HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text Full Text (PDF) All Versions of this Article: M500034-JLR200v1 46/8/1660    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Searls, T. Articles by Schofield, C. J. Search for Related Content PubMed PubMed Citation Articles by Searls, T. Articles by Schofield, C. J. Social Bookmarking                      What's this?

Journal of Lipid Research, Vol. 46, 1660-1667, August 2005
Copyright © 2005 by American Society for Biochemistry and Molecular Biology

Studies on the specificity of unprocessed 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^1,*

^* Chemistry Research Laboratory, University of Oxford, Oxford OX1 3TA, UK
Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK

Published, JLR Papers in Press, June 1, 2005. DOI 10.1194/jlr.M500034-JLR200

¹ To whom correspondence should be addressed. e-mail: christopher.schofield{at}chem.ox.ac.uk

The mature form of phytanoyl-coenzyme A 2-hydroxylase (PAHX), a nonheme Fe(II)- and 2-oxoglutarate-dependent oxygenase, catalyzes the -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.

Abbreviations: ARD, adult Refsum's disease; mat-PAHX, mature phytanoyl-coenzyme A 2-hydroxylase; 2OG, 2-oxoglutarate; PAHX, phytanoyl-coenzyme A 2-hydroxylase; pro-PAHX, unprocessed phytanoyl-coenzyme A 2-hydroxylase; PTS2, peroxisomal targeting sequence-2; TCEP, tris(carboxyethyl)phosphine

Supplementary key words chemical rescue • oxygenase • 2-oxoglutarate • phytanoyl-coenzyme A 2-hydroxylase • phytanic acid • Refsum's disease

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. A. McDonough, K. L. Kavanagh, D. Butler, T. Searls, U. Oppermann, and C. J. Schofield Structure of Human Phytanoyl-CoA 2-Hydroxylase Identifies Molecular Mechanisms of Refsum Disease J. Biol. Chem., December 9, 2005; 280(49): 41101 - 41110. [Abstract] [Full Text] [PDF]