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Plant biochemistry II; Lipid Anabolism

Plant biochemistry II; Lipid Anabolism. Andy Howard Biochemistry Lectures, Fall 2010 8 November 2010. Plant biochemistry; lipid anabolism. We’ll conclude our study of plant biochemistry Then we’ll discuss the anabolic pathways associated with lipids. Plant biochemistry CAM control

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Plant biochemistry II; Lipid Anabolism

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  1. Plant biochemistry II; Lipid Anabolism Andy HowardBiochemistry Lectures, Fall 20108 November 2010 Plants II; lipid anabolism

  2. Plant biochemistry;lipid anabolism • We’ll conclude our study of plant biochemistry • Then we’ll discuss the anabolic pathways associated with lipids Plants II; lipid anabolism

  3. Plant biochemistry CAM control Bacterial compartmentation Fatty Acid Synthesis Activation Elongation Special topics Other anabolic pathways for lipids Phospholipid synthesis Synthesis of other glycerol-dependent lipids What we’ll discuss Plants II; lipid anabolism

  4. iClicker question 1 Starch can be degraded two ways. The most important difference between them is: • (a) Starch phosphorylase requires energy; amylase produces it • (b) Starch phosphorylase breaks off one sugar unit at a time; amylase splits amylopectin into oligosaccharides • (c) Amylase is a larger enzyme • (d) Starch phosphorylase is never found in animals; amylase is • (e) None of the above Plants II; lipid anabolism

  5. iClicker quiz question 2 Why would you not expect to find crassulacean acid metabolism in tropical plants? • (a) Tropical plants do not photosynthesize. • (b) Tropical plants cannot develop the stomata that close off the chloroplast-containing cavities • (c) Water conservation is less critical in areas of high rainfall • (d) The waxy coating required to close off the leaves’ access to O2 would dissolve in the high humidity and high temperature of the tropics • (e) None of the above Plants II; lipid anabolism

  6. Answer: (c) • The primary significance of CAM is conservation of water in regions of low humidity, where evaporation rates are high and water is scarce. Neither of these conditions pertains in the tropics. Plants II; lipid anabolism

  7. Control of CAM • PEP carboxylase inhibited by malate and low pH • That prevents activity during daylight, which would lead to futile cycling and competition for CO2 between PEP carboxylase and RuBisCO Plants II; lipid anabolism

  8. Compartmentation in bacteria • In photosynthetic bacteria, RuBisCO is concentrated in protein microcompartment called a carboxysome • Active carbonic anhydrase there: catalyzes HCO3- OH- + CO2 • That tends to keep the CO2 / O2 ratio high Plants II; lipid anabolism

  9. Carboxysome structure • Icosahedral particle • Contains many copies of RuBisCO: many hexamers, some pentamers • Some auxiliary proteins present also • Resembles viral capsid Plants II; lipid anabolism

  10. Making and Breaking Lipids • Lipid biosynthesis is a significant route to the creation of energy-storage molecules, membrane components, and hormones; • Lipid catabolism is a critical energy-producing pathway, and we also need to understand degradation of functional lipids Plants II; lipid anabolism

  11. Lipids:What we won’t cover today • Special Cases • Locations for synthesis • Regulation by hormones • Absorption and mobilization • Ketone bodies • Catabolism Plants II; lipid anabolism

  12. Lipid Anabolism Malonyl CoA • Generally the starting point for building up lipids are acetyl CoA and malonyl CoA, and their variants acetyl ACP and malonyl ACP • Fatty acids • Steroids • These are energy-requiring reactions: the compounds we’re making are reduced Plants II; lipid anabolism

  13. Overview(cf. fig. 16.1) Acetoacetyl ACP • Bacteria:acetyl CoA + malonyl ACP acetoacetyl ACP + CO2 + CoASH • Eukaryotes: • acetyl CoA + ACP acetyl ACP + CoASH • Acetyl ACP + malonyl ACP  acetoacetyl ACP + CO2 + ACP Plants II; lipid anabolism

  14. PDB 1W96 (biotin carboxylase domain) 183 kDa trimer YeastEC 6.4.1.2, 1.8Å Making malonyl CoA • Acetyl CoA incorporates an extra carboxyl via acetyl CoA carboxylase:HCO3- + ATP + acetyl CoA  ADP + Pi + malonyl CoA • Biotin- and ATP-dependent ligase enzyme; similar to pyruvate carboxylase 1UYR (carboxyl-transferase domain), 2.5Å 162 kDa dimer; yeast Plants II; lipid anabolism

