1. Biosynthesis of Membrane Lipids, Cholesterol, Steroids and Isoprenoids CH353 February 5, 2008

2. Summary Review of membrane lipid structures and nomenclature –Lehninger Chapter 10 Biosynthesis of membrane lipid components –Lehninger Chapters 21.3, 21.4 Membrane Lipids –glycerolipids –sphingolipids –sterols (cholesterol) Other Complex Lipids –steroids –isoprenoids

3. Membrane Lipids: Glycerolipids Glycerophospholipid Plasmalogen (ether lipid)

4. Glycerophospholipid Head Groups

5. Membrane Lipids: Sphingolipids Sphingomyelins with phosphocholine or phosphoethanolamine Neutral Glycolipids cerebrosides (1 sugar) globosides (> 2 sugars) Gangliosides complex carbohydrates with sialic acid (Neu5Ac)

6. Membrane Lipids: Sterols Sterols are polymerized from isoprene units Rigid 4-ring structure Membrane sterols: –Cholesterol (animals) –Stigmasterol (plants) –Ergosterol (fungi) –None in bacteria

7. Biosynthesis of Membrane Lipids Glycerolipids Sphingolipids Cholesterol

8. Biosynthesis of Phosphatidic Acid ATPADP glycerol kinase NADH NAD + glycerol 3-phosphate dehydrogenase

9. Biosynthesis of Glycerophospholipids Strategy 1: Prokaryotes: all glycerophospholipids Eukaryotes: phosphatidylinositol phosphatidyglycerol cardiolipin phosphatidylserine (yeast) Strategy 2: phosphatidylcholine phosphatidylethanolamine

10. Glycerophospholipid Biosynthesis in E. coli Strategy 1 (CDP-DAG) phosphatidylserine (PS) by serine replacing CMP phosphatidylethanolamine (PE) by decarboxylation of PS phosphatidylcholine (PC) by methylation (3x) of PE phosphatidylglycerol (PG) by glycerol 3-phosphate replacing CMP, then phosphatase cardiolipin by one PG replacing glycerol on other PG

11. Biosynthesis in Eukaryotes of Anionic Glycerophospholipids Strategy 1 (CDP-DAG) phosphatidylglycerol (PG) by glycerol 3-phosphate replacing CMP, then phosphatase cardiolipin by PG replacing CMP on CDP-DAG [CDP-DAG instead of PG] phosphatidylinositol (PI) by inositol replacing CMP phosphorylation of PI at positions 4 and 5

12. Cardiolipin Biosynthesis Summary Phosphatidylglycerol Glycerol CDP-diacylglycerol CMP cardiolipin synthase (prokaryotic) cardiolipin synthase (eukaryotic)

13. Biosynthesis of Phosphatidylcholine and Phosphatidylethanolamine in Mammals Strategy 2: CDP-alcohol choline is phosphorylated and cytidylated to form CDP-choline phosphatidylcholine (PC) formed by diacylglycerol replacing CMP phosphatidylethanolamine (PE) formed by analogous pathway starting with ethanolamine salvage pathways for choline and ethanolamine in yeast

14. Biosynthesis of Phosphatidylserine in Mammals Head group exchange Mammals cannot directly make phosphatidylserine (PS) PS formed by exchanging serine for ethanolamine on PE (endoplasmic reticulum) Mammals can decarboxylate PS to form PE (mitochondria) PC can be made from PE in mammalian liver Salvage pathways in yeast

15. Summary of Pathways to Phosphatidylcholine and Phosphatidyethanolamine Enzymes for PE and PC: kinases cytidylate transferases DAG transferases methyltransferases (in liver) Also in Mammals: PE ↔ PS exchange PS → PE decarboxylation Not in Mammals: direct PS biosynthesis from CDP-DAG + serine

16. Biosynthesis of Glycerophospholipids Summary of Strategies: CDP-diacylglycerol + alcohol (head group) CDP-alcohol + diacylglycerol Head group exchange Head group modification (methylation, decarboxylation)

17. Biosynthesis of Ether Lipids & Plasmalogens 2 NADPH required for reducing carboxylate to alcohol 1 NADPH for reducing dihydroxyacetone phosphate

18. Biosynthesis of Ether Lipids and Plasmalogens CDP-Ethanolamine substrate for CDP- ethanolamine transferase (correction) Long chain alcohol in ether linkage oxidized with mixed-function oxidase (monooxygenase) CDP-ethanolamine transferase CDP-ethanolamine CDP

19. Serine decarboxylated and condensed on acyl-CoA NADPH reduces resulting ketone Mixed-function oxygenase forms double bond of sphingosine UDP-glucose for cerebroside PC exchange for sphingomyelin Sphingolipid Biosynthesis

20. Ceramide UDP- Glucose UDP UDP- Galactose UDP GlcGal Ceramide UDP- N-Acetylgalactoseamine UDP GlcGal Ceramide GlcGal NAc Neu NAc CMP- Sialic Acid CMP GM2, a ganglioside

