 9799096
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Additional Document Info

start page

  • 1

end page

  • 10

volume

  • 257

issue

  • 1

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