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Molecular characterization of a novel His333Arg variant of human protoporphyrinogen oxidase IX

Zora Novakova1, Jana Mikesova1, Marketa Ondrakova1

  • 1Laboratory of Structural Biology, Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, Vestec, 252 50, Czech Republic.

Biochemical and Biophysical Research Communications
|December 30, 2021
PubMed

Insights

The His333Arg mutation in the protoporphyrinogen oxidase IX (PPOX) gene causes Variegate porphyria by preventing proper protein folding and enzymatic activity. This mutation

Area of Science:

  • Biochemistry
  • Genetics
  • Molecular Biology

Background:

  • Variegate porphyria results from mutations in the protoporphyrinogen oxidase IX (PPOX) gene, leading to decreased enzyme activity.
  • A specific His333Arg mutation (PPOX(H333R)) was identified as a potential founder mutation in the Moroccan Jewish population.

Purpose of the Study:

  • To perform molecular characterization of the PPOX(H333R) mutation in vitro and in cellular models.
  • To elucidate the molecular mechanisms underlying the pathogenicity of the PPOX(H333R) mutation.

Main Methods:

  • In vitro enzymatic activity assays of purified recombinant PPOX(H333R).
  • Biophysical experiments and molecular modeling to assess protein structure and folding.
  • Live-cell confocal microscopy to evaluate subcellular distribution of PPOX(H333R).

Main Results:

  • Recombinant PPOX(H333R) exhibited no significant enzymatic activity in vitro.
  • Molecular modeling and biophysical data indicated PPOX(H333R) misfolding due to steric clashes near the active site.
  • Subcellular distribution of PPOX(H333R) within mitochondria was unaffected, suggesting fold-independent transport.

Conclusions:

  • The PPOX(H333R) mutation leads to Variegate porphyria through impaired protein folding and loss of enzymatic function.
  • Proper protein conformation is not essential for mitochondrial import of PPOX.
  • These findings provide a molecular basis for PPOX(H333R) pathogenicity and a framework for evaluating other PPOX variants.
Keywords:
Heme synthesisProtoporphyrinogen oxidase IXVariegate porphyria

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