2.3. The requirement for auxiliary enzymes in β -oxidation
2.3.1. β -Oxidation of (poly)unsaturated fatty acids
Since trans-2 is the only unsaturated intermediate in the classical β -oxidation cycle and most of the double bonds in the dietary fatty acids are in cis-configuration, the additional double bonds in (poly)unsaturated fatty acids must be converted to the trans-2 configuration in order for the β -oxidation to proceed. This is accomplished by the auxiliary enzymes of β -oxidation (Eaton et al. 1996, Hiltunen & Qin 2000). The cis-double bonds at even-numbered positions in fatty acids are degraded via the reductase-dependent route (Fig. 3A). When the double bond reaches position cis-4, a trans-2 double bond is added by acyl-CoA dehydrogenase/oxidase, creating 2,4-dienoyl-CoA. This compound is reduced in a NADPH-dependent manner by 2,4-dienoyl-CoA reductase, resulting in eucaryotes in 3-enoyl-CoA, which is further isomerized by Δ3-Δ2-enoyl-CoA isomerase to trans-2-enoyl-CoA, the substrate of the β -oxidation spiral (Wang & Schulz 1989).
The metabolism of cis-5 and other unsaturated fatty acids with double bonds at odd-numbered positions was initially thought to occur via the isomerase-dependent pathway: chain shortening in β -oxidation would result in cis-3-enoyl-CoA, which would be converted to trans-2-enoyl-CoA by the Δ3-Δ2-enoyl-CoA isomerase (Fig. 3B). Tserng and Jin (1991), however, showed that the cis-5 double bonds are removed by NADPH-dependent reduction in mitochondria. This alternative route was found to contain a novel enzyme, Δ3,5-Δ2,4-dienoyl-CoA isomerase, which acts together with 2,4-dienoyl-CoA reductase and Δ3-Δ2-enoyl-CoA isomerase to produce trans-2 double bonds (Fig. 3C) (Smeland et al. 1992). This pathway involves the isomerization of trans-2-cis-5-dienoyl-CoA to Δ3-cis-5-dienoyl-CoA by the Δ3-Δ2-enoyl-CoA isomerase and a second isomerization to Δ2-Δ4-dienoyl-CoA by the novel dienoyl-CoA isomerase followed by the reductase-dependent route. The rat mitochondrial Δ3,5-Δ2,4-dienoyl-CoA isomerase was subsequently purified from rat liver (Chen et al. 1994, Luo et al. 1994). Shortly after this, it was discovered that peroxisomes also contain a Δ3,5-Δ2,4-dienoyl-CoA isomerase and are thus capable of metabolizing fatty acids with double bonds in odd-numbered carbons (He et al. 1995, Luthria et al. 1995). Two studies, using different experimental approaches, were published to discuss whether the isomerase-dependent pathway (Fig. 3B) or the route requiring dienoyl-CoA isomerase, enoyl-CoA isomerase and dienoyl-CoA reductase (Fig. 3C) (referred to as the reductase-dependent pathway) is the predominant one in metabolizing cis-5 double bonds in mitochondria. Tserng and co-workers (1996) found that cis-5-decenoate was completely degraded via the reductase-dependent pathway in liver mitochondria, while when the chain length was elongated to cis-5-tetradecenoate, 86 % of the fatty acid was metabolized via the reductase-dependent route. Shoukry and Schulz (1998) reported an opposite finding, stating that 80 % of 2,5-octadienoyl-CoA is oxidized via the isomerase-dependent pathway. They also suggested that a small portion of trans-2-trans-4-dienoyl-CoA formed by the dienoyl-CoA isomerase could enter directly the process of β -oxidation at the site of hydratase-1 action without reduction by the dienoyl-CoA reductase.
All the auxiliary enzymes required for the metabolism of unsaturated fatty acids exist in both the peroxisomes and the mitochondria of mammalian cells. The molecular characterization of the Δ3,5-Δ2,4-dienoyl-CoA isomerase and the Δ3-Δ2-enoyl-CoA isomerase will be described in more detail in the chapter “Hydratase/isomerase superfamily of enzymes”. The existence of a mitochondrial 2,4-dienoyl-CoA reductase was first demonstrated by Kunau and Dommes (1978). Since then, reductases from various sources have been characterized, including dienoyl-CoA reductases from bovine (Dommes et al. 1982), rat (Hakkola & Hiltunen 1993) and human (Koivuranta et al. 1994) mitochondria as well as rat (Dommes et al. 1981, Kimura et al. 1984), human (De Nys et al. 2001) and mouse (Geisbrecht et al. 1999a) peroxisomes. As an example, the human mitochondrial 120 kDa 2,4-dienoyl-CoA reductase is composed of four 36-kDa subunits, each containing a N-terminal mitochondrial targeting sequence (Koivuranta et al. 1994). At the amino acid sequence level, it shows 82.7 % similarity to the corresponding rat mitochondrial dienoyl-CoA reductase (Hirose et al. 1990). All the characterized eucaryotic 2,4-dienoyl-CoA reductases belong to the functionally heterologous short-chain alcohol dehydrogenase/reductase (SDR) superfamily with a characteristic NADPH-binding site called the Rossmann fold (Hiltunen & Qin 2000).
