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Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease.

M Mukherji1, W Chien, N J Kershaw

  • 1The Oxford Centre for Molecular Science and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK.

Human Molecular Genetics
|September 14, 2001
PubMed
Summary

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Refsum's disease research shows mutations in phytanoyl-CoA 2-hydroxylase (PAHX) affect phytanic acid degradation. PAHX enzyme assays alone are insufficient for diagnosing Refsum's disease.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Refsum's disease is a neurological disorder linked to phytanic acid accumulation.
  • Mutations in phytanoyl-CoA 2-hydroxylase (PAHX) are a common cause of Refsum's disease.
  • PAHX catalyzes the initial alpha-oxidation step in phytanic acid degradation.

Purpose of the Study:

  • To characterize the enzymatic activity of wild-type and mutant PAHX.
  • To investigate the role of specific amino acid residues in PAHX function.
  • To evaluate the diagnostic utility of PAHX enzyme assays for Refsum's disease.

Main Methods:

  • Recombinant pro and mature PAHX were produced and purified from E. coli.
  • Enzyme assays were performed using phytanoyl-CoA and 2-oxoglutarate.

Related Experiment Videos

  • Site-directed mutagenesis was used to create specific PAHX variants.
  • Crystallographic data and sequence analysis guided structural predictions.

    • Main Results:

    • PAHX requires iron(II) and 2-oxoglutarate for activity.
    • Mutations H175A and D177A abolished phytanoyl-CoA hydroxylation, identifying iron-binding ligands.
    • The P29S mutation led to a fully active enzyme, suggesting impaired peroxisomal targeting.
    • Arg-275 mutations impaired 2-oxoglutarate binding, while other mutations caused uncoupling of reactions.

    Conclusions:

    • PAHX enzyme activity is crucial for phytanic acid metabolism.
    • Specific mutations affect different aspects of PAHX function, including substrate binding and reaction coupling.
    • PAHX enzyme assays alone are not sufficient for definitive Refsum's disease diagnosis due to complex mutation effects.