*2.3. Mechanism*

Conversion of free FAs into CoA esters constitutes an initial activation step before peroxisomal β-oxidation. This reaction is catalyzed by specific acyl-CoA synthetase connected to the cytosolic side of the peroxisomal membrane [69]. It was proved, using protease protection assays, that acyl-CoAs but not free FAs bind to the TMD of the transporter [70]. It is therefore only after activation that the fatty acyl-CoAs are transported to the peroxisomal matrix through peroxisomal ABC transporters. Fatty acyl-CoA are captured on the cytosolic side by the TMD, enhancing the affinity of NBD for ATP. ATP molecules are then hydrolyzed, thus producing the energy needed to switch the conformation of TMD and eventually allowing the translocation of substrates from the cytosol into the peroxisomal

matrix [22,71]. However, the exact mechanism of transport remains controversial. Two models are commonly considered. The first implies that esterified FAs are delivered directly to the peroxisomal matrix, whereas in the other model, free FAs are transported into the peroxisomal matrix after the hydrolysis of acyl-CoAs, which are re-esterified by acyl-CoA synthetase when in the peroxisomal lumen.

Although the process of cleavage and reactivation of acyl-CoAs seems to be a waste of energy as two ATP molecules are needed for the activation reaction, such a mechanism is crucial for the specific permeabilization of the substrates of β-oxidation [18]. Several studies have been done in an attempt to figure out the correct model for the transportation mechanism. Early studies on yeast models have demonstrated that fatty acyl-CoAs are hydrolyzed before being transported. This hydrolysis occurs when acyl-CoAs interacts with the heterodimer Pxa1p-Pxa2p at the cytosolic side of the peroxisomal membrane [72]. The peroxisomal ABC transporters would release a free fatty acid that should be re-esterified inside the peroxisome before its catabolic processing. In addition, intrinsic acyl-CoA thioesterase activity has been found in COMATOSE (CTS), a homolog of human ABCD1 in *Arabidopsis thaliana*, proving again that VLCFA-CoA is hydrolyzed prior to transport [73]. Very recently, the work of Kawaguchi et al. provided further proof of the transport mechanism [74]. After expressing human His-tagged ABCD1 in methylotrophic yeast, they directly demonstrated that ABCD1 transports the FA moiety after the hydrolysis of VLCFA-CoA and that acyl-CoA synthetase is required before the β-oxidation of VLCFA-CoA within the peroxisomes. When it comes to the fate of the free CoA, they are released in the peroxisomal lumen, as revealed using isolated peroxisomes from *Saccharomyces cerevisiae* [75].

Finally, after their re-esterification, substrates are directly delivered to specific acyl-CoA oxidases to initiate the β-oxidation process. Peroxisomal acyl-coenzyme A oxidase 1 (ACOX1) catalyzes the first and rate-limiting step of the β-oxidation pathway dedicated to straight-chain fatty acids, which includes LCFAs, VLCFAs, PUFAs, and dicarboxylic acids [76]. Other acyl-CoA oxidases also exist, ACOX2 and ACOX3. ACOX2 is specific to bile acid intermediates [76] whereas the oxidation of branched-chain FAs depends on both ACOX2 and ACOX3 enzymes [77,78]. Of note, mitochondria catalyze the β-oxidation of the majority of short, medium, and long chain FAs but not that of VLCFAs [79]. In yeast and plants, this process of FA β-oxidation occurs exclusively in peroxisomes, whereas in higher eukaryotes, the catabolism of VLCFAs is initiated solely in the peroxisomes [7,80].