Bacteria also contain the machinery for the β -oxidation of unsaturated fatty acids, but it will not be discussed here. The β -oxidation of the yeast S. cerevisiae has been subject to intensive investigations, including this study. The pathways for the metabolism of unsaturated fatty acids in yeast will be described in the “Discussion” section.
2.3.2. β -Oxidation of α-methyl branched-chain fatty acids
Pristanic acid (2,6,10,14-tetramethylpentadecanoic acid) can be derived from two sources, either directly from the diet or as the α-oxidation product of phytanic acid (3,7,11,15-tetramethylhexadecanoic acid) (reviewed by Hiltunen & Qin 2000, Wanders et al. 2001) (Fig. 4). Phytanic acid cannot be directly β -oxidized because the 3-methyl group inhibits the second dehydrogenation step. In order for phytanic acid to be suitable for β -oxidation, it has to be shortened by a process called α-oxidation. In this pathway, phytanoyl-CoA is first hydroxylased to 2-hydroxyphytanoyl-CoA, then cleaved to pristanal and formyl-CoA and finally oxidized to pristanic acid, which can be activated to pristanoyl-CoA and metabolized by peroxisomal β -oxidation (Mihalik et al. 1995, Croes et al. 1997, Verhoeven et al. 1997). Pristanoyl-CoA undergoes three cycles of β -oxidation in peroxisomes, after which it is transported into mitochondria for complete oxidation (Verhoeven et al. 1998). Pristanic acid is a racemic mixture containing the (2S,6R,10R) and (2R,6R,10R) diastereomers. The branched-chain acyl-CoA oxidase of β -oxidation, however, can only act on the 2S-isomer (Van Veldhoven et al. 1996). An enzyme overcoming this problem was purified from rat liver by Schmitz and co-workers (1994). The enzyme was named α-methylacyl-CoA racemase, and it catalyzed the conversion of the 2R-isomers to the 2S-conformation suitable for β -oxidation (Fig. 4). In addition to pristanoyl-CoA acid, other α-methylacyl-CoAs, the branched side chains of bile acid intermediates, namely di- and trihydroxycholestanoyl-CoA, and arylpropionic acids also serve as substrates for α-methylacyl-CoA racemase (Schmitz et al. 1994). During bile acid biosynthesis, (25R)-di- and (25R)-trihydroxycholestanoyl-CoA are produced from cholesterol by mitochondrial 27-hydroxylation (Shefer et al. 1978, Batta et al. 1983). Because of the S-stereospecificity of the branched-chain acyl-CoA oxidase, α-methylacyl-CoA racemase has to convert di- and trihdydroxycholestanoyl-CoAs to their 25S-stereoisoforms, after which the side chain can be shortened by peroxisomal β -oxidation (Fig. 4) (Van Veldhoven et al. 1996) leading to the formation of choloyl-CoA.
In addition to rat liver, racemase has also been purified from human liver, and the cDNA sequences of the rat and mouse racemases have been determined (Schmitz et al. 1995, 1997). Also, the gene structure of the mouse racemase has been resolved (Kotti et al. 2000). The rat α-methylacyl-CoA racemase cDNA encodes for a polypeptide of 39.7 kDa, in good agreement with the molecular mass of the purified enzyme (Schmitz et al. 1994, 1997). All the α-methylacyl-CoA racemases studied have dual subcellular localization, both in mitochondria and in peroxisomes (Van Veldhoven et al. 1997, Kotti et al. 2000). At least in mouse, the same gene encodes both mitochondrial and peroxisomal racemases and the gene product is targeted to two different locations without modifications (Kotti et al. 2000). The physiological function of the peroxisomal racemase is clear: to convert the (R)-α-methyl groups of pristanoyl-CoA and trihydroxycholestanoyl-CoA to the S-conformation. In mitochondria, the degradation product of pristanoyl-CoA, 2,6-dimethylheptanoyl-CoA, is also a substrate for racemase, since both of its methyl groups occur in the R-conformation (Ferdinandusse et al. 2000).