  15. Making malonyl ACP • Malonyl CoA:ACPtransacylasetransfers the malonate group from coenzyme A to the acyl carrier protein • Ferredoxin-like protein • Similar enzyme converts acetyl CoA to acetyl ACP E.coli Malonyl CoA: ACP transacylaseE.C. 2.3.1.39PDB 1MLA, 1.5Å 32kDa monomer Plants II; lipid anabolism

  16. Acyl carrier protein itself • Acts as a template on which acyl chain elongation can occur • Simple protein: 83 amino acids, mostly helical • This is actually an NMR structure PDB 1OR59.1 kDa monomerStreptomyces Plants II; lipid anabolism

  17. Initiation reaction • We want to start with a four-carbon unit attached to acyl carrier protein • We get that by condensing acetyl CoA or acetyl ACP with malonyl ACP with ketoacyl ACP synthase (KAS) to form acetoacetyl ACP • Intermediate has KAS covalently attached to both substrates • Decarboxylation of enzyme-bound intermediates leads to 4-carbon unit attached to ACP3 + 2  1 + 4 Plants II; lipid anabolism

  18. Is this typical? Yes! • We’ve carboxylated acetyl CoA to make malonyl ACP and then decarboxylated the product of malonyl ACP with acetyl CoA / ACP • This provides a favorable free-energy change (at the expense of ATP) for the overall reaction • Similar approach happens in gluconeogenesis(pyruvate  oxaloacetate  PEP) E.coli Ketoacyl ACP synthasePDB 1HNJ70 kDa dimer;monomer shownEC 2.3.1.41, 1.46Å Plants II; lipid anabolism

  19. Elongations in FA synthesis: overview • Acetoacetyl ACP: starting point for elongations • Pattern in each elongation isreduction  dehydration  reduction,resulting in a saturated product • Reenter pathway by condensing with malonyl ACP • Elongated product plays the same role that acetyl CoA or acetyl ACP plays in the initial -ketoacyl ACP synthase reaction: C2n + C3 CO2 + C2n+2 Plants II; lipid anabolism

  20. 1st step: reduce ketone  sec-alcohol • Enzyme:3-ketoacylACP reductase • Ketone reacts with NADPH+ H+ to produce sec-alcohol + NADP+ • D-isomer of sec-alcohol always forms;by contrast, during degradation,L-isomer forms • Enzyme is typical NAD(P)-dependent oxidoreductase PDB 2C07125 kDa tetramer; Monomer shownPlasmodium falciparumEC 1.1.1.100, 1.5Å Plants II; lipid anabolism

  21. 2nd step: alcohol to enoyl ACP • 3-hydroxyacyl ACP dehydratase • Eliminates water at beta, alphapositions to producetrans-2-enoyl ACP:R–CHOH–CH2-CO-S-ACP R–CH=CH–CO-S-ACP + H2O • Note that this is a derivative of atrans-fatty acid; but it’s complexed to ACP! • This form is primarily helical;there is an alternative found in Aeromonas that is an alpha-beta roll structure PDB 1DCI182 kDa hexamertrimer shownRat mitochondriaEC 4.2.1.17, 1.5Å Plants II; lipid anabolism

  22. 3rd step:enoyl CoA to saturated ACP PDB 2Z6I73 kDa dimerStreptococcus pneumoniaeEC 1.3.1.19, 1.7Å • Enzyme: enoyl-ACP reductase • Leaves behind fully saturated FAcomplexed to acyl carrier protein:R–CH=CH–CO-S-ACP R–CH2CH2CO-S-ACP • This can then condense with malonyl ACP with decarboxylation to form longer beta-ketoacyl ACP:Rn-ACP + malonyl-CoA -keto-Rn+2-ACP + CO2 + CoASH • Enzyme is FMN-dependent Plants II; lipid anabolism

  23. How does this end? • Generally starts at C4 and goes to C16 or C18. • Condensing enzyme won’t fit longer FAs • Completed fatty acid is cleaved from ACP by action of a thioesterasewith a 3-layer Rossmann fold Palmitoyl thioesterase IPDB 1EI9 31 kDa monomerbovineEC 3.1.2.22, 2.25Å Plants II; lipid anabolism

