2.2. The β -oxidation cycle
In this section, the β -oxidation of saturated fatty acids in mammalian mitochondria and peroxisomes as well as in yeast peroxisomes is described. The metabolism of unsaturated fatty acids is discussed in the chapter titled ”The requirement for auxiliary enzymes of β -oxidation”.
2.2.1. β -Oxidation in mitochondria
The bulk of dietary long-chain fatty acids are metabolized by the β -oxidation enzymes of mitochondria. Mitochondrial β -oxidation degrades fatty acids completely to acetyl-CoA, which is then oxidized by the citric acid cycle or, during starvation, condensed into ketone bodies. In mitochondria, β -oxidation enzymes are organized into two separate functional systems: the inner membrane-bound complex for long-chain fatty acid oxidation and the soluble matrix system for the degradation of medium- and short-chain fatty acids (for reviews see Eaton et al. 1996, Hiltunen & Qin 2000, Liang et al. 2001, Reddy & Hashimoto 2001). One cycle of β -oxidation consists of four subsequent reactions (Fig. 2). First, the acyl-CoA molecule is dehydrogenated by an acyl-CoA dehydrogenase leading to the generation of trans-2-enoyl-CoA and FADH2. Second, 2–enoyl-CoA hydratase-1 (hydratase-1, crotonase) adds water to the trans-2 double bond and L-3-hydroxyacyl-CoA is formed. Third, another dehydrogenase, L-3-hydroxyacyl-CoA dehydrogenase catalyzes the formation of 3-ketoacyl-CoA and NADH. As the fourth step, 3-ketoacyl-CoA thiolase cleaves 3-ketoacyl-CoA to acetyl-CoA and an acyl-CoA molecule shortened by two carbon atoms, which can re-enter the β -oxidation cycle.
In rat liver mitochondria, four straight-chain acyl-CoA dehydrogenases with different chain length specificities have been characterized. The very-long-chain acyl-CoA dehydrogenase is a homodimer located on the inner mitochondrial membrane and active towards acyl-CoA molecules up to 24 carbon atoms in length (Izai et al. 1992). The mitochondrial matrix contains separate short-, medium- and long-chain acyl-CoA dehydrogenases (Ikeda et al. 1985). These enzymes are homotetramers with substrate specificities of <C8, C6-C12 and C10-C22, respectively. For 2-methyl-branched fatty acyl-CoAs, a separate acyl-CoA dehydrogenase exists (Ikeda et al. 1983).
The very-long and long-chain 2-enoyl-CoAs formed by the respective dehydrogenases are fed into the long-chain-specific inner membrane-bound trifunctional β -oxidation complex for the second, third and fourth steps of the β -oxidation cycle (Carpenter et al. 1992, Uchida et al. 1992). The mitochondrial trifunctional enzyme (MTP) is an oligomer formed of four 79-kDa α-subunits and four 48-kDa β -subunits. The α-subunit contains the long-chain 2-enoyl-CoA hydratase-1 and long-chain L-3-hydroxyacyl-CoA dehydrogenase activities, whereas the β -subunit catalyzes the long-chain 3-ketoacyl-CoA thiolase reaction (Kamijo et al. 1993).
Once the long-chain acyl-CoAs have been shortened enough by the membrane-bound complex, the acyl-CoAs are further oxidized by the monofunctional medium- and short-chain-specific enzymes in the mitochondrial matrix. The short-chain 2-enoyl-CoA hydratase-1, also called crotonase, is a homohexamer and most active towards C4 substrates, but can also metabolize, at a much lower rate, substrates up to C16 (Hass & Hill 1969, Furuta et al. 1980). Another matrix-associated 2-enoyl-CoA hydratase-1, which is most active towards medium- and long-chain substrates, has been detected in human and pig mitochondria (Jackson et al. 1995).
The soluble L-3-hydroxyacyl-CoA dehydrogenase in the matrix acts on substrates with chain lengths from C4 to C16, but prefers substrates with shorter chain length (Osumi & Hashimoto 1980, He et al. 1989, Eaton et al. 1996). For the final step of the β -oxidation cycle, the mitochondrial matrix contains two short- and medium-chain-specific 3-ketoacyl-CoA thiolases. One is specific for acetoacetyl-CoA and 2-methylacetoacetyl-CoA and the other for substrates ranging from C6 to C16 (Eaton et al. 1996).
2.2.2. β -Oxidation in peroxisomes
The presence of β -oxidation in peroxisomes was first discovered by Cooper and Beevers in 1969. They found that fatty acids could be oxidized in glyoxysomes, which are plant organelles closely related to peroxisomes, of germinating castor bean seedlings. Lazarow and De Duve (1976) were able to further show that mammalian peroxisomes were also capable of fatty acid β -oxidation. The peroxisomal β -oxidation cycle consists principally of the same four reactions as the mitochondrial pathway: dehydrogenation/oxidation, hydration, another dehydrogenation and thiolytic cleavage (Fig. 2). The main differences lie in the substrate and stereo specificities of these pathways (reviewed by Hiltunen & Qin 2000, Reddy & Hashimoto 2001, Van Veldhoven et al. 2001, Wanders et al. 2001). Peroxisomes degrade fatty acids and fatty acid derivatives that cannot be oxidized by mitochondrial enzymes. The main role of peroxisomal β -oxidation is to shorten or otherwise convert fatty acids into a form that can be accepted by the mitochondrial enzymes. The substrates of peroxisomal β -oxidation include very-long-chain fatty acids, dicarboxylic fatty acids, branched-chain fatty acids (e.g. pristanic acid), intermediates of C27 bile acid synthesis (di- and trihydroxycholestanoic acid), prostaglandins, leucotrienes and some xenobiotics. Very-long-chain fatty acids (>C20) are not oxidized effectively in mitochondrial β -oxidation for two reasons. First, mitochondria do not contain very-long-chain specific acyl-CoA synthetase (Uchiyama et al. 1996) and very-long-chain fatty acids are not substrates for the mitochondrial long-chain acyl-CoA synthetase (Lazo et al. 1990). Second, very-long-chain fatty acids are not substrates for the human carnitine palmitoyltransferase required for the mitochondrial import of long-chain fatty acids (Wanders et al. 2001). The reason why many of the branched-chain fatty acids, such as phytanic and pristanic acid, are oxidized mainly in peroxisomes is the fact that carnitine palmitoyltransferase does not accept branched-chain fatty acids as substrates, either (Singh et al. 1994). After being chain-shortened or sufficiently modified in peroxisomes, the acyl moieties are linked to carnitine inside peroxisomes and transported into mitochondria for complete oxidation. Also the acetyl-CoA molecules formed in peroxisomes after every β -oxidation cycle are taken into mitochondria for oxidation in the citric acid cycle.
The first step of peroxisomal β -oxidation is catalyzed by acyl-CoA oxidase instead of acyl-CoA dehydrogenase in mitochondria. Acyl-CoA oxidase, being linked to FAD, donates the electrons obtained from the oxidation reaction directly to molecular oxygen, thus generating H2O2. The product of the oxidation reaction is the same as the product of mitochondrial dehydrogenation, trans-2-enoyl-CoA. The oxidation is catalyzed by multiple acyl-CoA oxidases using either straight-chain or branched-chain substrates. Two acyl-CoA oxidases have been characterized in human peroxisomes (Casteels et al. 1990, Vanhove et al. 1993) and three in rat peroxisomes (Osumi et al. 1980, Schepers et al. 1990, Van Veldhoven et al. 1991, 1992, 1994).
Mammalian peroxisomes contain two distinct multifunctional enzymes, type 1 and type 2, both of which catalyze the second and third reactions of β -oxidation, but with opposite chirality. No monofunctional enzymes catalyzing these reactions in mammalian peroxisomes exist. Multifunctional enzyme type 1 (MFE-1) was first purified and characterized from rat liver peroxisomes by Osumi and Hashimoto (1979). The purified 77-kDa polypeptide contained the 2-enoyl-CoA hydratase-1 and L-3-hydroxyacyl-CoA dehydrogenase activities (Osumi & Hashimoto 1979), the hydratase-1 activity being located in the amino terminal (N-terminal) domain and the dehydrogenase activity in the carboxyl terminal (C-terminal) domain (Ishii et al. 1987). Later, however, it was found that instead of being a bifunctional protein, MFE-1 is trifunctional and also catalyzes the isomerization of cis-3-enoyl-CoAs to trans-2-enoyl-CoA (Palosaari & Hiltunen 1990). The hydratase-1 and isomerase reactions occur in the same catalytic domain in the N-terminal part of MFE-1 (Palosaari et al. 1991). The MFE-2 of rat liver peroxisomes is a 79 kDa polypeptide catalyzing two reactions, hydration of trans-2-enoyl-CoA to D-3-hydroxyacyl-CoA and dehydrogenation of D-3-hydroxyacyl-CoA to 3-ketoacyl-CoA (Dieuaide-Noubhani et al. 1997a, Qin et al. 1997a). The D-3-hydroxyacyl-CoA dehydrogenase activity is located in the N-terminal part and the 2-enoyl-CoA hydratase-2 activity in the middle portion. The C-terminal part is similar to the rat sterol carrier protein 2 (Mori et al. 1991, Seedorf & Assman 1991). The amino acid sequences of MFE-1 and MFE-2 are not homologous and they use different substrates in peroxisomal β -oxidation. MFE-1 catalyzes the hydration and dehydrogenation of very-long-chain and other straight-chain fatty acids, such as dicarboxylic acids and eicosanoids, whereas MFE-2 is more active towards branched-chain substrates, such as pristanoyl-CoA and intermediates of bile acid synthesis (Dieuaide-Noubhani et al. 1997a, Dieuaide-Noubhani et al. 1997b, Qin et al. 1997a, Qin et al. 1997b).
The final reaction of the β -oxidation cycle is catalyzed by a 3-ketoacyl-CoA thiolase, which creates acetyl-CoA and an acyl-CoA chain shortened by two carbon atoms. For this, multiple enzyme isoforms also exist in mammalian peroxisomes. In rat, two closely related straight-chain ketoacyl-CoA thiolases could be found, one being inducible by peroxisome proliferators and the other not (Miyazawa et al. 1981, Hijikata et al. 1990). In humans, only one gene is similar to the rat genes (Bout et al. 1988). In human and rat peroxisomes, there is yet another 3-ketoacyl-CoA thiolase called peroxisomal thiolase 2 or SCPx. In addition to the thiolase domain it also contains a sterol carrier protein (SCP) domain (Seedorf et al. 1994). SCPx is capable of metabolizing branched-chain fatty acids and bile acid intermediates, whereas the original ketoacyl-CoA thiolase cleaves only straight-chain ketoacyl-CoAs (Antonenkov et al. 1997, Wanders et al. 1997).
The straight-chain-metabolizing L-specific β -oxidation pathway and the D-specific route that also metabolizes branched-chain fatty acids similarly differ in their inducibility. The expression of the “classical” enzymes is strongly induced in rodents by peroxisome proliferators, such as clofibrate, whereas the enzymes with branched-chain specificity are not (Lazarow & De Duve 1976, Van Veldhoven et al. 1992).
2.2.3. β -Oxidation in the yeast Saccharomyces cerevisiae
In the yeast S. cerevisiae, β -oxidation is restricted to peroxisomes (Kunau et al. 1988). The enzymes of the β -oxidation cycle consist of an acyl-CoA oxidase, a multifunctional enzyme and a thiolase, as in mammalian peroxisomes (for a review see Trotter 2001). These enzymes are encoded by the yeast genes POX1 (Dmochowska et al. 1990), FOX2 (Hiltunen et al. 1992) and FOX3/POT1 (Einerhand et al. 1991, Igual et al. 1991), respectively. The disruption of any of these genes results in the inability of yeast cells to grow on fatty acids as their sole carbon source (Dmochowska et al. 1990, Igual et al. 1991, Hiltunen et al. 1992), indicating that there are no isoforms for these enzymes in S. cerevisiae and that the enzymes must be able to metabolize substrates of all chain lengths. The enzyme encoded by FOX2 catalyzes the same D-specific reactions as the mammalian MFE-2. In fact, fox2p was the first multifunctional enzyme characterized as catalyzing the D-specific route of hydration and dehydrogenation (Hiltunen et al. 1992). This finding ruled out the previous assumption of yeast MFE catalyzing three reactions: L-specific hydration, L-specific dehydrogenation and epimerization, which would convert L-3-hydroxyacyl-CoA to D-3-hydroxyacyl-CoA and vice versa (Hiltunen et al. 1992). Thus, S. cerevisiae completely lacks the L-specific 2-enoyl-CoA hydratase-1 and 3-hydroxyacyl-CoA dehydrogenase. In vivo studies have shown, however, that the L-specific route can also be introduced into yeast cells (Filppula et al. 1995). This was accomplished by expressing the rat MFE-1 in yeast cells devoid of the endogenous D-specific MFE and then testing the complementation of growth on fatty acids. Indeed, although MFE-1 and yeast MFE are not related in terms of the amino acid sequence, they can functionally complement each other in S. cerevisiae (Filppula et al. 1995).
The NADH formed in the 3-hydroxyacyl-CoA dehydrogenation reaction is recycled back to NAD+ by malate dehydrogenase (van Roermund et al. 1995). The other end product of β -oxidation, acetyl-CoA, is transported to the mitochondria as an acetyl-carnitine derivative and oxidized by the citric acid cycle. Alternatively, acetyl-CoA may be metabolized by the glyoxylate cycle that produces isocitrate and succinate, which can be further metabolized in mitochondria (van Roermund et al. 1995).