21. Cholesterol Biosynthesis Cholesterol is made in 4 stages: 1.Condensation of Mevalonate from 3 Acetates 2.Conversion of Mevalonate into Two Activated Isoprenes 3.Polymerization of 6 Activated Isoprenes into Squalene 4.Cyclization of Squalene and Modification of Lanosterol

22. Cholesterol Biosynthesis Stage 1: Condensation of Mevalonate from Acetate 1.Final step in β- oxidation of fatty acids in reverse (cytosolic) 2.Aldol condensation at C3 carbonyl to form HMG-CoA 3.Reduction of HMG-CoA Committed step in biosynthesis of isoprenes Requires 2 NADPH for reduction of carboxylate to alcohol

23. Cholesterol Biosynthesis Stage 2: Conversion of Mevalonate to Activated Isoprenes Requires 3 ATP’s in 4 enzymatic steps

24. Stage 3: Polymerization of Activated Isoprenes Farnesyl-PP requires: –1 Dimethylallyl-PP –2 Δ 3 -Isopentenyl-PP ( head to tail polymerization) Squalene requires: –2 farnesyl-PP ( head to head polymerization) 1 NADPH required Cholesterol Biosynthesis

25. Stage 4: Cyclization of Squalene and Modification of Lanosterol Monooxygenase forms squalene 2,3-epoxide Cyclase reaction: –H + opens epoxide ring –Cascade of 4 carbocation additions to C=C’s form the 4 rings –2 hydride migrations, 2 methyl migrations, and H + loss gives lanosterol Modification of lanosterol (19 steps) gives cholesterol

26. Cholesterol Biosynthesis Stage 4: Conversion of Lanosterol to Cholesterol 19-Step process involves: Oxidative removal of 3 methyl groups as HCO 2 H or CO 2 10 Monooxygenase reactions Oxidation of 15 NAD(P)H Reduction of 2 NAD + Overall Cholesterol Biosynthesis: 18 ATP hydrolyzed 27 NAD(P)H oxidized (net) from Risley 2002, J. Chem. Educ. 79: 377 This Slide FYI only – Not on Final Exam

27. Metabolic Fates of Cholesterol OHOH 7α-hydroxycholesterol cholesterol pregnenolone 7α-hydroxylasedesmolase 7-dehydrocholesterol reductase hνhν cholecalciferol (Vitamin D 3 ) Bile (Salts) Acids Catabolism Steroid HormonesVitamin D 7α-hydroxylase and desmolase are cytochrome P-450 monooxygenases

28. Cytochrome P-450 Monooxygenases usually located in smooth endoplasmic reticulum involved in hydroxylation of steroids or xenobiotics General Reaction: AH + BH 2 + O–O → A–OH + B + H 2 O

29. Biosynthesis of Pregnenolone Steroid hormone synthesis from cholesterol side chain removed in mitochondria of steroidogenic tissues Desmolase is a cytochrome P-450 mixed-function oxidase (monooxygenase) 2 O 2 introduce diols at C20, C22 3 rd oxidation cleaves the C–C bond with ketone and aldehyde products

30. Steroid Hormones Pregnenolone

31. Vitamin D Metabolism in skin: 7-dehydrocholesterol absorbs ultraviolet B (~300 nm) previtamin D 3 isomerizes to cholecaliferol (vitamin D 3 ) in liver: vitamin D 3 → 1-hydroxyvitamin D 3 [1-(OH)D 3 ] in kidney: 1-(OH)D 3 → 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] Final 2 steps involve cytochrome P-450 monooxygenases

32. Bile (Salts) Acids 7 hydroxycholesterol hydroxylated and oxidized carboxylate is activated with CoA amino groups of glycine or taurine attack activated carboxylate trihydroxycoprostanoate7α-hydroxycholesterol glycine taurine cholyl CoA glycocholate taurocholate OHOH

33. Isoprenoid Compounds and Derivatives

34. Isoprenoid Biosynthesis Dimethylallyl-PPΔ 3 -Isopentenyl-PP Geranyl-PP Farnesyl-PP Geranylgeranyl-PP Oligoprenyl-PP Squalene Stigmasterol Lanosterol Ergosterol Cholesterol Carotenoids x (3 – 7) Polyprenyl-PP x (8 – 21) Phytyl-PP Chlorophyll Tocopherols (Vitamin E) Phylloquinone (Vitamin K) Plastoquinone Ubiquinone (Coenzyme Q) Dolichol x 2 Retinoids (Vitamin A) x 2 Mevalonate Vitamin DBile Salts Steroid Hormones HMG-CoA Statins C10 C15 C20 C30-50 C55-120

35. Inhibitors of HMG-CoA Reductase Statins: synthetic analogs of mevalonate Competitive inhibitors of HMG-CoA reductase For inhibiting cholesterol synthesis

36. Study Problem Statins are widely prescribed drugs for lowering high cholesterol which may lead to atherosclerosis They are effective in preventing synthesis of cholesterol by inhibiting HMG-CoA reductase Since statins effectively block the entire isoprenoid pathway, there are concerns of potential side effects What possible metabolic consequences may statins have by inhibiting isoprenoid biosynthesis? What dietary supplements may be prescribed for overcoming possible side effects of statins?