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

Refsum's disease is a neurological syndrome characterized by adult-onset re tinitis pigmentosa, anosmia, sensory neuropathy and phytanic acidaernia. Ma ny cases are caused by mutations in peroxisomal oxygenase phytanoyl-CoA 2-h ydroxylase (PAHX) which catalyses the initial a-oxidation step in the degra dation of phytanic acid. Both pro and mature forms of recombinant PAHX were produced in Escherichia coli, highly purified, and shown to have a require ment for iron(II) as a co-factor and 2-oxoglutarate as a co-substrate. Sequ ence analysis in the light of crystallographic data for other members of th e 2-oxoglutarate-dependent oxygenase super-family led to secondary structur al predictions for PAHX, which were tested by site-directed mutagenesis. Th e H175A and D177A mutants did not catalyse hydroxylation of phytanoyl-CoA, consistent with their assigned role as iron(II) binding ligands. The clinic ally observed P29S, Q176K, G204S, N269H, R275Q and R275W mutants were assay ed for both 2-oxoglutarate and phytanoyl-CoA oxidation. The P29S mutant was fully active, implying that the mutation resulted in defective targeting o f the protein to peroxisomes. Mutation of Arg-275 resulted in impaired 2-ox oglutarate binding. The Q176K, G204S and N269H mutations caused partial unc oupling of 2-oxoglutarate conversion from phytanoyl-CoA oxidation. The resu lts demonstrate that the diagnosis of Refsum's disease should not solely re ly upon PAHX assays for 2-oxoglutarate or phytanoyl-CoA oxidation.