Studies on phytanoyl-CoA 2-hydroxylase and synthesis of phytanoyl-Coenzyme A

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Abstract

Phytanoyl-CoA 2-hydroxylase (PAHX), an iron(II) and 2-oxoglutarate-dependent oxygenase, catalyses an essential step in the mammalian metabolism of β-methylated fatty acids. Phytanoyl-CoA was synthesised and used to develop in vitro assays for PAHX. The product of the reaction was confirmed as 2-hydroxyphytanoyl-CoA by NMR and mass spectrometric analyses. In accord with in vivo analyses, hydroxylation of both 3R and 3S epimers of the substrate was catalysed by PAHX. Both pro- and mature- forms of PAHX were fully active.

Section snippets

Chemistry

Phytanic acid (Scheme 1, Scheme 2) was synthesised from an E/Z mixture of phytol (6) according to a modification of the published procedure.11 Hydrogenation of the double bond to (3RS)-phytanol (7) was achieved using Rh/alumina in methanol. This procedure reduced the proportion of hydrogenolysis of the allylic alcohol obtained using PtO2 in ethanol.11 In contrast to the reported use of Jones reagent, oxidation of (3RS)-phytanol (7) to phytanic acid (Scheme 1, Scheme 2) was achieved by initial

Incubation of phytanoyl-CoA with PAHX

The pahx gene was cloned from a human liver cell cDNA library and expressed in E. coli. Pro-PAHX (i.e., with an N-terminal PTS-2 signal sequence) was produced as an N-terminal MBP-fusion,3 whilst mature PAHX was prepared without a fusion ‘tag’. Both forms of the enzyme were purified to >95% purity (by SDS-PAGE analysis).20

Initial assays measured 2-oxoglutarate oxidation21 in order to test with highly purified PAHX if phytanic acid (Scheme 1, Scheme 2) was a substrate and to confirm the

Acknowledgements

We thank Dr. R. T. Aplin for mass spectrometric analyses and Dr. V. Lee for helpful discussions and also the BBSRC, MRC, EPSRC, Felix foundation (scholarship to MM), E.U. Biotechnology project and the Wellcome Trust for financial support of the work.

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  • Cited by (0)

    Nadia J. Kershaw and Mridul Mukherji contributed equally to this work.

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