Studies on the selectivity of both pro- and mature- forms of phytanoyl-CoA 2-hydroxylase and mutation of the iron binding ligands

Timothy Searls, Danica Butler, Winnie Chien, Mridul Mukherji, Matthew D. Lloyd, and Christopher J. Schofield

Chemistry Dept., Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA

Corresponding Author: tim.searls{at}chem.ox.ac.uk

Phytanoyl-CoA 2-hydroxylase (PAHX), a non-haem iron(II) and 2-oxoglutarate (2OG)-dependent oxygenase, catalyzes the a-hydroxylation of phytanoyl-CoA within peroxisomes. Mutations in PAHX result in some forms of Adult Refsum’s Disease. The unprocessed protein (pro-PAHX) contains an N-terminal peroxisomal targeting sequence, which is cleaved to give the mature form of PAHX (mature-PAHX). Previous studies have implied a difference in the substrate selectivity of the pro- and mature- forms of PAHX. We demonstrate that both forms accept a range of coenzyme A (CoA) derivatives, but that the pro-form was consistently less active than the mature form. The lack of activity previously reported for some of these (e.g. isovaleryl-CoA) for the pro-form of PAHX probably arises from differences in the assay conditions in some previous studies, including the presence of ATP. Site-directed mutagenesis was used to test proposals for the identity of the iron-binding ligands (His-175, Asp-177 and His-264) of PAHX. Mutation of other histidine residues (His-213, His-220, His-259) to alanine suggested that these residues were not involved in iron(II) binding, but that His-213 and His-259 may play a structural role. With the Q176A and Q176K mutants, the latter of which is observed in Refsum’s patients, uncoupling of 2OG oxidation from phytanoyl-CoA hydroxylation was observed.

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