CHAPTER 21 Lipid Biosynthesis Key topics: Biosynthesis of fatty acids and eicosanoids Biosynthesis of triacylglycerols Biosynthesis of cholesterol 白麗美 醫學一8F, 5520 pai@mail.cgu.edu.tw

Lipids Fulfill a Variety of Biological Functions Storage of energy Constituents of cellular membranes Anchors for membrane proteins Cofactors for enzymes Signaling molecules Pigments Detergents Transporters Antioxidants ‘Forehead to forehead I meet thee, this third time, Moby Dick!’ [Ahab (Melville, 1851)] Head-butting during male–male aggression is a basal behavior for cetaceans Sinking of Essex (238 ton ship) in 1821 is the first documented case of a sperm whale deliberately striking a ship (Chase, 1821). http://jeb.biologists.org/cgi/content/full/205/12/1755 2

Catabolism and Anabolic of Fatty Acids Proceed via Different Pathways Catabolism of fatty acids produced acetyl-CoA reducing power to NADH location: mitochondria Anabolism of fatty acids - requires malonyl-CoA and acetyl-CoA - reducing power from NADPH - location: cytosol in animals, chloroplast in plants

ACC is a bifunctional enzyme Biotin carboxylase Transcarboxylase Fig. 21.1 The Acetyl-CoA carboxylase reaction ACC is a bifunctional enzyme Biotin carboxylase Transcarboxylase FIGURE 21-1 (part 1) The acetyl-CoA carboxylase reaction. Acetyl-CoA carboxylase has three functional regions: biotin carrier protein (gray); biotin carboxylase, which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction (see Figure 16-16); and transcarboxylase, which transfers activated CO2 (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA. The long, flexible biotin arm carries the activated CO2 from the biotin carboxylase region to the transcarboxylase active site. The active enzyme in each step is shaded blue. 4

FIGURE 21-1 The acetyl-CoA carboxylase reaction FIGURE 21-1 The acetyl-CoA carboxylase reaction. Acetyl-CoA carboxylase has three functional regions: biotin carrier protein (gray); biotin carboxylase, which activates CO2 by attaching it to a nitrogen in the biotin ring in an ATP-dependent reaction (see Figure 16-16); and transcarboxylase, which transfers activated CO2 (shaded green) from biotin to acetyl-CoA, producing malonyl-CoA. The long, flexible biotin arm carries the activated CO2 from the biotin carboxylase region to the transcarboxylase active site. The active enzyme in each step is shaded blue. 5

Fig. 21.2 Addition of two carbons Four steps FIGURE 21-2 (part 1) Addition of two carbons to a growing fatty acyl chain: a four-step sequence. Each malonyl group and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a multienzyme system described later in the text. 1 Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonyl-CoA, with elimination of CO2 from the malonyl group, extends the acyl chain by two carbons. The mechanism of the first step of this reaction is given to illustrate the role of decarboxylation in facilitating condensation. The β-keto product of this condensation is then reduced in three more steps nearly identical to the reactions of β oxidation, but in the reverse sequence: 2 the β-keto group is reduced to an alcohol, 3 elimination of H2O creates a double bond, and 4 the double bond is reduced to form the corresponding saturated fatty acyl group. 6

FIGURE 21-2 (part 2) Addition of two carbons to a growing fatty acyl chain: a four-step sequence. Each malonyl group and acetyl (or longer acyl) group is activated by a thioester that links it to fatty acid synthase, a multienzyme system described later in the text. 1 Condensation of an activated acyl group (an acetyl group from acetyl-CoA is the first acyl group) and two carbons derived from malonyl-CoA, with elimination of CO2 from the malonyl group, extends the acyl chain by two carbons. The mechanism of the first step of this reaction is given to illustrate the role of decarboxylation in facilitating condensation. The β-keto product of this condensation is then reduced in three more steps nearly identical to the reactions of β oxidation, but in the reverse sequence: 2 the β-keto group is reduced to an alcohol, 3 elimination of H2O creates a double bond, and 4 the double bond is reduced to form the corresponding saturated fatty acyl group. 7

