Chapter 21 Lipid Biosynthesis 1. Fatty acids; 2. Eicosanoids; 3. Triacylglycerols; 4. Membrane phospholipids; 5. Cholesterol, steroids, and isoprenoids; 1. Fatty acid synthesis takes a different pathway from its degradation Occurs in the cytosol (chloroplasts in plants). Acetyl-CoA provides the first two carbons, which is elongated by sequential addition of two-carbon units donated from malonyl-CoA. Intermediates are attached to the -SH groups of an acyl carrier protein (ACP). NADPH is the reductant. The enzymes are associated as a multi-enzyme complex or even being in one polypeptide chain in higher organisms (fatty acid synthase). Elongation by the fatty acid synthase complex stops upon formation of palmitate (C16), further elongation and desaturation are carried out by other enzyme systems. 2. Malonyl-CoA is formed from acetylCoA and bicarbonate discovered that HCO3- is required for fatty acid synthesis. Acetyl-CoA carboxylase (being trimeric in bacteria, monomeric in animals and both in plants) catalyzes this carboxylation reaction. The enzyme has three functional parts: a biotin carrier protein; an ATP-dependent biotin carboxylase; and a transcarboxylase. The enzyme exemplifies a ping-pong reaction mechanism. Salih Wakil This irreversible reaction commits acetyl-CoA to fatty acid synthesis. biotin carboxylase Transcarboxylase Acetyl-CoA carboxylase catalyzes the two-step carboxylation reaction of acetyl-CoA in two active sites. 3. The acetyl and malony groups are first transferred to two –SH groups of the fatty acid synthase complex The acetyl group of acetyl-CoA is first transferred to the –SH group of a Cys residue on the b-ketoacylACP synthase (KS) in a reaction catalyzed by acetyl-CoA-ACP transacetylase (AT). The malonyl group is transferred from malonyl-CoA to the –SH group of the 4`-phosphopantetheine covalently attached to a Ser residue of the acyl carrier protein (ACP). The acyl carrier protein (ACP) is very similar to CoA (thus can be regarded as “macro CoA”) 4. Fatty acids are synthesized by a repeating four-step reaction sequence In the condensation reaction (step 1), catalyzed by b-ketoacyl-ACP synthase, the methylene group of malonyl-CoA (linked to ACP) undergoes a nucleophilic attack on the carbonyl carbon of the acetyl group linked to KS, forming the b-ketobutyryl-ACP with simultaneous elimination of CO2. the b-ketobutyryl-ACP is then reduced to D-bhydroxybutyryl-ACP (step 2), using NADPH and the b-ketobutyryl-ACP reductase (KR). molecule is then removed from the bhydroxybutyryl-ACP to produce trans-2butenoyl-ACP in a reaction catalyzed by bhydroxybutyryl-ACP dehydratase (step 3). A further reduction (step 4), also using NADPH, of the carbon-carbon double in trans-2-butenoyl-ACP, catalyzed by enoylACP reductase produces a saturated acyl on ACP (butyryl-ACP). The butyryl group is then transferred to the Cys –SH group of b-ketoacyl-ACP synthase for another round of four reactions, which will extend the chain by two more carbons. A water Seven rounds of the four-step lengthening reactions produces palmitoyl-ACP, which will be hydrolyzed to release a free palmitate. The flexible 4`-phosphopantetheine group covalently attached to ACP is believed to act as a switch arm to move the intermediates from one active site to the next on the enzyme complex (i.e., the substrates are channeled). A total of 7 ATP and 14 NADPH will be consumed for making one palmitate molecule. 5. The seven activities of fatty acid synthesis from different organisms have different level of integration Each activity resides in a separate polypeptide chain in bacteria and higher plants. The seven activities reside in two separate polypeptide chains, with the synthase present as dodecamers (a6b 6). The seven activities reside in one large polypeptide chain in vertebrates, with the synthase present as dimers. The seven activities of fatty acid synthase are integrated to different levels in different organisms. 