1. Reginald Garrett and Charles Grisham
Chapter 24
Lipid Biosynthesis
Biochemistry by
Reginald Garrett and Charles Grisham

2. Outline How Are Fatty Acids Synthesized?
How Are Complex Lipids Synthesized?
How Are Eicosanoids Synthesized, and What Are Their Functions?
How Is Cholesterol Synthesized?
How Are Lipids Transported Throughout the Body?
How Are Bile Acids Biosynthesized?
How Are Steroid Hormones Synthesized and Utilized?

3. 24.1 – How Are Fatty Acids Synthesized?
The Biosynthesis and Degradation Pathways are Different
As in cases of glycolysis/gluconeogenesis and glycogen synthesis/breakdown, fatty acid synthesis and degradation go by different routes
There are five major differences between fatty acid breakdown and biosynthesis

4. The Differences Between fatty acid biosynthesis and breakdown
Intermediates in synthesis are linked to -SH groups of acyl carrier proteins (as compared to -SH groups of CoA)
Synthesis in cytosol; breakdown in mitochondria
Enzymes of synthesis are one polypeptide (fatty acid synthase) in animals (bacteria and plants employ separate enzymes)
Biosynthesis uses NADPH/NADP+; breakdown uses NADH/NAD+ & FAD
Stereochemistry (D-b-hydroxyacyl)

5. Activated form: Malonyl-CoA
Acetate Units are Activated for Transfer in Fatty Acid Synthesis by Malonyl-CoA
Fatty acids are built from 2-C units -- acetyl-CoA
Acetyl-CoA are activated by formation of malonyl-CoA
Decarboxylation of malonyl-CoA and reducing power of NADPH drive chain growth
Chain grows to 16-carbons (palmitate)
Other enzymes add double bonds and more Cs

6. The sources of Acetyl-CoA
Amino acid degradation produces cytosolic acetyl-CoA
Fatty acid oxidation produces mitochondrial acetyl-CoA
Glycolysis yields cytosolic pyruvate which is converted to acetyl-CoA in mitochondria
Citrate-malate-pyruvate shuttle provides cytosolic acetate units and reducing equivalents for fatty acid synthesis

7. Citrate-malate-pyruvate shuttle

8. The reducing power: NAPDH
Produced in pentose phosphate pathway (chapter 22)
Produced by malic enzyme
Malate + NADP+ → pyruvate + CO2 + NADPH + H+

9. The "ACC enzyme" commits acetate to fatty acid synthesis
Acetyl-CoA Carboxylase (ACC)
Carboxylation of acetyl-CoA to form malonyl-CoA is the irreversible and committed step in fatty acid biosynthesis
ACC uses bicarbonate and ATP (& biotin)
E.coli enzyme has three subunits
Animal enzyme is one polypeptide with all three functions - biotin carboxyl carrier protein, biotin carboxylase and transcarboxylase
Acetyl-CoA + ATP + HCO3- → malonyl-CoA + ADP + Pi + H+

10. Malonyl-CoA
Figure 24.2 (a) The acetyl-CoA carboxylase reaction produces malonyl-CoA for fatty acid synthesis. (b) A mechanism for the acetyl-CoA carboxylase reaction.

11. Figure In the acetyl-CoA carboxylase reaction, the biotin ring, on its flexible tether, acquires carboxyl groups from carbonylphosphate on the carboxylase subunit and transfers them to acyl-CoA molecules on the transcarboxylase subunits.

12. Acetyl-CoA Carboxylase
ACC forms long, active filamentous polymers from inactive protomers
As a committed step, ACC is carefully regulated
Palmitoyl-CoA (product) favors monomers
Citrate favors the active polymeric form
Phosphorylation modulates citrate activation and palmitoyl-CoA inhibition
Citrate
Inactive protomers active polymer
Acyl-CoA

13. Figure Schematic of the acetyl-CoA carboxylase, with domains and phosphorylation sites indicated, along with the protein kinases responsible.