Fig. 21.3 Multifunctional Polypeptide Fatty acid synthase thioesterase FIGURE 21-3a The structure of fatty acid synthase type I systems. The low-resolution structures of (a) the mammalian (porcine; derived from PDB ID 2CF2) and (b) fungal enzyme systems (derived from PDB IDs 2UV9, 2UVA, 2UVB, and 2UVC) are shown. (a) All of the active sites in the mammalian system are located in different domains within a single large polypeptide chain. The different enzymatic activites are: β-ketoacyl-ACP synthase (KS), malonyl/acetyl-CoA—ACP transferase (MAT), β-hydroxyacyl-ACP dehydratase (DH), enoyl-ACP reductase (ER), and β-ketoacyl-ACP reductase (KR). ACP is the acyl carrier protein. The linear arrangement of the domains in the polypeptide is shown in the lower panel. The seventh domain (TE) is a thioesterase that releases the palmitate product from ACP when the synthesis is completed. The ACP and TE domains are disordered in the crystal and are therefore not shown in the structure. thioesterase 8

Palmitate synthesis FIGURE 21-4 (part 1) The overall process of palmitate synthesis. The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step. The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0). 9

First acyl FIGURE 21-4 (part 2) The overall process of palmitate synthesis. The fatty acyl chain grows by two-carbon units donated by activated malonate, with loss of CO2 at each step. The initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. After each two-carbon addition, reductions convert the growing chain to a saturated fatty acid of four, then six, then eight carbons, and so on. The final product is palmitate (16:0). 10

Acyl carrier protein FIGURE 21-5 Acyl carrier protein (ACP). The prosthetic group is 4′-phosphopantetheine, which is covalently attached to the hydroxyl group of a Ser residue in ACP. Phosphopantetheine contains the B vitamin pantothenic acid, also found in the coenzyme A molecule. Its —SH group is the site of entry of malonyl groups during fatty acid synthesis. 11

FIGURE 21-6 Sequence of events during synthesis of a fatty acid FIGURE 21-6 Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 12

FIGURE 21-6 (part 1) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 13

FIGURE 21-6 (part 2) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 14

Condensation with acetate -ketoacyl-ACP synthase (KS) FIGURE 21-6 (part 3) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 15

Reduction of carbonyl to hydroxyl -ketoacyl-ACP reductase (KR) FIGURE 21-6 (part 4) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 16

Dehydration of alcohol to alkene -hydroxyacyl-ACP dehydratase (DH) FIGURE 21-6 (part 5) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 17

Reduction of alkene to alkane enoyl-ACP reductase (ER) FIGURE 21-6 (part 6) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 18

Malonyl/acetyl-CoA ACP transferase Chain transfer Malonyl/acetyl-CoA ACP transferase FIGURE 21-6 (part 7) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 19

FIGURE 21-6 (part 8) Sequence of events during synthesis of a fatty acid. The mammalian FAS I complex is shown schematically, with catalytic domains colored as in Figure 21-3. Each domain of the larger polypeptide represents one of the six enzymatic activities of the complex, arranged in a large, tight "S" shape. The acyl carrier protein (ACP) is not resolved in the crystal structure shown in Figure 21-3, but is attached to the KS domain. The phosphopantetheine arm of ACP ends in an —SH. After the first panel, the enzyme shown in color is the one that will act in the next step. As in Figure 21-4, the initial acetyl group is shaded yellow, C-1 and C-2 of malonate are shaded pink, and the carbon released as CO2 is shaded green. Steps 1 to 4 are described in the text. 20

Figure 21-7 Second round of the fatty acid Synthesis cycle

Figure 21-8 Subcellular localization of lipid metabolism Glycolysis produce NADH, in cytosol NADH/NAD is small