6. Fatty acid synthesis occurs in cellular compartments having a high NADPH/NADP+ ratio NAD and NADP have selected for functioning as electron carriers in oxidative catablism and reductive anabolism respectively. In the hepatocytes and adipocytes, NADPH is mainly produced in the cytosol via the pentose phosphate pathway and by the malic enzyme. In photosynthetic plants, fatty acid synthesis occur in the chloroplast stroma, using NADPH made from photophosphorylation. Malic enzyme Pentose phosphate pathway NADPH in the cytosol of animal cells is largely produced by the oxidative decarboxylation of malate and the pentose phosphate pathway 7. The acetyl groups of the mitochondrion are transported into the cytosol in the form of citrate The acetyl-CoA molecules are made from glucose and amino acids in mitochondria. The are shuttled into the cytosol in the form of citrate via the citrate transporter of the inner membrane. Acetyl-CoA is regenerated by the action of ATP-dependent citrate lyase in the cytosol. Oxaloacetate is shuttled back into the mitochondria as malate or pyruvate. 8. The rate of fatty acid biosynthesis is controlled by acetyl-CoA carboxylase Excess fuel is generally converted to fatty acids/triacylglycerol for longer term storage. Acetyl-CoA carboxylase, catalyzing the committing and rate-limiting step of fatty acid synthesis, is allosterically inhibited by palmitoylCoA and activated by citrate. Glucagon and epinephrine triggers the phosphorylation and disassociation of the polymeric enzyme subunits, which inactivates the enzyme. Citrate partially activate the phosphorylated acetyl-CoA carboxylase (similar to how AMP partially active the dephosphorylated glycogen phosphorylase). In plants, acetyl-CoA carboxylase is activated by a increase of Mg 2+ concentration and decrease of H+ concentration that accompany illumination. (Malonyl-CoA inhibits carnitine acyltransferase I) Dephosphorylated acetyl-CoA Carboxylase (active) Acetyl-CoA carboxylase is regulated by allosteric effectors and reversible phosphorylation Citrate partially activate the phosphorylated acetyl-CoA carboxylase 9. Palmitate can be further elongated and desaturated in smooth ER Palmitoyl-CoA can be further elongated by the fatty acid elongation system present mainly in the smooth endoplasmic reticulum, with twocarbon units also donated by malonyl-CoA. Palmitoyl-CoA and Stearoyl-CoA can be desaturated between C-9 and C-10 to produce palmitoleate, 16:1(9), and oleate, 18:1(9) respectively. The double bonds are introduced by the catalysis of fatty acyl-CoA desaturase (a mixed-function oxidase), where both the fatty acyl group and NADPH are oxidized by O2. The electrons of NADPH are transferred to O2 via Cyt b5 reductase and cytochrome b5. Further desaturation of oleate occur on phosphatidylcholine and is catalyzed by another desaturase, which is present only in plant cells. Linoleate and linolenate, needed to make other polyunsaturated fatty acids like arachidonate are essential fatty acids for mammals. Palmitate is the Precursor for the biosynthesis of other fatty acids Fatty acyl-CoA is desaturated (oxidized) by O2 and NADPH. Oleate can be desaturated on Phosphatidylcholine (often attaching to C-2) to form linoleate and linolenate 10. Eicosanoids are derived from arachidonate, 20:4 (5,8,11,14) The arachidonate (花生四烯酸) is first cleaved off from membrane phospholipids by phospolipase A2, in response to hormonal or other stimuli. Arachidonate is then converted to PGH2 by the catalysis of the bifunctional cyclooxygenase (COX): the cyclooxygenase activity converts arachidonate to PGG2; the peroxidase activity then converts PGG2 to PGH2. PGH2 is the immediate precursor of other prostaglandins and thromboxanes. Aspirin (acetylsalicylate) irreversibly inhibits the cyclooxygenase by acetylating an active site Ser, thus blocking the synthesis of prostglandins and thromboxanes; Ibuprofen also inhibit the same enzyme. Arachidonate can also be modified by adding hydroperoxy groups at various positions to form various hydroperoxyeicosatetraenoates (HPETEs) in