14. Phosphorylation of ACC modulates activation by citrate and inhibition by palmitoyl-CoA
The animal enzyme is phosphorylated at 8 to 10 sites on each enzyme subunit (Figure 24.4)
Unphosphorylated E
Has high affinity for citrate and is active at low [citrate]
Has low affinity for palmitoyl-CoA and needs high [palmitoyl-CoA] to inhibit
Phosphorylated E
Has low affinity for citrate and needs high [citrate] to activate
Has high affinity for palmitoyl-CoA and is inhibited at low [palmitoyl-CoA]

15. Figure 24.5 The activity of acetyl-CoA carboxylase is modulated by phosphorylation and dephosphorylation.
The dephospho form of the enzyme is activated by low [citrate] and inhibited only by high levels of fatty acyl-CoA. In contrast, the phosphorylated form of the enzyme is activated only by high levels of citrate, but is very sensitive to inhibition by fatty acyl-CoA.

16. The Acyl Carrier Protein (ACP)
The acyl carrier protein carry the intermediates in fatty acid synthesis
ACP is a 77 residue protein in E. coli - with a phosphopantetheine
The same functional group of CoA

17. Fatty Acid Synthase (FAS)
Fatty acid synthesis in mammals occurs on homodimeric fatty acyl synthase I (FAS I)
consists of 270 kD polypeptides which contain all reaction centers required to produce a fatty acid
In yeast and fungi (lower eukaryotes), the activities of FAS are distributed on two multifunctional peptide chains (a6b6)
In plants and bacteria, the enzymes of FAS are separated and independent, and this collection of enzymes is referred to as fatty acid synthase II (FAS II)

18. Structure of the Fatty Acid Synthase

19. Fatty Acid Synthesis
The individual steps of fatty acid synthesis are similar across all organisms
The mammalian pathway (Figure 24.7) is a cycle of elongation that involves six enzyme activities
Elongation is initiated by transfer of the acyl moiety of acetyl-CoA to the acyl carrier protein by the malonyl-CoA-acetyl-CoA-ACP transacylase (MAT)
This enzyme also transfers the malonyl group of malonyl-CoA to ACP

20. Fatty Acid Synthase Malonyl-CoA-acetyl-CoA-ACP transacylases (MAT)
b-ketoacyl-ACP synthase (KS)
b-ketoacyl-ACP reductase (KR)
b-hydroxyacyl-ACP dehydratase (DH)
Enoyl-ACP reductase (ER)
Thioesterase (TE) in animals

21. The pathway of palmitate synthesis from acetyl-CoA and malonyl-CoA.
Acetyl and malonyl building blocks are introduced as acyl carrier protein conjugates.
Decarboxylation drives the b-ketoacyl-ACP synthase and results in the addition of two-carbon units to the growing chain.
The first turn of the cycle begins at “1” and goes to butyryl-ACP; subsequent turns of the cycle are indicated as “2” through “6”.
Figure 24.7

22. Acetyl-ACP
Malonyl -ACP
Acetoacetyl -ACP
Butyryl -ACP
Figure 24.8 An acetyl group is transferred from CoA to MAT, then to the acyl carrier protein, and then to the ketoacyl synthase. Next a malonyl group is tranferred to MAT and then to the acyl carrier protein.

23. Fatty Acid Synthesis Acetyl-CoA + 7 malonyl-CoA + 14 NAPDH + 14 H+
Palmitoyl-CoA + 7 HCO NAPD+ + 7 CoA-SH
The formation of malonyl-CoA:
7 Acetyl-CoA + 7 HCO ATP4-
7 malonyl-CoA + 7 ADP3- + 7Pi2- +7H+
The overall reaction:
8 Acetyl-CoA + 7 ATP NAPDH + 14 H+
Palmitoyl-CoA + 14 NAPD+ + 7 CoA-SH + 7 ADP3- + 7Pi2-

24. Further Processing of C16 FAs
Additional elongation & Unsaturation
Additional elongation occurs in mitochondria and the surface of ER
In ER
Similar to fatty acid synthase
Involving malonyl-CoA
In mitochondrion (Figure 24.12)
A reversal of fatty acid oxidation
The reducing coenzyme for the second step is NADH, whereas the reductant for the fourth step is NADPH.