  24. The overall reaction • Acetyl CoA + 7 Malonyl CoA + 14NADPH + 14 H+ 14 NADP + Palmitate + 7CO2 + 8HS-CoA + 6H2O • In bacteria we have separate enzymes:a type II fatty acid synthesis system • In animals we have a type I FA synthesis system: a large, multi-functional enzyme including the phosphopantatheine group by which the ACP attaches Plants II; lipid anabolism

  25. iClicker question 3 What advantage, if any, might be associated with type I fatty acid synthesis systems? • (a) None • (b) Lowered probability of undesirable oxidations of metabolites • (c) Lowered probability of undesirable reductions of metabolites • (d) Reactants remain associated with the enzymatic complex, reducing diffusive inefficiencies • (e) improved solubility of products Plants II; lipid anabolism

  26. Answer: (d) • If the enzyme doesn’t have to find the substrate at the beginning of each reaction, things will proceed more readily. Plants II; lipid anabolism

  27. Activating fatty acids • Activate stearate or palmitatevia acyl CoA synthetase: • R–COO- + CoASH + ATP R–CO–SCoA + AMP + PPi • As usual, PPi hydrolysis drivesthe reaction to the right • PLP-dependent reaction • Bacteria have one acyl CoA synthetase • Mammals: four isozymes for different FA lengths (small, medium, long, very long) PDB 1BS042 kDa monomerE.coliEC 2.3.1.47, 1.65Å Plants II; lipid anabolism

  28. Extending and unsaturating fatty acids • There are applications for FAs with more than 18 carbons and FAs with ≥ 1 cis double bonds • Elongases and desaturases exist to handle these needs (fig. 16.7) • Desaturase adds a cis-double bond; if the FA already has unsaturations, the new one is added three carbons closer to the carboxyl • Elongases condense FA with malonyl CoA; decarboxylation means we add two carbons Plants II; lipid anabolism

  29. Bacterial Desaturases • Acyl ACP desaturases in bacteria simply add a cis double bond in place of the normal trans double bond at the second phase of elongation; the cis double bond thus created remains during subsequent rounds • Ferritin-like structure PDB 1ZA0;30 kDa monomerMycobacterium tuberculosisEC 1.14.19.2, 2Å Plants II; lipid anabolism

  30. Eukaryotic Desaturases • Desaturases like stearoyl ACP desaturase in eukaryotes act on the completed saturated fatty acyl CoA species • Enzyme is ferritin-like or RNR-like • Mammals can’t synthesize linoleate and they need it, so it has to be part of the diet PDB 1OQ9 80 kDa dimermonomer showncastor beanEC 1.14.19.2, 2.4Å Plants II; lipid anabolism

  31. Making arachidonate • We can convert dietary linoleate to archidonyl CoA via desaturation and elongations (fig. 16.7) • The fact that the new double bonds start 3 carbons away from the previous one means they’re not conjugated Plants II; lipid anabolism

  32. We’re done with fatty acid synthesis! • We’ll study FA degradation Wednesday after the quiz • There are several other important anabolic pathways associated with lipids, though. Plants II; lipid anabolism

  33. Phosphatidates • Phosphatidates are intermediates in making triacylglycerol & glycerophospholipids • Fatty acyl groups esterifying 1 and 2 positions of glycerol, phosphate esterifying 3 position Plants II; lipid anabolism

  34. Making phosphatidates • Glycerol-3-phosphate acyltransferase transfers acyl CoA to 1 position of glycerol-3-phosphate; prefers saturated chains • 1-acylglycerol-3-phosphate acyl transferase transfers acyl CoA to 2 position of resulting molecule; prefers unsaturated chains Glycerol-3-P acyltransferasePDB 1IUQ40 kDa monomer CucurbitaEC 2.3.1.15, 1.55Å Plants II; lipid anabolism

  35. Making triacylglycerols and phospholipids • Phosphatidate phosphatase gets rid of the phosphate at the 3 position by hydrolysis to make 1,2-diacylglycerol • A bit counterintuitive in making phospholipids: why get rid of the phosphate when you’re going to put a phosphorylated compound back at 3 position? • But the groups you add already have phosphate on them! Plants II; lipid anabolism

  36. Further steps in making triacylglycerols • Diacylglycerol acyltransferase (EC 2.3.1.20; no structures currently available) catalyzes reaction between 1,2-diacylglycerol and acyl CoA to form triacylglycerol • See fig. 16.9, left-hand side Plants II; lipid anabolism

