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Plant biochemistry II; Lipid Anabolism
Andy Howard Biochemistry Lectures, Fall November 2010 11/08/2010 Plants II; lipid anabolism
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Plant biochemistry; lipid anabolism
We’ll conclude our study of plant biochemistry Then we’ll discuss the anabolic pathways associated with lipids 11/08/2010 Plants II; lipid anabolism
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What we’ll discuss Other anabolic pathways for lipids
Phospholipid synthesis Synthesis of other glycerol-dependent lipids Plant biochemistry CAM control Bacterial compartmentation Fatty Acid Synthesis Activation Elongation Special topics 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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. 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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Carboxysome structure
Icosahedral particle Contains many copies of RuBisCO: many hexamers, some pentamers Some auxiliary proteins present also Resembles viral capsid 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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Lipids: What we won’t cover today
Special Cases Locations for synthesis Regulation by hormones Absorption and mobilization Ketone bodies Catabolism 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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PDB 1W96 (biotin carboxylase domain)
183 kDa trimer Yeast EC , 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 11/08/2010 Plants II; lipid anabolism
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Making malonyl ACP Malonyl CoA:ACP transacylase transfers 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 transacylase E.C PDB 1MLA, 1.5Å 32kDa monomer 11/08/2010 Plants II; lipid anabolism
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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 1OR5 9.1 kDa monomer Streptomyces 11/08/2010 Plants II; lipid anabolism
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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 ACP 1 + 4 11/08/2010 Plants II; lipid anabolism
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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 synthase PDB 1HNJ 70 kDa dimer; monomer shown EC , 1.46Å 11/08/2010 Plants II; lipid anabolism
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Elongations in FA synthesis: overview
Acetoacetyl ACP: starting point for elongations Pattern in each elongation is reduction 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 11/08/2010 Plants II; lipid anabolism
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1st step: reduce ketone sec-alcohol
Enzyme:3-ketoacyl ACP 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 2C kDa tetramer; Monomer shown Plasmodium falciparum EC , 1.5Å 11/08/2010 Plants II; lipid anabolism
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2nd step: alcohol to enoyl ACP
3-hydroxyacyl ACP dehydratase Eliminates water at beta, alpha positions to produce trans-2-enoyl ACP: R–CHOH–CH2-CO-S-ACP R–CH=CH–CO-S-ACP + H2O Note that this is a derivative of a trans-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 1DCI 182 kDa hexamer trimer shown Rat mitochondria EC , 1.5Å 11/08/2010 Plants II; lipid anabolism
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3rd step: enoyl CoA to saturated ACP
PDB 2Z6I 73 kDa dimer Streptococcus pneumoniae EC , 1.7Å Enzyme: enoyl-ACP reductase Leaves behind fully saturated FA complexed 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 11/08/2010 Plants II; lipid anabolism
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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 thioesterase with a 3-layer Rossmann fold Palmitoyl thioesterase I PDB 1EI9 31 kDa monomer bovine EC , 2.25Å 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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Answer: (d) If the enzyme doesn’t have to find the substrate at the beginning of each reaction, things will proceed more readily. 11/08/2010 Plants II; lipid anabolism
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Activating fatty acids
Activate stearate or palmitate via acyl CoA synthetase: R–COO- + CoASH + ATP R–CO–SCoA + AMP + PPi As usual, PPi hydrolysis drives the 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 1BS0 42 kDa monomer E.coli EC , 1.65Å 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 monomer Mycobacterium tuberculosis EC , 2Å 11/08/2010 Plants II; lipid anabolism
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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 dimer monomer shown castor bean EC , 2.4Å 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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. 11/08/2010 Plants II; lipid anabolism
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Phosphatidates Phosphatidates are intermediates in making triacylglycerol & glycerophospholipids Fatty acyl groups esterifying 1 and 2 positions of glycerol, phosphate esterifying 3 position 11/08/2010 Plants II; lipid anabolism
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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 acyltransferase PDB 1IUQ 40 kDa monomer Cucurbita EC , 1.55Å 11/08/2010 Plants II; lipid anabolism
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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! 11/08/2010 Plants II; lipid anabolism
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Further steps in making triacylglycerols
Diacylglycerol acyltransferase (EC ; no structures currently available) catalyzes reaction between 1,2-diacylglycerol and acyl CoA to form triacylglycerol See fig. 16.9, left-hand side 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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How do we get CDP-alcohols?
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 11/08/2010 Plants II; lipid anabolism
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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 This route to phosphatidylserine is found only in bacteria 11/08/2010 Plants II; lipid anabolism
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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 3HSI 161kDa homotrimer EC , 2.2Å Haemophilus 11/08/2010 Plants II; lipid anabolism
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Phosphatidylserine 11/08/2010 Plants II; lipid anabolism
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Phosphatidylinositol
Phosphatidylinositol is made by this CDP-diacylglycerol pathway in bacteria and eukaryotes 11/08/2010 Plants II; lipid anabolism
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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- ! 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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! 11/08/2010 Plants II; lipid anabolism
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Synthesizing prostaglandins
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 ) PDB 1Q4G 132 kDa dimer Sheep EC Å 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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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 11/08/2010 Plants II; lipid anabolism
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Leukotrienes Leukotriene B4 Lipoxygenases convert arachidonate to these compounds, which contain 3 conjugated double bonds These compounds interact with GPCRs Involved in inflammatory and allergic reactions Also involved in the pathophysiology of asthma PDB 2P0M 146 kDa dimer rabbit EC Å 11/08/2010 Plants II; lipid anabolism
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Synthesis of ether lipids
Remember: these are lipids with ether linkages instead of acyl linkages Begins with dihydroxyacetone phosphate Acyltransferase acylates DHAP C-1 1-alkyl-DHAP synthase swaps an alcohol for the acyl group at C-1 Keto group at C2 of DHAP is reduced to an alcohol (NADPH-dependent reaction) 11/08/2010 Plants II; lipid anabolism
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Ether lipids, continued
1-alkylglycerophosphate acyltransferase adds another acyl group at C-2 Dephosphorylated at C-3 (as with phospholipids … take the P off, put it back on …) Phosphocholine or other phosphate-based ligand added at C-3 Plasmalogens earn a double bond between the two carbons adjacent to the ether oxygen on C-1 11/08/2010 Plants II; lipid anabolism
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Sphingolipid synthesis
ceramide Sphingolipid synthesis These are based formally on sphingosine, a C18 unsaturated amino alcohol (fig.16.14) Condense serine with palmitoyl CoA to make 3-ketosphinganine and CO2 NADPH-reduce this to sphinganine Acetylate the amine group to make N-acylsphinganine Beta-unsaturate the palmitoyl group to make ceramide, the basis for all other sphingolipids 11/08/2010 Plants II; lipid anabolism
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Serine palmitoyl-transferase
Catalyzes 1st step in this pathway: Serine + palmitoyl CoA 3-ketodihydrosphingosine + CO2 + CoASH Can accept other amino acids as substrates PLP-dependent enzyme PDB 2JG2 Sphingomonas paucimobilis EC , 1.3Å 91kDa dimer; monomer shown 11/08/2010 Plants II; lipid anabolism
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spingomyelin Other sphingolipids React ceramide with phosphatidylcholine; products are sphingomyelin and 1,2-diacylglycerol React ceramide with UDP-galactose to form a galactocerebroside Additional UDP-sugars or CMP-N-acetyl-neuraminic acid can be added 11/08/2010 Plants II; lipid anabolism
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