Abstract
Phytanic acid and pristanic acid are derived from phytol, which enter the body via the diet. Phytanic acid contains a methyl group in position three and, therefore, cannot undergo beta-oxidation directly but instead must first undergo alpha-oxidation to pristanic acid, which then enters beta-oxidation. Both these pathways occur in peroxisomes, and in this study we have identified a novel peroxisomal acyl-CoA thioesterase named ACOT6, which we show is specifically involved in phytanic acid and pristanic acid metabolism. Sequence analysis of ACOT6 revealed a putative peroxisomal targeting signal at the C-terminal end, and cellular localization experiments verified it as a peroxisomal enzyme. Subcellular fractionation experiments showed that peroxisomes contain by far the highest phytanoyl-CoA/pristanoyl-CoA thioesterase activity in the cell, which could be almost completely immunoprecipitated using an ACOT6 antibody. Acot6 mRNA was mainly expressed in white adipose tissue and was co-expressed in tissues with Acox3 (the pristanoyl-CoA oxidase). Furthermore, Acot6 was identified as a target gene of the peroxisome proliferator-activated receptor alpha (PPARalpha) and is up-regulated in mouse liver in a PPARalpha-dependent manner.
Highlights
The 2-methyl group in pristanic acid is present in both the 2R and 2S configuration, and it has been shown that the peroxisomal pristanoyl-CoA oxidase (ACOX3) is only active on the 2S stereoisomer [8]
We have characterized in detail the sixth gene in this cluster, acyl-CoA thioesterase 6 (Acot6), and show that this gene codes for a peroxisomal thioesterase, which hydrolyzes the CoA esters of the methylbranched fatty acids phytanic acid and pristanic acid
Elucidation of the metabolism of phytanic acid during the last few years has demonstrated the importance of peroxisomes, which is obvious from the severe elevation in phytanic acid and pristanic acid seen in various peroxisomal disorders [30]
Summary
To verify that ACOT6 is a peroxisomal pristanoyl-CoA thioesterase, we performed complementary experiments utilizing purified peroxisomes from mouse liver and kidney from control and clofibrate-treated animals. The activity in purified peroxisomes is very similar with phytanoyl-CoA and pristanoyl-CoA, with Km values of 32 and 35 M and Vmax values of 176 and 215 nmol/min/mg, respectively, suggesting that ACOT6 could be active on both these substrates (Fig. 4, A and B).
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