The Identification of a Succinyl-CoA Thioesterase Suggests a Novel Pathway for Succinate Production in Peroxisomes

Abstract

Dicarboxylic acids are formed by -oxidation of fatty acids in the endoplasmic reticulum and degraded as the CoA ester via ␤-oxidation in peroxisomes. Both synthesis and degradation of dicarboxylic acids occur mainly in kidney and liver, and the chain-shortened dicarboxylic acids are excreted in the urine as the free acids, implying that acyl-CoA thioesterases (ACOTs), which hydrolyze CoA esters to the free acid and CoASH, are needed for the release of the free acids. Recent studies show that peroxisomes contain several acyl-CoA thioesterases with different functions. We have now expressed a peroxisomal acyl-CoA thioesterase with a previously unknown function, ACOT4, which we show is active on dicarboxylyl-CoA esters. We also expressed ACOT8, another peroxisomal acyl-CoA thioesterase that was previously shown to hydrolyze a large variety of CoA esters. Acot4 and Acot8 are both strongly expressed in kidney and liver and are also target genes for the peroxisome proliferator-activated receptor ␣. Enzyme activity measurements with expressed ACOT4 and ACOT8 show that both enzymes hydrolyze CoA esters of dicarboxylic acids with high activity but with strikingly different specificities. Whereas ACOT4 mainly hydrolyzes succinyl-CoA, ACOT8 preferentially hydrolyzes longer dicarboxylyl-CoA esters (glutaryl-CoA, adipyl-CoA, suberylCoA, sebacyl-CoA, and dodecanedioyl-CoA). The identification of a highly specific succinyl-CoA thioesterase in peroxisomes strongly suggests that peroxisomal ␤-oxidation of dicarboxylic acids leads to formation of succinate, at least under certain conditions, and that ACOT4 and ACOT8 are responsible for the termination of ␤-oxidation of dicarboxylic acids of medium-chain length with the concomitant release of the corresponding free acids.

Compartmentation of Metabolism of the C12-, C9-, and C5-n-dicarboxylates in Rat Liver, Investigated by Mass Isotopomer Analysis

“…This question arises because of the identification of a peroxisomal thioesterase, ACOT4, specific for succinyl-CoA hydrolysis (with some activity toward glutaryl-CoA) (30). M4 succinate, released by peroxisomes, could be transferred to mitochondria, as suggested by Tserng and Jin (11) and Westin et al (30).…”

Novel functions of acyl-CoA thioesterases and acyltransferases as auxiliary enzymes in peroxisomal lipid metabolism

“…Further characterization of the ACOT8 enzyme in mouse and rat identified it as an acylCoA thioesterase, which hydrolyzes a very wide variety of CoASH esters [25,37,38]. In fact ACOT8 hydrolyzes all acyl-CoA esters tested so far, indicating that this enzyme likely recognizes the CoA moiety of the substrate for binding and not the acyl-chain.…”

“…These four proteins end -AKL (ACOT3 and ACOT5), -CRL (ACOT4) and -SKL (ACOT6) (Fig. 2), which targets them to peroxisomes as confirmed in all cases using green fluorescent fusion protein experiments [25][26][27]. The open reading frames of all four genes are encoded by three exons and the exon/intron boundaries are conserved in this gene family.…”

“…One particular gene family that was identified at that time was named the Type-I acyl-CoA thioesterase gene family, which based on enzyme activity measurements and Western blot analysis was suggested to code for closely related 8 enzymes in cytosol, mitochondria and peroxisomes [20,22]. Subsequent molecular cloning of this gene family identified a cluster of six related genes, localized on chromosome 12 D3 in mouse [24][25][26][27] (Fig. 2).…”

“…The ACOT4 shows the lowest percentage sequence similarity to the other peroxisomal thioesterases (65%) and indeed was the first thioesterase to reveal a more 'specific' substrate specificity, being active only with succinyl-CoA (K m ≈13 µM) and glutaryl-CoA (K m ≈37 µM) [25], suggesting a role in dicarboxylic acid metabolism. The mouse Acot4 mRNA is expressed mainly in liver, kidney and proximal intestine, with the former two organs being involved in dicarboxylic acid formation and metabolism.…”