Lipid Biosynthesis I

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Lectured by Dr. Qin Yongmei (???) Dec. 5, 2007
Lipid Biosynthesis I

• Biosynthesis of fatty acids and eicosanoids
• 2. Biosynthesis of other lipids
• a. Triacyloglycerols
• b. Membrane phopholipids
• c. Cholesterol, steroids and
• isoprenoids

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carbohydrate
pyruvate
pyruvate
?-oxidation
Ketone bodies
Acetyl-CoA
Acetyl-CoA
ketogenesis
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H

H
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• Fatty acid synthesis is not simply a reversal of
  
• the degradation pathway.
• Fatty acid synthesis and degradation pathways
• again exemplify the principle that synthetic
  and
• degradation pathways are almost always
  distinct.

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• Biosynthesis of fatty acids
• Occurs through the condensation of C2 units
• Occurs largely in adipocytes and liver

• Acetyl-CoA is required to be transported from
• mitochondrial matrix to cytosol
• 2. Malonyl-CoA is formed from carboxylation of
  acetyl-CoA

3. Loading step transfer of acetyl-CoA and
malonyl-CoA to form acetyl-ACP and
malonly-ACP Successive rounds of
condensation, reduction, dehydration and
reduction
Fatty acid synthase
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Biosynthesis of fatty acid
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ACP
Acyl Carrier Protein (ACP)
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Similarities between ACP and CoA
3,5-ADP
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ACP
Ser
An adenosine nucleotide
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Mitochondrial matrix
Cytosol
Citrate lyase
Citrate synthase
Malate DH
Malate DH
Pyr.carboxylase
Malic enzyme
shuttle for transfer of acetyl groups from Mit.
to the cytosol
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Synthesis of fatty acids may be stimulated when
there is A. high levels of acetyl CoA in
mitochondria.B. high levels of palmitate in the
cytosol.C. high levels of citrate in
mitochondria.D. high levels of malate in
mitochondria.E. activation of acyl CoA
synthetase in the cytosol.
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The accumulation of precursors for fatty acid
synthesis is a wonderful example of the
coordinated use of multiple processes to fulfill
a biochemical need. The citric acid cycle,
subcellular compartmentalization, and the
pentose phosphate pathway provide the carbon
atoms and reducing power, whereas glycolysis and
oxidative phosphorylation provide the ATP to meet
the needs for fatty acid synthesis.
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Shuttle for transfer of acetyl-CoA from
mitochondrial matrix to the cytosol

• The mitochondrial membranes are not permeable to
• acetyl-CoA
• It is shuttled out of the mitochondria in the
  form
• of citrate
• 3. Acetyl-CoA is regenerated by the action of
• ATP-citrate lyase

Citrate ATP CoA H2O ? acetyl-CoA ADP Pi
oxaloacetate
Acetyl CoA and oxaloacetate are transferred from
mitochondria to the cytosol at the expense of the
hydrolysis of one molecule of ATP.
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Sources of NADPH for fatty acid synthesis

• NADPH in adipocyte cytosol is largely generated
  from the oxidative decarboxylation of malate
  (catalyzed by malic enzyme, a reaction of acetyl
  shuttling)
• NADPH in hepatocytes and mammary glands is
  supplied primarily from the pentose phosphate
  pathway
• NADPH in chloroplast stroma is mainly produced by
  the light reactions.

NADP NADH ATP H2O ? NADPH NAD ADP
Pi H (malate DH, malic enzyme and
pyruvate carboxylase)
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Formation of malonyl-CoA by acetyl-CoA
Carboxylase

• In bacteria,
• Acetyl-CoA carboxylase has three separate
• polypeptide subunits, biotin carboxylase,
• biotin carrier protein, and transcarboxylase
• In animal cells,
• All three activities of acetyl-CoA carboxylase
  are part of a single multifunctional polypeptide

