Studies on phytanoyl-CoA 2-hydroxylase and synthesis of phytanoyl-Coenzyme A
Graphic
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.
References (28)
- et al.
Biochem. Biophys. Res. Commun.
(1999) - et al.
Biochem. Biophys. Res. Commun.
(1997) - et al.
J. Mol. Biol.
(1999) - et al.
J. Lipid Res.
(1966) - et al.
J. Lipid Res.
(1969) Anal. Biochem.
(1973)- et al.
Biochem. Biophys. Res. Commun.
(2000) - et al.
J. Lipid Res.
(2000) - et al.
J. Lipid Res.
(1999) - Wanders, R. J. A.; Jakobs, C.; Skjelda, O. M. Refsum's disease. In The Metabolic and Molecular Basis of Inherited...
J. Clin. Invest.
Nat. Genet.
Nat. Genet.
Proc. Natl. Acad. Sci. U.S.A.
Cited by (0)
- †
Nadia J. Kershaw and Mridul Mukherji contributed equally to this work.