  37. Making phospholipids from 1,2-diacylglycerol • 1,2-diacylglycerol reacts with CDP-choline to form phosphatidylcholine with liberation of cytidine monophosphate • 1,2-diacylglycerol reacts with CDP-ethanolamine to form phosphatidylethanolamine • this can be methylated 3 times to make phosphatidylcholine • S-adenosylmethionine is the methyl donor in that case Plants II; lipid anabolism

  38. CDP-ethanolamine How do we get CDP-alcohols? • Easy:CTP + alcohol phosphate CDP-alcohol + PPi • As usual, reaction is driven to the right by hydrolysis of PPi • Enzymes are CTP:phosphoethanolamine cytidylyltransferase and CTP:phosphocholine cytidylyltransferase Plants II; lipid anabolism

  39. Making acidic phospholipids • Phosphatidate activated to CDP-diacylglycerol as catalyzed by CTP:phosphatidate cytidylyltransferase with release of PPi (see previous reactions) • This can react with serine or inositol to form the relevant phospholipids; see fig. 16.10. • This route to phosphatidylserine is found only in bacteria Plants II; lipid anabolism

  40. Bacterial approach • Phosphatidylserine synthase:CDP-diacylglycerol + serine CMP + phosphatidylserine • Illustrates the fact that each of the four RNA nucleotides has its own special role in biosynthesis PDB 3HSI161kDa homotrimerEC 2.7.8.8, 2.2ÅHaemophilus Plants II; lipid anabolism

  41. Phosphatidylserine Plants II; lipid anabolism

  42. Phosphatidylinositol • Phosphatidylinositol is made by this CDP-diacylglycerol pathway in bacteria and eukaryotes Plants II; lipid anabolism

  43. Making phosphatidylserine • Alternative approach to phosphatidylserine found in eukaryotes:make phosphatidylethanolamine, then phosphatidylethanolamine:serine transferase swaps serine for ethanolamine • When we do it that way, we can recover phosphatidylethanolamine back by a decarboxylation (or another exchange) • Ethanolamine is just serine without COO- ! Plants II; lipid anabolism

  44. Where does this happen? • Mostly in the endoplasmic reticulum in eukaryotes • Biosynthesis enzymes are membrane bound but have their active sites facing the cytosol so they can pick up the water-soluble metabolites from which they can build up phospholipids and other lipids Plants II; lipid anabolism

  45. Making eicosanoids • Classes of eicosanoids: • Prostaglandins and thromboxanes • Leukotrienes • Remember that we make arachidonate from linoleoyl CoA; eiconsanoids made from arachidonate • Reactions involve formation of oxygen-containing rings; thus the enzymes are cyclooxygenases Plants II; lipid anabolism

  46. What eiconsanoids do • They’re like hormones, but they act very locally: within µm of the cell in which they’re produced • Involved in platelet aggregation, blood clots, constriction of smooth muscles • Mediate pain sensitivity, inflammation, swelling • Therefore enzymes that interconvert them are significant drug targets! Plants II; lipid anabolism

  47. Prostaglandin H2 Synthesizing prostaglandins • Prostaglandin H synthase (PGHS) binds on inner surface of ER • Cyclooxygenase activity makes a hydroperoxide; this is converted to PGH2 • PGH2 gets converted to other prostaglandins, prostacyclin, thromboxane A2 (fig. 16.12) PDB 1Q4G132 kDa dimer SheepEC 1.14.99.12.0Å Plants II; lipid anabolism

  48. How aspirin works • Aspirin blocks irreversibly inhibits the COX activity of PGHS by transferring an acetyl group to an active-site Ser • That blocks eiconsanoid production, which reduces swelling and pain • But there are side effects because some PGHS isozymes are necessary Plants II; lipid anabolism

  49. Cyclooxygenase inhibition • Cox-1 is constitutive and regulates secretion of mucin in the stomach • Cox-2 is inducible and promotes inflammation, pain, fever • Aspirin inhibits both: the mucin-secretion inhibition means that causes bleeding or ulcers in the stomach lining • Other nonsteroidal anti-inflammatories (NSAIDs) besides aspirin compete with arachidonate rather than binding covalently to COX-1 and COX-2 Plants II; lipid anabolism

  50. Could we find a COX-2 inhibitor? • This would eliminate the stomach irritation that aspirin causes • Some structure-based inhibitors have been developed • They work as expected; but • They also increase risk of cardiovascular disease • Prof. Prancan (Rush U) discussed these issues in his February 2007 colloquium Plants II; lipid anabolism

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