In
Refsum disease, disorders of peroxisome biogenesis, and
rhizomelic chondrodysplasia punctata, pathological accumulation of
phytanic acid results from impaired alpha-oxidation of this branched-chain
fatty acid. Previous studies from this laboratory indicated that activation of
phytanic acid to its
CoA derivative precedes its alpha-oxidation in peroxisomes. It was reported that this reaction is catalyzed by a unique
phytanoyl-CoA synthetase in human peroxisomes. We wanted to determine whether
phytanic acid activation in rats required
long-chain acyl-CoA synthetase (LCS),
very long-chain acyl-CoA synthetase (VLCS), or a different
enzyme. To test directly whether LCS could activate
phytanic acid, rat liver
cDNA encoding this
enzyme was transcribed and translated in vitro. The expressed
enzyme had both LCS activity (assayed with
palmitic acid, C16: 0) and
phytanoyl-CoA synthetase activity; VLCS activity (assayed with
lignoceric acid, C24: 0) was not detectable. The ratio of
phytanoyl-CoA synthetized activity to
palmitoyl-CoA synthetase activity for LCS synthetized in vitro (approximately 205) was higher than that observed in peroxisomes isolated from rat liver (5-10%), suggesting that the expressed
enzyme contained sufficient
phytanoyl-Coa synthetase activity to account for all activity observed in intact peroxisomes. Further experiments were carried out to verify that
phytanic acid was activated by LCS in rat liver peroxisomes. Attempts to separate LCS from
phytanoyl-CoA synthetase by chromatography on several matrices were unsuccessful. Preparative isoelectric focusing revealed that
phytanoyl-CoA synthetase and LCS had indistinguishable isoelectric points.
Phytanoyl-CoA synthetase activity was inhibited by unlabeled
palmitic acid but not by
lignoceric acid. Heat treatment inactivated both
phytanoyl-CoA and
palmitoyl-CoA synthetase activities at similar rates.
5,8,11,14-Eicosatetraynoic acid inhibited activation of
phytanic acid and
palmitic acid in a parallel dose-dependent manner, whereas activation of
lignoceric acid was not affected. These data support our conclusion that rat liver LCS, an
enzyme known to be present in peroxisomal membranes, has
phytanoyl-CoA synthetase activity.