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

Hum Mol Genet. 2001 Sep 1;10(18):1971-82. doi: 10.1093/hmg/10.18.1971.


Refsum's disease is a neurological syndrome characterized by adult-onset retinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaemia. Many cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-hydroxylase (PAHX) which catalyses the initial alpha-oxidation step in the degradation of phytanic acid. Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a requirement for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate. Sequence analysis in the light of crystallographic data for other members of the 2-oxoglutarate-dependent oxygenase super-family led to secondary structural predictions for PAHX, which were tested by site-directed mutagenesis. The H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands. The clinically observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assayed for both 2-oxoglutarate and phytanoyl-CoA oxidation. The P29S mutant was fully active, implying that the mutation resulted in defective targeting of the protein to peroxisomes. Mutation of Arg-275 resulted in impaired 2-oxoglutarate binding. The Q176K, G204S and N269H mutations caused partial uncoupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation. The results demonstrate that the diagnosis of Refsum's disease should not solely rely upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites / genetics
  • Cloning, Molecular
  • Enzyme Precursors / metabolism
  • Escherichia coli / genetics
  • Gene Expression Regulation, Enzymologic
  • Humans
  • Iron / metabolism
  • Ketoglutaric Acids / metabolism
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics*
  • Mixed Function Oxygenases / metabolism
  • Molecular Sequence Data
  • Molecular Structure
  • Mutation
  • Oxidation-Reduction
  • Protein Binding
  • Recombinant Proteins / isolation & purification
  • Refsum Disease / enzymology
  • Refsum Disease / genetics*
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Stereoisomerism
  • Structure-Activity Relationship
  • Substrate Specificity


  • Enzyme Precursors
  • Ketoglutaric Acids
  • Recombinant Proteins
  • Iron
  • Mixed Function Oxygenases
  • PHYH protein, human

Associated data

  • OMIM/266500