Human Molecular Genetics, 2001, Vol. 10, No. 18 1971-1982
© 2001 Oxford University Press
Structure–function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum’s disease
The Oxford Centre for Molecular Science and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK and 1Department of Chemical Pathology, King’s, Guy’s and St Thomas’ Medical School, St Thomas’ Hospital Campus, Lambeth Palace Road, London SE1 7EH, UK
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 
-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.
+ To whom correspondence should be addressed. Tel: +44 1865 275677; Fax: +44 1865 275674; Email: matthew.lloyd@chem.ox.ac.uk
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