1. Chapter 14 Lipid Biosynthesis Lipid Biosynthesis 1. Fatty acids; 2. Eicosanoids类花生酸类; 3. Triacylglycerols; 4. Membrane phospholipids膜磷脂; 5. Cholesterol, steroids, and isoprenoids;

2. 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丙二酰. 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. Lipid Biosynthesis

3. 2. Malonyl-CoA is formed from acetyl-CoA and bicarbonate Salih Wakil 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 carboxyl carrier protein, BCCP; an ATP-dependent biotin carboxylase, BC羧化酶; and a transcarboxylase (carboxyl transferase, CT)羧基转移酶. The enzyme exemplifies a ping-pong reaction mechanism. This irreversible reaction commits acetyl-CoA to fatty acid synthesis. Lipid Biosynthesis

4. Acetyl-CoA carboxylase catalyzes the two-step carboxylation reaction of acetyl-CoA in two active sites. Lipid Biosynthesis Trans- carboxylase biotin carboxylase

5. 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 β-ketoacyl-ACP synthase (KS) in a reaction catalyzed by acetyl-CoA-ACP transacetylase (AT,乙酰基转移酶). The malonyl group is transferred from malonyl-CoA to the –SH group of the 4`-phosphopantetheine磷酸泛酰巯基乙胺covalently attached to a Ser residue of the acyl carrier protein (ACP). Lipid Biosynthesis

6. The acyl carrier protein (ACP) is very similar to CoA (thus can be regarded as “macro CoA”) Lipid Biosynthesis

7. 4. Fatty acids are synthesized by a repeating four-step reaction sequence In the condensation reaction (step 1), catalyzed by β-ketoacyl-ACP酮脂酰synthase, the methylene亚甲基group of malonyl丙二酰-CoA (linked to ACP) undergoes a nucleophilic亲核的attack on the carbonyl羰基carbon of the acetyl group linked to KS, forming the β-ketobutyryl-ACP with simultaneous elimination of CO2. the β -ketobutyryl-ACP is then reduced to D- β -hydroxybutyryl-ACP (step 2), using NADPH and the β -ketobutyryl-ACP reductase (KR). Lipid Biosynthesis

8. A water molecule is then removed from the β -hydroxybutyryl-ACP to produce trans-2-butenoyl-ACP in a reaction catalyzed by β-hydroxybutyryl-ACP dehydratase (step 3). A further reduction (step 4), also using NADPH, of the carbon-carbon double in trans-2-butenoyl-ACP, catalyzed by enoyl-ACP reductase produces a saturated acyl on ACP (butyryl-ACP). The butyryl group is then transferred to the Cys –SH group of β -ketoacyl-ACP synthase for another round of four reactions, which will extend the chain by two more carbons. 4. Fatty acids are synthesized by a repeating four-step reaction sequence Lipid Biosynthesis

9. Seven rounds of the four-step lengthening reactions produces palmitoyl软脂酰-ACP, which will be hydrolyzed to release a free palmitate. The flexible 4`-phosphopantetheine磷酸泛酰巯基乙胺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. 4. Fatty acids are synthesized by a repeating four-step reaction sequence Lipid Biosynthesis

10. Table Proteins of the fatty acid synthase complex of E.coli Lipid Biosynthesis

11. 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 do decamers (α6β6, yeast). The seven activities reside in one large polypeptide chain in vertebrates, with the synthase present as dimers. Lipid Biosynthesis

12. The seven activities of fatty acid synthase are integrated todifferent levels in different organisms. Lipid Biosynthesis

13. 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肝细胞and adipocytes脂细胞, NADPH is mainly produced in the cytosol via the pentose phosphate pathway and by the malic苹果的enzyme. In photosynthetic plants, fatty acid synthesis occur in the chloroplast stroma, using NADPH made from photophosphorylation. Lipid Biosynthesis

14. NADPH in the cytosol of animal cells is largely produced by the oxidative decarboxylation of malate and the pentose phosphate pathway Lipid Biosynthesis Malic enzyme Pentose phosphate pathway

15. 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. Lipid Biosynthesis

16. 7. The acetyl groups of the mitochondrion are transported into the cytosol in the form of citrate Lipid Biosynthesis