reactions catalyzed by various lipooxygenases with the incorporation of O2. The HPETEs will be further converted to leukotrienes (白细胞三烯). Prostaglandins and thromboxanes are synthesized from arachidonate Cyclooxygenase activity of COX Peroxidase activity of COX Tyr385, a key residue for the cyclooxygenase activity Heme for the peroxidase active site Ser530 flurbiprofen The dimeric bifunctional cyclooxygenase (COX-1) 11. Newly synthesized fatty acids have mainly two alternative fates in cells Fate I: be incorporated into triacylglycerols as a form to store metabolic energy in long terms. Fate II: be incorporated into membrane phospholipids (during rapid growth). 12. Phosphatidic acid is the common precursor for the syntheses of both triacylglycerols and glycerophospholipids Phosphatidic acid (or diacylglycerol 3-phosphate) is made by transferring two acyl groups from two acylCoAs to L-glycerol 3-phosphate, which is derived from either glycerol or dihydroxyacetone phosphate. A phosphatidic acid is converted to a triacylglycerol via a dephosphorylation reaction (catalyzed by phosphatidic acid phosphatase) and a acyl transferring reaction. Phosphatidic acid is derived from L-glycerol 3phosphate and two acyl-CoAs. Often saturated Often unsaturated Phosphatidic acid is the common precursor for both triacylglycerols and glycerophospholipids Phosphatidic acid phosphatase 13. Insulin stimulates conversion of dietary carbohydrates/proteins into fat Diabetes patients due to lack of insulin would neither be able to use glucose properly, nor to synthesize fatty acids from carbohydrates and amino acids. They show increased rates of fatty acid oxidation and ketone body formation, thus losing weight. 14. Two strategies are taken for converting phosphatidic acid to glycerophospholipid Eugene Kennedy revealed in 1960s that either the –OH group of the diacylglycerol (strategy 1) or that of the polar head (strategy II) is first activated by attaching to cytidine nucleotide. The CMP moiety is displaced by the other – OH group in a nucleophilic attack reaction to synthesize a glycerophospholipid. Both strategies are used in eukaryotic cells, but only strategy I is use in bacterial cells. Eukaryotic cells use both strategies (occurring on sER and inner membrane of mitochondria) Bacteria mainly use this strategy Phospholipid synthesis in E. coli employs CDP-diacylglcerol phosphatase decarboxylase 15. Acidic (anionic) phospholipids in eukaryotic cells are synthesized using CDP-diacylglycerol These include phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylserine. eukaryotic cardiolipin is synthesized from one phosphatidylglycerol and one CDPdiacylglycerol (from two phosphatidylglycerols in bacteria). 4 5 16. Phosphatidyl choline (PC) and phosphatidyl ethanolamine (PE) are often made from the salvage (reuse) pathway in mammals Diet choline and ethanolamine are first converted to CDP-choline and CDPethanolamine after an initial phosphorylation step. The CMP moiety is then replaced by a diacylglycerol, forming PC and PE. Phosphatidylserine (PS) is often made from PE by a head exchange reaction (reversible). PC can be made from PE by three methylation reactions using S-adenosylmethionine (adoMet) in the liver cells. PS can also be converted to PE by a decarboxylation reaction. (ethanolamine) (Phosphoethanolamine) (CDP-ethanolamine) PC and PE are made from the salvage pathway in mammals (Phosphatidylthanolamine) The synthesis of PE, PC, PS in eukaryotic cells. 17. The synthesis of ether lipids involves a displacement of fatty acyl by fatty alcohol step and a desaturation step Both plasmalogen (缩醛磷脂) and plateletactivating factor are made using this pathway. The acyl group on 1-acyldihydroxyacetone 3phosphate is replaced by a long chain alcohol group to form the ether linkage. The double bond in plasmalogen is introduced at the end by the catalysis of a mixed-funciton oxidase. Synthesis of the ether lipids (醚脂类） 1-alkylglycerol 3-phosphate 18. The sphingosine backbone of spingolipids is derived from palmitoylCoA and Ser Palmitoyl-CoA condenses with serine (PLP is needed for decarboxylate serine) to form bketosphinganine, which is then reduced to sphinganine (二氢鞘氨醇). Sphinganine is then acylated and desaturated to form ceramide (containing sphingosine). Addition of sugar(s) or phosphocholine heads leads to the synthesis of cerebroside, gangliosides, or sphingomyelin. The ways for the membrane lipids (glycerolphospholipids and spingolipids) synthesized at smooth endoplasmic reticulum or inner membrane of Mitochondria to be transported to specific cellular locations are not well understood yet. PLP Spingolipid synthesis begins with the condensation between palmitoyl-CoA and Ser. A glycolipid, not a phospholipid (not CDP-choline!) 19. Radioisotope tracer experiments revealed that all the 27 carbons of cholesterol is derived from acetyl-CoA The origin of the carbon atoms of cholesterol was deduced from tracer experiments where either with the methyl carbon or the carboxyl carbon in acetate is labeled with 14C (1940s). The pattern of labeling provided the blueprint for revealing the detail enzymatic steps for cholesterol biosynthesis occurring in mammals. The 30-carbon squalene (of six isoprene units) and later on mevalonate were found to be intermediates of cholesterol biosynthesis. The biosynthetic pathway of cholesterol, being the most complex known, was elucidated mainly by Konrad Bloch and Feodor Lynen in the 1950s. The carbon origins of cholesterol as revealed by radioisotope labeling studies. 20. The cholesterol biosynthesis pathway can be divided into four stages Stage I: three acetyl-CoA molecules condense to form the 6-carbon mevalonate (甲羟戊酸). Stage II: mevalonate is converted to activated 5-carbon isoprene (异戊二烯) units. Stage III: Six isoprene units condense to form the linear 30-carbon squalene(鲨烯). Stage IV: The linear squalene is cyclized to form a four-ring structure, which is eventually converted to the 27-carbon cholesterol through a series of complicated reactions. (2C) (6C) (5C) (30C) (27C) Reactions assembling cholesterol from 18 molecules of acetyl-CoA can be divided into four stages. 21. Mevalonate commits the acetyl groups for cholesterol synthesis molecule of b-hydroxy-b-methylglutarylCoA (HMG-CoA) is formed from three acetylCoA molecules in the cytosol via the same reactions as occurring in mitochondria for ketone body formation. HMG-CoA reductase (an integrated membrane protein in the smooth ER) catalyzes the irreversible reduction of HMG-CoA (using two molecules of NADPH) to form mevalonate: committing the acetyl groups for cholesterol synthesis (thus being a major regulation step). One One mevalonate is synthesized from three acetyl-CoA molecules. HMG-CoA lyase in mitochondria Acetyl-CoA + acetoacetate HMG-CoA Reductase In cytosol The irreversible committing step for cholesterol biosynthesis 22. Two activated isoprenes are formed from mavelonate after going through three phosphorylation steps Three phosphate groups are transferred from three ATP molecules to mevalonate to form 3phospho-5-pyrophosphomevalonate. The leaving of both the carboxyl and the 3phosphate groups leads to the formation of 3isopentenyl pyrophosphate. 3-Isopentenyl pyrophosphate is isomerized to form the second activated isoprene: dimethylallyl pyrophosphate. Two activated isoprenes are formed from mavelonate. 23. The 30-carbon linear squalene is formed from the condensation of six activated isoprene units A dimethylallylpyrophosphate is joined to an isopentenylpyrophosphate (head-to-tail) to form the 10-carbon geranyl pyrophosphate. A geranyl pyrophosphate is joined to another 3isopentenyl pyrophosphate (head-to-tail) to form the 15-carbon farnesyl pyrophosphate(法呢基焦 磷酸). Two farnesyl pyrophosphate join (head-to-head) to form the 30-carbon squalene. Farnesyl pyrophosphate is formed from three activated isoprene units 15-carbon Squalene is formed from the condensation of two farnesyl pyrophosphates 24. The rings of cholesterol are formed via a concerted reaction across four double bonds of the linear squalene epoxide intermediate Squalene 2,3-epoxide, is first formed in a reaction catalyzed by squalene monooxygenase using O2 and NADPH. Concerted movement of electrons through four double bonds and the migration of two methyl groups generates lanosterol (羊毛固醇). Lanosterol is converted to cholesterol via about 20 enzymatic reactions including many double bond reduction and demethylations. Oxygenation induced ring closing converts the linear squalene to lanosterol of four rings, which is Squalene monooxygenase converted to cholesterol after going through another 20 or so reactions! cyclase ~ 20 reactions 25. Cholesterols made in vertebrate livers can be converted to bile acids and cholesterol esters before exporting Cholesterol can be converted to bile acids and bile salts, which will be secreted to the intestine for emulsifying lipids. Cholesterol can also be converted to the more hydrophobic cholesterol esters, which will be stored in the liver or transported to other tissues after being incorporated into lipoprotein particles. Cholesterol can be converted to bile acids (salts) ： glycocholate and taurocholate 牛黄胆酸盐 （甘胆酸盐） Acyl-CoA-Cholesteryl acyl transferase (ACAT) catalyzes the addition of an acyl group to the hydroxyl group of cholesterol 26. Lipids (including cholesterols) are transported in the vertebrate plasmia as various lipoprotein particles The different lipoprotein particles, having different combinations of lipids and apolipoproteins, can be separated by untracentrifugation due to different densities and sizes. The human plasma lipoproteins include chylomicrons (which transports lipids from intestine to various tissues), VLDL (very low density lipoproteins), LDL(Low density lipoproteins), HDL (high density lipoproteins). At least nine apolipoproteins (named as apo A, B, C, D, E) have been revealed in human, which act as signals to target the lipoprotein particles to various tissues or activating enzymes that will act on the lipoproteins. Endogenous lipids made in liver are transported to other tissues as VLDL particles (~ 87% lipids and 12% proteins). The apoC-II protein in VLDL activates the lipoprotein lipase in muscle and adipocyte tissues, thus releasing free fatty acids there. Some VLDL remnants is then converted to LDL (with 23% proteins and 75% lipids) and with the others being absorbed by the liver cells via receptor-mediated endocytosis. LDL delivers cholesterols to extrahepatic tissues, where its apoB-100 protein (4636 residues) is recognized by specific LDL receptors and LDL is endocytosed. HDL, with its precursors formed in the liver or intestine cells, collects the cholesterols in the plasma and deliver them to the liver cells. (A negative correlation between blood HDL level and arterial diseases has been observed.) Lipids are transported as various lipoprotein particles in vertebrate plasma LDL is uptaken by cells via the LDL receptors, LDL receptors are recycled to the cell surfaces 27. The de novo synthesis of cholesterol is regulated to complement dietary intake HMG-CoA reductase, catalyzing the ratelimiting step of the de novo cholesterol synthesis, has an activity variable over 100 fold! An yet characterized sterol promotes proteolytic degradation of HMG-CoA reductase and inhibits the transcription of the genes of HMGCoA reductase and LDL receptor. Hormones (insulin and glucagon) regulate the activity of the HMG-CoA reductase via reversible phosphorylation. Genetic defect of the LDL receptor was found to cause the familial hypercholesterolemia and atherosclerosis: the LDL cholesterols can not enter the cells, while de novo synthesis continues despite the high cholesterol level in the blood. Mevalonate analogs (e.g., compactin and lovastatin) can be used to treat patients with familial hypercholesterolemia. The de novo synthesis of cholesterol is regulated to complement the dietary uptake (For storage) The mevalonate analogs are used to treat hypercholesterolemia patients. 