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The acetyl-CoA carboxylase reaction
The reactions occur at two active sites, via a
Ping-Pong mechanism.
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Loading step transfer of acetyl-CoA and
malonyl-CoA to form acetyl-ACP and
malonly-ACP
Malonyl-CoA-ACP transacylase
Acetyl-CoA-ACP transacylase
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Condensation
?-ketoacyl-ACP synthase
?-ketoacyl-ACP reductase
Reduction
?-hydroxyacyl-ACP dehydratase
Dehydration
Enoyl-ACP reducatse inhibited by triclosan (a
broad-spectrum antibacterial agent)
Reduction
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?-Ketoacyl-ACP synthase Acetyl-ACP
malonyl-ACP ? acetoacetyl-ACP ACP CO2
Why is the four-carbon unit not formed from 2
two-carbon units ? The equilibrium is
favorable if malonyl ACP is a reactant because
its decarboxylation contributes a substantial
decrease in free energy. ATP drives the
condensation reaction, though ATP does not
directly participate in the condensation
reaction. Rather, ATP is used to carboxylate
acetyl-CoA to malonyl-CoA. The free energy stored
in malonyl-CoA is released in the decarboxylation
accompanying the formation of acetoacetyl ACP.
Similar mechanism Pyruvate
PEP(gluconeogenesis)
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Sequence of events during synthesis of a fatty
acid
Acetyl-CoA- ACP transacetylase
Translocation of butyryl group to Cys on KS
Malonyl-CoA -ACP transacylase
Enoyl-ACP reductase
?-Ketoacyl-ACP synthase
?-Ketoacyl-ACP reductase
?-Dedroxyacyl-ACP dehydratase
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Beginning of the second round of the fatty
acid synthesis cycle
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The overall process of palmitate synthesis
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Overall chemical reactions leading to fatty acid
synthesis
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• Seven cycles of condensation and reduction
• 1Acetyl-CoA 7 malonyl-CoA 14NADPH
  14H
• palmitate 7CO2 14 NADP 8CoA
  6H2O
• 2. Formation of seven malonyl-CoA molecules
• 7 Acetyl-CoA 7CO2 7ATP
  7 malonyl-CoA 7ADP 7Pi
• 3. Palmitate-ACP H2O Palmitate
  ACP H2O

Palmitoyl thioesterase
The overall process 8 Acetyl-CoA 7ATP
14NADPH 14H palmitate 14 NADP
8CoA 6H2O 7ADP 7Pi
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The formation of a fatty acid catalyzed by fatty
acid synthase

• The complex in E. coli consists of seven
  polypeptide chains
• Intermediates are covalently attached to an ACP,
  which structurally and functionally similar to
  coenzyme A
• The phosphopantethenine prosthetic group of ACP
  is believed to serve as a flexible arm, moving
  the attached intermediates from one active site
  to another
• Catalyzed by six enzymes on the complex two
  transfer the acyl and malonyl groups from
  coenzyme A to ACP, the other four catalyzing the
  four repeating reactions
• NADPH is used as the reducing agent.

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Domain 1 acyl and malonyl binding
and condensation
Domain 2 Reduction of domain 1
intermediate
KS
Domain 3 Liberation of palmitate
product
Substrate entry Acetyl-CoA Malonyl-CoA
The proposed ways for the single polypeptide
mammalian fatty acid synthase to fold and to act
Substrate entry Acetyl-CoA Malonyl-CoA
KS
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Mammalian fatty acid synthase (FAS) -260
kDa subunits (forming dimer) -Domain 1, the
substrate entry and condensation unit (acetyl
transferase, malonyl transferase, and ?-ketoacyl
synthase ) -Domain 2, the reduction
unit (acyl carrier protein, ? -ketoacyl
reductase, dehydratase, and enoyl reductase )
-Domain 3, the palmitate release unit
(thioesterase) Advantages of FAS -
synthetic activity of different enzymes is
coordinated. - a multienzyme complex
(multifunctional enzyme) consisting of
covalently joined enzymes is more stable
than one formed by noncovalent attractions -
intermediates can be efficiently handed from one
active site to another without leaving the
assembly
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The flexible phosphopantetheinyl unit (20-Å
maximal length ) of ACP carries substrate from
one active site to another The enzyme subunits
do not need to undergo large structural
rearrangements to interact with the substrate.
Instead, the substrate is on a long, flexible arm
that can reach each of the numerous active sites.
The organization of the fatty acid synthases of
higher organisms enhances the efficiency of the
overall process because intermediates are
directly transferred from one active site to the
next (substrate channeling).

• