Degradation of FA (b-oxidation)

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Fatty acids are the largest energy reserve in the body, supplying energy-yielding substrates by β-oxidation in mitochondria and peroxisomes. Mitochondrial Fatty Acid β-oxidation (FAO) generates acetyl-CoA and reducing equivalents (NADH and FADH2), which are linked to the Krebs cycle and the mitochondrial respiratory chain, leading to ATP production by oxidative phosphorylation in aerobic tissues. During fasting, FAO provides 80% to 90% of cellular energy requirements, with ketone bodies generated from acetyl-CoA being excellent energy source for peripheral tissues, especially the brain (Mitchell et al. 1995). Whilst almost all tissues rely essentially on FAO for their energy supply during prolonged fasting, heart and skeletal muscle derive most of their required energy from long-chain fatty acid oxidation at all times (Neely et al. 1972).

Figure 9. The mitochondrial carnitine shuttle
Figure 9. The mitochondrial carnitine shuttle
Long-chain fatty acyl-CoA esters are transported across the mitochondrial membrane by the carnitine cycle shuttle mechanism, involving the enzymes carnitine palmitoyltransferase I (CPT1), carnitine palmitoyltransferase II (CPT2), and carnitine acylcarnitine translocase (CACT), each with different sub-mitochondrial localisations, and with carnitine as a cofactor (McGarry et al. 1997, Kerner et al. 2000). Free L-carnitine crosses the plasma membrane against a high concentration gradient with the aid of the plasma membrane carnitine transporter (CT) encoded by the OCTN2 gene (Tamai et al. 1998).

The CPT1 protein, which exists in two genetically distinct isoforms - a liver type (CPT1A) and a muscle type (CPT1B) - is located on the outer mitochondrial membrane, and catalyses the formation of long-chain acylcarnitine from acyl-CoA ester and free L-carnitine. The CACT in the inner mitochondrial membrane carries the acylcarnitine into the mitochondrial matrix in exchange for free L-carnitine, and the CPT2, situated in the inner mitochondrial membrane, reesterifies the fatty acylcarnitine to fatty acyl-CoA ester, the substrate for β-oxidation (Fig.9).

Within the mitochondria, the fatty acyl-CoA esters undergo repeat cycles of four sequential reactions, catalysed by enzymes with overlapping chain length specificities.

This begins with flavoprotein-linked (FAD) dehydrogenation catalysed by the acyl-CoA dehydrogenases (ACD), followed by hydration by the 2-enoyl-CoA hydratases (ECH), NAD+-linked dehydrogenation by the L-3-hydroxyacyl-CoA dehydrogenases (HAD), and lastly, thiolytic cleavage by the 3-ketoacyl-CoA thiolases (KAT), generating an acetyl- CoA and an acyl-CoA ester 2 carbon atoms shorter at end of each cycle (Eaton et al. 1996). The membrane-bound mitochondrial trifunctional protein (MTP) comprises the last 3 three consecutive steps (Kamijo et al. 1994). The electrons generated during the FAD-linked dehydrogenation are transferred via the electron transfer flavoprotein (ETF) and ETF dehydrogenase (ETFDH) to ubiquinone, and those from NADH-linked dehydrogenation are passed to complex I in the respiratory chain leading to production of ATP. Unsaturated fatty acids with cis double bonds are also degraded by mitochondrial β-oxidation, with the pre-existing double bonds being catalysed by auxiliary enzymes such as enoyl-CoA isomerase and dienoyl-CoA reductase (Wanders et al. 1999). Long-chain acyl-CoA dehydrogenase serves an important function in the mitochondrial β-oxidation of unsaturated fatty acids (Lea et al. 2000). Acyl-Coenzyme A oxidase 1 (ACOX1) is the first enzyme of the fatty acid β-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs. ACOX1 donates electrons directly to molecular oxygen, thereby producing hydrogen peroxide.

Defects in the ACOX1 gene result in pseudoneonatal adrenoleukodystrophy, a disease that is characterized by accumulation of very long chain fatty acids.

Figure 11. Mitochondrial ATP generation
Figure 11. Mitochondrial ATP generation

The multisubunit NADH:ubiquinone oxidoreductase (complex I) is the first enzyme complex in the electron transport chain of mitochondria where adenosine triphosphate (ATP) is generated via oxidative phosphorylation in the mitochondria of almost all cells. The protein components consist of 4 respiratory chain complexes (I-IV) and an ATP synthase. Complex I is the largest at 900 kD and appears to be the most commonly affected in adult human mitochondrial diseases and include the NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 2 (8 kD) and 1 beta subcomplex, 1, 7 kDa.