17. 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 palmitoyl-CoA and activated by citrate. Glucagon高血糖素and epinephrine肾上腺素triggers the phosphorylation and disassociation of the polymeric enzyme subunits, which inactivates the enzyme. Lipid Biosynthesis

18. 8. The rate of fatty acid biosynthesis is controlled by acetyl-CoA carboxylase 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) Lipid Biosynthesis

19. Acetyl-CoA carboxylase is regulated by allosteric effectors and reversible phosphorylation Lipid Biosynthesis

20. Citrate partially activate the phosphorylated acetyl-CoA carboxylase Lipid Biosynthesis

21. 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 two-carbon 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. Lipid Biosynthesis

22. Palmitate is the Precursor for the biosynthesis of other fatty acids Lipid Biosynthesis

23. Fatty acyl-CoA is desaturated (oxidized) by O2 and NADPH. Lipid Biosynthesis

24. Oleate can be desaturated on Phosphatidylcholine(oftenattaching to C-2) to form linoleate and linolenate Lipid Biosynthesis

25. 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. Lipid Biosynthesis

26. 10. Eicosanoids are derived from arachidonate, 20:4 (5,8,11,14) 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). Lipid Biosynthesis

27. Prostaglandins and thromboxanes are synthesized from arachidonate Lipid Biosynthesis Cyclooxygenase activity of COX Peroxidase activity of COX

28. The dimeric bifunctional cyclooxygenase(COX-1) Tyr385, a key residue for the cyclooxygenase activity Heme for the peroxidase active site Lipid Biosynthesis Ser530 flurbiprofen

29. 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). Lipid Biosynthesis

30. 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 acyl-CoAs 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. Lipid Biosynthesis

31. Phosphatidic acid is derived from L-glycerol 3- phosphate and two acyl-CoAs. Lipid Biosynthesis Often saturated Oftenunsaturated

32. 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. Lipid Biosynthesis

33. 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. Lipid Biosynthesis

34. Eukaryotic cells use both strategies (occurring on sER and inner membrane of mitochondria) Lipid Biosynthesis Bacteria mainly use this strategy

35. Phospholipid synthesis in E. coli employs CDP-diacylglcerol Lipid Biosynthesis phosphatase decarboxylase

36. 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 CDP-diacylglycerol (from two phosphatidylglycerols in bacteria). Lipid Biosynthesis

37. 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 CDP-ethanolamine 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. Lipid Biosynthesis

38. PC and PE are made from the salvage pathway in mammals Lipid Biosynthesis (ethanolamine) (Phosphoethanolamine) (CDP-ethanolamine) (Phosphatidylthanolamine)

39. The synthesis of PE, PC, PS in eukaryotic cells. Lipid Biosynthesis

40. 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 platelet-activating factor are made using this pathway. The acyl group on 1-acyldihydroxyacetone 3-phosphate 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. Lipid Biosynthesis

41. Synthesis of the ether lipids (醚脂类） Lipid Biosynthesis 1-alkylglycerol 3-phosphate

42. 18. The sphingosine backbone of spingolipids is derived from palmitoyl-CoA and Ser Palmitoyl-CoA condenses with serine (PLP is needed for decarboxylate serine) to form b-ketosphinganine, 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. Lipid Biosynthesis

43. Spingolipid synthesis begins with the condensation between palmitoyl-CoA and Ser. Lipid Biosynthesis A glycolipid, not a phospholipid PLP (not CDP-choline!)

44. 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. Lipid Biosynthesis

45. The carbon origins of cholesterol as revealed byradioisotope labeling studies. Lipid Biosynthesis

46. The Nobel Prize in Physiology or Medicine, 1964 Lipid Biosynthesis

47. 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. Lipid Biosynthesis

48. Reactions assembling cholesterol from 18 molecules of acetyl-CoA can be divided into four stages. Lipid Biosynthesis

49. 21. Mevalonate commits the acetyl groups for cholesterol synthesis One molecule of β-hydroxy-b-methylglutaryl-CoA (HMG-CoA) is formed from three acetyl-CoA 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). Lipid Biosynthesis

50. One mevalonate is synthesized from three acetyl-CoA molecules. Lipid Biosynthesis HMG-CoA lyase in mitochondria Acetyl-CoA + acetoacetate HMG-CoA Reductase In cytosol The irreversible committing step for cholesterol biosynthesis