28. Pregnenolone, the common precursor of all steroid hormones, is derived from cholesterol The tail chain of cholesterol is first hydroxylated at C20 and C22, and then cleaved between these two carbons to remove a 6carbon unit, forming pregnenolone(孕烯醇酮). The hydroxylation is catalyzed by cytochrome P450 monooxygenases (a mitochondrial enzyme) that utilize NADPH and O2. Cytochrome P450 is a large family of enzymes with different substrate specificity, hydroxylates many hydrocarbon chains. Prognenolone, the common precursor of steroid hormones, is synthesized from cholesterol cytochrome P450 monooxygenases Desmolase 碳链裂解酶 29. All steroid hormones are derived from cholesterol Progesterone (孕酮) is synthesized from pregnenolone by oxidizing the 3-OH group and the isomerization of the double bond (from 5 to 4 position). Cortisol (a major glucocorticoid) is synthesized from progesterone by hydroxylation at C-17, C21, and C-11. Aldosterone (a mineralocorticoid) is synthesized from progesterone by hydroxylation at C-21, C-11, and oxidation of C-18 to an aldehyde. Testosterone (an androgen, or male hormone) is synthesized from progesterone by the removal of 2-carbon unit and hydroxylation at C-17. Estradiol ( an estrogen, or female hormone) is synthesized from testosterone by the removal of C-19 and formation of the aromatic A ring. Progesterone is synthesized from pregnenolone by oxidizing the 3-OH group and the isomerization of the double bond from 5 to 4 position Cortisol and aldosterone are synthesized from progesterone by several oxygenation reactions (forming hydroxyl and aldehyde groups) Testosterone is synthesized from progesterone by the removal of a 2-carbon unit and hydroxylation at C-17 Estradiol is synthesized from testosterone by the removal of C-19 and formation of the aromatic A ring 30. A hugh array of biomolecules, all called isoprenoids are synthesized using activated isoprenes These include many pigments (carotenoids, phytol chain of chlorophylls), fragrant principles, vitamines (A, D, E, K), rubber, dolichols, quinones (ubiquinone, plastoquinone), juvenile hormones of insects. Prenylation of proteins (attaching of geranylgeranyl and farnesyl groups) leads to membrane association. Some plant pigments are isoprenoids Some fragrant molecules are isoprenoids (called terpenes) Natural rubber is cis-polyisoprene Isoprenoid tails function to anchor proteins to membranes “Perfumes, colors and sounds echo one another.” Charles Baudelaire Correspondances Summary Fatty acid biosynthesis takes a different pathway from the reverse of its degradation and takes place in different cellular compartments. The aceytl-CoA units are transported out of mitochondrial matrix as citrate. Acetyl-CoA carboxylase catalyzes the ratelimiting step of fatty acid synthesis and is highly regulated by allosteric and covalent modifications. Palmitate, the usual final product of fatty acid synthesis, can be further elongated and desaturated in sER. Eicosanoids are derived from arachidonate by the action of cyclooxygenases and peroxidases. Phosphatidic acid (diacylglycerol 3-phosphate) is the common precursor of both triacylglycerol and glycerophospholipids. Glycerophospholipids are made using two alternative strategies of CDP modification. The backbone of sphingolipids are made from palmitoyl-CoA and Ser. Radioisotope tracer experiments revealed that all the 27 carbons of cholesterol are from acetyl-CoA. The biosynthesis of cholesterol takes a long pathway, with the reaction catalyzed by HMGCoA reductase being the rate-limiting step for de novo synthesis of cholesterol. Activated isoprene untis, mevalonate, squalene were found to be important intermediates of cholesterol biosynthesis. The lipids are transported as lipoprotein particles (including chylomicrons, VLDL, LDL, and HDL). The de novo biosynthesis of cholesterol is regulated to complement the dietary uptake. All streroid hormones are derived from choleterol. A huge arrays of isoprenoids are made using activated isoprene units.