25. ( in mitochondria)
Figure Elongation of fatty acids in mitochondria is initiated by the thiolase reaction.

26. Introduction of cis double bonds
Prokaryotes use an O2-independent process
Eukaryotes use an O2-dependent process
E.coli add double bonds during the fatty acid synthesis
After four normal cycles, b-hydroxydecanoyl thioester dehydrase forms a double bond b, g to the thioester
O2-independent

27. Figure Double bonds are introduced into the growing fatty acid chain in E. coli by specific dehydrases.

28. Eukaryotes add double bond until the fatty acyl chain has reached its full length (usually 16 to 18 carbons)
Stearoyl-CoA desaturase
Cytochrome b5 reductase & Cytochrome b5
All three proteins are associated with the ER membrane
NADH & O2 are required; O2-dependent

29. Mammals cannot synthesize most polyunsaturated fatty acids
Plants Can manufacture double bonds between the D9 and the methyl end of the chain, but mammals cannot
Mammals can introduce double bonds between the double bond at the 8- or 9- position and the carboxyl group
Form a double bond at 5-position (6) if one already exists at the 8-position (9)
Essential fatty acids: mammals require polyunsaturated fatty acids (PUFAs) but must acquire them in their diet.

30. Arachidonic acid is systhesized from linoleic acid
Linoleic acid is acquired from diet
Mammals can add double bonds to unsaturated fatty acids
Is the precursor for prostaglandins and other biologically active derivatives
Figure Arachidonic acid is synthesized from linoleic acid in eukaryotes.

31. ω3 and ω6 – Essential Fatty Acids with Many Functions

32. Regulation of Fatty Acid Synthesis
Allosteric regulation
Citrate activates acetyl-CoA carboxylase
Fatty acyl-CoAs inhibit acetyl-CoA carboxylase
Malonyl-CoA blocks the carnitine acyltransferase and thus inhibits b-oxidation

33. Regulation of FA Synthesis
Hormone signals regulate ACC and fatty acid biosynthesis
Glucagon activates lipases/inhibits ACC
Insulin inhibits lipases/activates ACC
Phosphorylation causes inhibition of fatty acid biosynthesis
Inactivated ACC can be reactivated by a specific phosphatase induced by insulin

34. Figure Hormonal signals regulate fatty acid synthesis, primarily through actions on acetyl-CoA carboxylase. Availability of fatty acids also depends upon hormonal activation of triacylglycerol lipase.

35. 24.2 – How Are Complex Lipids Synthesized?
Complexed lipids:
Glycerolipid
Triacylglycerols
Glycerophospholipids
Sphingolipids
Phospholipids (membrane components)
Different organisms posses greatly different complements of lipids

36. Synthetic pathways depend on different organism
Sphingolipids and triacylglycerols only made in eukaryotes
Phosphatidylethanolamine (PE) accounts for 75% of phospholipids in E.coli
With phosphatidylglycerol (PG) and cardiolipin
No PC, PI, sphingolipids, cholesterol in E.coli
But some bacteria do produce PC

37. Glycerolipid Biosynthesis
Glycerolipids are synthesized by phosphorylation & acylation of glycerol
Phosphatidic acid (PA) is the precursor for all other glycerolipids in eukaryotes
Eukaryotic systems can also utilize DHAP as a starting point

38. (PA)
Figure 24.19

39. Satuated Fatty Acid
Unsatuated Fatty Acid
Figure Synthesis of glycerolipids in eukaryotes begins with the formation of phosphatidic acid, which may be formed from dihydroxyacetone phosphate or glycerol as shown.

40. Glycerolipid Biosynthesis
Phosphatidic acid (PA) is converted either to DAG or CDP-DAG
DAG is a precursor for synthesis of TAG, phosphatidylethanolamine (PE) and phosphatidylcholine (PC)
TAG is synthesized mainly in adipose tissue, liver, and intestines
CDP-DAG is a precursor for synthesis of phosphotidylserine (PS), phosphatidylglycerol (PG), cardiolipin and phosphatidylinositol (PI)

41. Figure Diacylglycerol and CDP-diacylglycerol are the principal precursors of glycerolipids in eukaryotes. Phosphatidylethanolamine and phosphatidylcholine are formed by reaction of diacylglycerol with CDP-ethanolamine or CDP-choline, respectively.

42. Glycerolipid Biosynthesis
PE synthesis
begins with phosphorylation of ethanolamine to form phosphoethanolamine
Transfer of a cytidylyl group from CTP to from CDP-ethanolamine
Phosphoethanolamine transferase link phosphoethanolamine to the DAG
Synthesis of PC is entirely analogous
PC can also be converted from PE by methylation reactions

43. Exchange of ethanolamine for serine converts PE to PS (phosphatidylserine)
Figure The interconversion of phosphatidylethanolamine and phosphatidylserine in mammals.

44. Other PLs from CDP-DAG
CDP-diacylglycerol is used in eukaryotes to produce:
Phosphatidylinositol (PI) in one step
2-8% in animal membrane
Breakdown to form inositol-1,4,5-triphosphate & DAG (second messengers)
Phosphatidylglycerol (PG) in two steps
Cardiolipin in three steps

45. Figure CDP-diacylglycerol is a precursor of phosphatidylinositol, phosphatidylglycerol, and cardiolipin in eukaryotes.

46. Plasmalogen Biosynthesis
Dihydroxyacetone phosphate (DHAP) is the precursor to the plasmalogens
Acylation of DHAP
Exchange reaction produces the ether linkage by long-chain alcohol (acyl-CoA reductase)
Ketone reduction
Acylation again
CDP-ethanolamine delivers the head group
A desaturase produces the double bond in the alkyl chain

47. Figure 24.23 Biosynthesis of plasmalogens in animals.
DHAP
1-Acyl-DHAP
1-Alkyl-DHAP
1-Alkyl-Glycero-3-P
1-Alkyl-2-acyl-Glycero-3-P
1-Alkyl-2-acyl-Glycero-3-phosphoethanolamine
Plasmalogen
Acyl-CoA
Acyl-CoA reductase

48. Figure Platelet-activating factor, formed from 1-alkyl-2-lysophosphatidylcholine by acetylation at C-2, is degraded by the action of acetylhydrolase.
Dilate blood vessels Reduce blood pressure Aggregate platelet

49. Sphingolipid Biosynthesis
High levels made in neural tissue
Initial reaction is a condensation of serine and palmitoyl-CoA (by 3-ketosphinganine synthase)
3-ketosphinganine synthase is PLP-dependent
Ketone is reduced with help of NADPH
Acylation to form N-acyl-sphinganine
Desaturated to form ceramide
Ceramide is precursor for other sphingolipids

51. Sphingolipid Biosynthesis
Ceramide is precursor for other sphingolipids
Sphingomyelin
Rich in myelin sheath
Insulates nerve axons
By transfer of phosphocholine from PC
Cerebrosides
Glycosylation of ceramide
Galactosylceramide makes up ~15% of the lipid of myelin sheath
Gangliosides
Cerebrosides contain one or more sialic acid

52. Figure Glycosylceramides (such as galactosylceramide), gangliosides, and sphingomyelins are synthesized from ceramide in animals.
Gangliosides
UDP-glucose
UDP-galactose
UDP-N-acetylgalactosamine
CMP-sialic acid
(N-acetylneuraminidate)

53. Fig. 8-10, p. 243

54. 24.3 – How Are Eicosanoid Synthesized and What Are Their Functions?
Eicosanoid are all derived from 20-carbon fatty acid (arachidonic acid)
Eicosanoids are local hormones
Exert their effect at very low concentration
Usually act near their sites of synthesis
PLA2 releases arachidonic acid from phospholipids (phosphatidylcholine)
May also be released by PLC & DAG lipase

55. Figure Arachidonic acid, derived from breakdown of phospholipids (PL), is the precursor of prostaglandins (PG), thromboxanes (Tx), and leukotrienes.
Specificities of phospholipases A1, A2, C, and D. (Figure 8.18, p. 251)

56. Eicosanoids are local hormones
Eicosanoids include
Prostaglandins (PG)
Thromboxanes (Tx)
Leukotrienes
Other hydroxyeicosanoic acid
Tissue injury and inflammation triggers arachidonate release and eicosanoid synthesis
Most PGs are cyclopentanoic acids
Initiated by PGH synthase associated with the ER

57. PGH synthase (Prostaglandin endoperoxide H synthase)
Known as Cyclooxygenase
The enzyme has two different activities:
Cyclooxygenase (COX)
Peroxidase (POX)

58. Aspirin & NSAIDs
Aspirin and other nonsteroid anti-inflammatory drugs (NSAIDs) inhibit the cyclooxygenase
Aspirin covalently
Others noncovalently
(Tylenol)
(Advil)

59. Two isoforms in animals COX-1: normal, physiological production of PG
COX-2: induced by cytokines, mitogens, and endotoxins in inflammatory cells
Aspirin and bromoaspirin bind covalently to COX-1 and COX-2.
Ibuprofen binds noncovalently to both.
Celebrex and Deramaxx bind selectively to COX-2.
COX-2 has Val (blue) at position 523, whereas COX-1 has Ile (red).

60. 24.4 – How Is Cholesterol Synthesized?
Occurs primarily in the liver
The most prevalent steroid in animal cells is cholesterol
Serve as cell membranes, precursor of bile acids and steroid hormones, and vitamin D3

61. First step is a thiolase reaction Second step makes HMG-CoA
Biosynthesis begins in the cytosol with the synthesis of mevalonate from acetyl-CoA
First step is a thiolase reaction
Second step makes HMG-CoA
Third step produces 3R-mevalonate
HMG-CoA reductase
The rate-limiting step in cholesterol biosynthesis
HMG-CoA reductase is site of regulation in cholesterol synthesis → the action site of cholesterol-lowering drugs

62. Figure 24. 32 A reaction mechanism for HMG-CoA reductase
Figure A reaction mechanism for HMG-CoA reductase. Two successive NADPH-dependent reductions convert the thioester, HMG-CoA, to a primary alcohol.

63. Regulation of HMG-CoA Reductase
As rate-limiting step, it is the principal site of regulation in cholesterol synthesis
Phosphorylation by cAMP-dependent kinases inactivates the reductase
Degradation of HMG-CoA reductase
Half-life is 3 hrs and depends on cholesterol level
High [cholesterol] means a short half-time
Gene expression (mRNA production) is controlled by cholesterol levels
If [cholesterol] is low, more mRNA is made

64. cAMP-dependent protein kinase
Figure HMG-CoA reductase activity is modulated by a cycle of phosphorylation and dephosphorylation.

65. Squalene is synthesized from Mevalonate
Six-carbon mevalonate makes 5-carbon isopentenyl pyrophosphate and dimethylallyl pyrophosphate
Condensation of these two 5-carbon intermediates produces geranyl pyrophosphate (10-carbon)
Addition of another isopentenyl pyrophosphate yields farnesyl pyrophosphate (15-carbon)
Two farnesyl pyrophosphates link to form squalene

66. Figure 24.34 The conversion of mevalonate to squalene.

68. Geranyl pyrophosphate

69. Cholesterol from Squalene
At the endoplasmic reticulum membrane
Squalene monooxygenase converts squalene to squalene-2,3-epoxide
2,3-oxidosqualene:lanosterol cyclase converts the epoxide to lanosterol
Though lanosterol looks like cholesterol, 20 more steps are required to form cholesterol
All at/in the endoplasmic reticulum membrane

70. Figure 24.35 Cholesterol is synthesized from squalene via lanosterol.
The primary route from lanosterol involves 20 steps, the last of which converts 7-dehydrocholesterol to cholesterol. An alternative route produces desmosterol as the penultimate intermediate.

71. Inhibiting Cholesterol Synthesis
HMG-CoA reductase is the key - the rate-limiting step in cholesterol biosynthesis
Lovastatin (mevinolin) blocks HMG-CoA reductase and prevents synthesis of cholesterol
Lovastatin is an inactive lactone
In the body, the lactone is hydrolyzed to mevinolinic acid, a competitive inhibitor of the reductase, Ki = 0.6 nM

72. For several years, Lipitor has been the best-selling drug in the world, with annual sales exceeding $10 Billion.
The structures of (inactive lactone) lovastatin, (active) mevinolinic acid, mevalonate, and synvinolin.

73. 24.5 – How Are Lipids Transported Throughout the Body?
Lipoproteins are the carriers of most lipids in the body
Lipoprotein - a cluster of lipids, often with a monolayer membrane (phospholipids), together with an apolipoprotein
Classify lipoproteins according to their densities
The densities are related to the relative amounts of lipid and protein
Proteins have densities of about 1.3 to 1.4 g/ml
Lipids have densities of about 0.8 g/ml

74. The various apoproteins have an abundance of hydrophobic amino acid residues, as is appropriate for interactions with lipids.

75. LDL (low density lipoprotein) not made directly, but is made from VLDL
HDL (high density lipoprotein) & VLDL (very low density lipoprotein) are assembled primarily in the ER of liver cells (some in intestines)
LDL (low density lipoprotein) not made directly, but is made from VLDL
LDL appears to be the major circulatory complex for cholesterol & cholesterol esters
Chylomicrons form in the intestines

76. VLDLs carry lipid from liver to other tissues
Chylomicrons carry TAG & cholesterol esters from intestine to other tissues
VLDLs carry lipid from liver to other tissues
In the capillaries of muscle and adipose cells, lipoprotein lipases hydrolyze triglycerides from lipoproteins, making the lipoproteins smaller and raising their density
Thus chylomicrons and VLDLs are progressively converted to IDL (intermediate-density lipoprotein) and then LDL, which either return to the liver for reprocessing or are redirected to adipose tissues and adrenal glands

77. Figure Lipoprotein components are synthesized predominantly in the ER of liver cells. Following assembly of lipoprotein particles (red dots) in the ER and processing in the Golgi, lipoproteins are packaged in secretory vesicles for export from the cell (via exocytosis) and released into the circulatory system.

78. Figure 24.39 Endocytosis and degradation of lipoprotein particles.
ACAT is acyl-CoA cholesterol acyltransferase.
Figure Endocytosis and degradation of lipoprotein particles.

79. Lipoproteins
LDLs are main carriers of cholesterol and cholesterol esters
Newly formed HDL contains virtually no cholesterol esters
Cholesterol esters are accumulated through the action lecithin:cholesterol acyltransferase (LCAT)
HDLs function to return cholesterol and cholesterol esters to the liver

80. The LDL Receptor
The LDL receptor in plasma membranes consists of 839 amino acid residues and is composed of five domains
D1: LDL binding domain on N-terminus
D2 & D3: N-linked and O-linked oligosaccharide domains
D4: A single TMS
D5: A cytosolic domain essential to aggregation of receptors in the membrane during endocytosis
Dysfunctions in or absence of LDL receptors lead to familial hypercholesterolemia

81. 24.6 – How Are Bile Acids Biosynthesized?
Carboxylic acid derivatives of cholesterol
Essential for the digestion of food, especially for solubilization of ingested fats
Synthesized from cholesterol in the liver, stored in the gallbladder, and secreted as need into the intestine
Cholic acid conjugates with taurine and glycine to form taurocholic and glycocholic acids
First step is oxidation of cholesterol by a mixed-function oxidase (p-450, 7a-hydroxylase)

84. 24.7 – How Are Steroid Hormones Synthesized and Utilized?
Steroid hormones are crucial signal molecules
Biosynthesis begins with the desmolase reaction (in mitochondria), which converts cholesterol to pregnenolone, precursor to all others
Pregnenolone migrates from mitochondria to ER where progesterone is formed
Progesterone is a branch point - it produces sex steroids (testosterone and estradiol), and corticosteroids (cortisol and aldosterone)

85. (27C)
(21C)
(19C)
aromatase
Figure The steroid hormones are synthesized from cholesterol
(18C)

86. Anabolic steroids are illegal and dangerous