title: Structure-function analysis of phytanoyl-CoA 2-hydroxylase mutations causing Refsum's disease.
abstract: 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.
authors.affiliation: The Oxford Centre for Molecular Science and The Dyson Perrins Laboratory, South Parks Road, Oxford OX1 3QY, UK.
authors.forename: M
authors.initials: M
authors.lastname: Mukherji
authors.forename: W
authors.initials: W
authors.lastname: Chien
authors.forename: N J
authors.initials: NJ
authors.lastname: Kershaw
authors.forename: I J
authors.initials: IJ
authors.lastname: Clifton
authors.forename: C J
authors.initials: CJ
authors.lastname: Schofield
authors.forename: A S
authors.initials: AS
authors.lastname: Wierzbicki
authors.forename: M D
authors.initials: MD
authors.lastname: Lloyd
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009154:Mutation; D010084:Oxidation-Reduction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000302:isolation & purification; D012035:Refsum Disease / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D004792:Enzyme Precursors; D007656:Ketoglutaric Acids; D011994:Recombinant Proteins; D007501:Iron; D006899:Mixed Function Oxygenases; C102623:PHYH protein, human