Studies on the specificity of unprocessed and mature forms of phytanoyl-CoA 2-hydroxylase and mutation of the iron binding ligands.
The mature form of phytanoyl-coenzyme A 2-hydroxylase (PAHX), a nonheme Fe(II)- and 2-oxoglutarate-dependent oxygenase, catalyzes the alpha-hydroxylation of phytanoyl-CoA within peroxisomes. Mutations in PAHX result in some forms of adult Refsum's disease. Unprocessed PAHX (pro-PAHX) contains an N-terminal peroxisomal targeting sequence that is cleaved to give mature PAHX (mat-PAHX). Previous studies have implied a difference in the substrate specificity of the unprocessed and mature forms of PAHX. We demonstrate that both forms are able to hydroxylate a range of CoA derivatives, but under the same assay conditions, the N-terminal hexa-His-tagged unprocessed form is less active than the nontagged mature form. Analyses of the assay conditions suggest a rationale for the lack of activity previously reported for some substrates (e.g. isovaleryl-CoA) for the (His)6pro-PAHX. Site-directed mutagenesis was used to support proposals for the identity of the iron binding ligands (His-175, Asp-177, His-264) of the 2-His-1-carboxylate motif of PAHX. Mutation of other histidine residues (His-213, His-220, His-259) suggested that these residues were not involved in Fe(II) binding.
Searls
Timothy
T
Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, UK.
Butler
Danica
D
Chien
Winnie
W
Mukherji
Mridul
M
Lloyd
Matthew D
MD
Schofield
Christopher J
CJ
D001665:Binding Sites / Q000235:genetics; D006801:Humans; D007501:Iron / Q000378:metabolism*; D008024:Ligands; D006899:Mixed Function Oxygenases / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011499:Protein Processing, Post-Translational; D013379:Substrate Specificity
D008024:Ligands; D007501:Iron; D006899:Mixed Function Oxygenases; C102623:PHYH protein, human