1. Plant biochemistry II; Lipid Anabolism
Andy Howard Biochemistry Lectures, Fall November 2010
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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
11/08/2010
Plants II; lipid anabolism

3. 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
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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
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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
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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.
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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
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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
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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
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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
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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
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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
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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
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Plants II; lipid anabolism

14. 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
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Plants II; lipid anabolism

15. 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
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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 1OR5 9.1 kDa monomer Streptomyces
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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 ACP  1 + 4
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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 synthase PDB 1HNJ 70 kDa dimer; monomer shown EC , 1.46Å
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Plants II; lipid anabolism

19. 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
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Plants II; lipid anabolism

20. 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
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Plants II; lipid anabolism

21. 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
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Plants II; lipid anabolism

22. 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
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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 thioesterase with a 3-layer Rossmann fold
Palmitoyl thioesterase I PDB 1EI9
31 kDa monomer bovine EC , 2.25Å
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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
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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
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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.
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Plants II; lipid anabolism

27. 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
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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
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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 monomer Mycobacterium
tuberculosis EC , 2Å
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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 dimer monomer shown castor bean EC , 2.4Å
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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
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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.
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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
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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 acyltransferase PDB 1IUQ 40 kDa monomer Cucurbita EC , 1.55Å
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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!
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Plants II; lipid anabolism

36. 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
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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
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Plants II; lipid anabolism

38. 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
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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
This route to phosphatidylserine is found only in bacteria
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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 3HSI 161kDa homotrimer EC , 2.2Å Haemophilus
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Plants II; lipid anabolism

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

47. 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
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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
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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
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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
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Plants II; lipid anabolism

51. 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
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Plants II; lipid anabolism

52. 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)
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Plants II; lipid anabolism

53. 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
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Plants II; lipid anabolism

54. 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
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Plants II; lipid anabolism

55. 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
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Plants II; lipid anabolism

56. 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
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Plants II; lipid anabolism