Abstract
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.
Highlights
The mature form of phytanoyl-coenzyme A 2-hydroxylase (PAHX), a nonheme Fe(II)- and 2-oxoglutarate-dependent oxygenase, catalyzes the ␣-hydroxylation of phytanoylCoA within peroxisomes
Observed mutations in PAHX [9] and activity analyses [22] suggest that His-175 and Asp-177 are two of the likely three residues involved in iron binding, but the identity of the C-terminal histidine is undefined. Using both unprocessed and mature forms of recombinant PAHX, we report that both forms of the enzyme have similar substrate specificities and that the differences in the previous reports probably result from the use of nonoptimal assay conditions relating to the iron cofactors and/or to the method of (His)6pro-PAHX purification
Another group has reported that for N-terminal (His)6pro-PAHX for substrates lacking a -methyl group on the acyl chain, hydroxylation does not occur [12]; pro-PAHX produced without an N-terminal His tag is produced in an insoluble form in E. coli [11]
Summary
The mature form of phytanoyl-coenzyme A 2-hydroxylase (PAHX), a nonheme Fe(II)- and 2-oxoglutarate-dependent oxygenase, catalyzes the ␣-hydroxylation of phytanoylCoA within peroxisomes. Because divalent metals have been shown to inhibit 2OG oxygenases [31] and a pro-PAHX mutant (P29S) lacking a His tag had full in vitro activity compared with wild-type mature PAHX as measured by both 2OG and phytanoyl-CoA conversion [22], it was considered possible that the presence of Ni(II) used in protein purification results in reduced activity.
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