Solute carrier family 27 (fatty acid transporter), member 2 is an isozyme of long-chain fatty-acid-coenzyme A ligase family. Although differing in substrate specificity, subcellular localization, and tissue distribution, all isozymes of this family convert free long-chain fatty acids into fatty acyl-CoA esters, and thereby play a key role in lipid biosynthesis and fatty acid degradation. This isozyme activates long-chain, branched-chain and very-long-chain fatty acids containing 22 or more carbons to their CoA derivatives. It is expressed primarily in liver and kidney, and is present in both endoplasmic reticulum and peroxisomes but not in mitochondria.

The carnitine biosynthesis pathway and chemical structures of the intermediates are shown in Fig.12 (A and B).

Figure 12. Carnitine biosynthesis pathway
Figure 12. Carnitine biosynthesis pathway

Carnitine is synthesized from the amino acids lysine and methionine (Vaz and Wanders 2002). Lysine provides the carbon backbone of carnitine and the 4-N-methyl groups originate from methionine (Fig.12, panel A). In mammals, certain proteins contain N-trimethyl-lysine (TML) residues (Fig.12, panel B). N-methylation of these lysine residues occurs as a post-translational event, which is catalysed by specific methyltransferases, which use S-adenosylmethionine as a methyl donor. Lysosomal hydrolysis of these proteins results in the release of TML, which is the first metabolite of carnitine biosynthesis (Fig.12, panel A). TML is first hydroxylated on the 3-position by TML dioxygenase to yield 3-hydroxy-TML (HTML). Aldolytic cleavage of HTML yields 4-trimethylaminobutyraldehyde (TMABA) and glycine, a reaction catalysed by HTML aldolase (HTMLA). Dehydrogenation of TMABA by TMABA dehydrogenase (TMABA-DH) results in the formation of 4-Ntrimethylaminobutyrate (butyrobetaine). In the last step, butyrobetaine is hydroxylated on the 3-position by c-butyrobetaine dioxygenase (BBD) to yield carnitine.

Carnitine acyltransferases (CRATs) are a group of enzymes that catalyze the reversible transfer of acyl groups from an acyl-CoA thioester to carnitine, thus forming the corresponding acylcarnitine. These enzymes can be distinguished according to their substrate specificity in carnitine palmitoyltransferase, carnitine octanoyltransferase, and carnitine acetyltransferase. CRAT is a key enzyme for metabolic pathways involved with the control of the acyl-CoA/CoA ratio in mitochondria, peroxisomes, and endoplasmic reticulum. Mitochondrial oxidation of long chain fatty acids is initiated by the sequential action of carnitine palmitoyltransferase I (CPT1), which is located in the outer membrane and is detergent-labile, and CPT II (CPT2), which is located in the inner membrane and is detergent-stable, together with a carnitine-acylcarnitine translocase.

Carnitine-acylcarnitine translocase Solute carrier family member 25 (carnitine/acylcarnitine translocase), member 20 is 1 of 10 closely related mitochondrial-membrane carrier proteins that shuttle substrates between cytosol and the intramitochondrial matrix space. The acyl-CoA dehydrogenases (ACADs) are a group of mitochondrial enzymes involved in the metabolism of fatty acids or branched-chain amino acids. Short-chain L-3-hydroxyacyl-CoA dehydrogenase plays an essential role in the mitochondrial β-oxidation of short chain fatty acids. It catalyzes the reversible dehydrogenation of 3-hydroxyacyl-CoAs to their corresponding 3-ketoacyl-CoAs with concomitant reduction of NAD to NADH and exerts it highest activity toward 3-hydroxybutyryl-CoA. Hydroxyacyl-Coenzyme A dehydrogenase/3-ketoacyl-Coenzyme A thiolase/enoyl-Coenzyme A hydratase encode the alpha and beta subunits of the mitochondrial trifunctional protein, respectively. The heterocomplex contains 4 alpha and 4 beta subunits and catalyzes 3 steps in the β-oxidation of fatty acids, including the long-chain 3-hydroxyl-CoA dehydrogenase step. Peroxisomal acyl-CoA thioesterase is an acyl-CoA thioesterase that shows a preference for medium-length fatty acyl-CoAs.

Genelist - Degradation of FA (b-oxidation)

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