Figure 21-9 Production of NADPH Hepatocyte and adipocyte

Figure 21-10 Not from fatty acid

Regulation of fatty acid synthesis Acetyl-CoA carboxylase Figure 21-11 Regulation of fatty acid synthesis Acetyl-CoA carboxylase Active-dephosphorylation 1. Allosteric regulation citrate, palmitoyl CoA 2. hormone- insulin-dP (activation) glucagon and epinephrine P (inactivation) 3. plant: Mg 2+ , pH, illumination 4. bacteria-cell growth guanine nucleotide 5. Gene level: polyunsaturated FA inhibit lipogenic enzyme in liver

Figure 21-12 synthesis of other fatty acids Elongation: smooth ER and mitochondria Acetyl CoA from Malonyl CoA Coenzyme A as acyl carrier Essential fatty acid

Figure 21-13 Desaturation of fatty acids in vertebrates Oxidation on both, on smooth ER

Box 21-1 Mixed function oxidases (fatty acyl-CoA desaturase), oxygenases, and cytochrome P450 Oxidase: O2 as electron acceptor e.g. fatty acid oxidation in peroxisomes cytochrome oxidase in mitochondria most: flavoproteins Oxygenase: O2 into substrate As hydroxyl or carboxyl group Dioxygenase

Box 21-1 Mixed function oxidases, oxygenases, and cytochrome P450 Monooxygenase (hydroxylases, mix-function oxidases (oxygenase)) Heme protein-cytochrome P-450 (SER) Hydroxylation –adrenocortical hormone Drug-soluble , Detoxification Figure, 21-13, 16, 30, 37, 47

Figure, 21-14 Action of plant desaturation ER and chloroplast Membrane fluidity

Figure, 21-15 (a) the cyclic pathway Eicosanoids: biological signal molecules Local hormone G-protein linked receptor (SER) COX: Prostaglandin H2 synthase

Figure, 21-15 (b) the cyclic pathway Cyclooxygenase: Cox-1: prostaglandins-gastric mucin (protect) Cox-2: prostaglandins-inflammation, pain, fever

Figure, 21-15 (b) nonsteroidal anti-inflammatory drug (NSAID) minicking the structure of the substrate or an intermediate Thromboxane synthase PGH2 to thromboxane A2-constriction of blood vessels and platelet aggregation (under COX1 pathway) Low doses of aspirin reduce heart attacks and strokes

Figure21-15C Cox-2-specific drugs , 1,000 times difference to Cox-1 and Cox-2 Reduce prostacyclin (COX2) Dilates blood vessels)

Figure, 21-16 the linear pathway lipooxygenase (mix-function oxidases) Leukocytes, heart, brain, lung, and spleen Use cytochrome P-450 (SER)

Figure, 21-17 Biosynthesis of phosphatidic acid Liver and kidney

Figure, 21-18 Biosynthesis of triacylglycerol

Figure, 21-19 Regulation of triacylglycerol synthesis by insulin Reduce cAMP- reduce lipolysis: reduce free fatty acid in blood Citrate lyase ACC

Figure, 21-20 the triacylglycerol cycle –low when other fuels available 75% released to form TAG in liver

Figure, 21-21Glyceroneogenesis In adipocyte: glucagon and epinephrine Suppress glycolysis Little DHAP is available no glycerol kinase in adipocyte No glucose synthesis DHAP

Glucocorticoil hormones regulate PEP carboxykinase In the liver and adipose tissue

Figure, 21-22 regulation of glyceroneogenesis Increase flux of TAG cycle

High levels of free fatty acids in the blood Interfere with glucose utilization in muscle Promote insulin resistance-type 2 diabetes Thiazolidinediones-reduce fatty acids in the blood Increase sensitivity to insulin

Figure, 21-22 regulation of glyceroneogenesis This drug binds to and activate a nuclear hormone receptor -peroxisome proliferator activated receptor g Increase PEP carboxykinase in adipose tissue

Synthesis of malonyl-CoA, and fatty acyl chain 2. Regulation of fatty acid synthesis 3. Mixed function oxidase 4. Eicosanoids (NSAID) 5. Biosynthesis of triacylglycerol and the TAG cycle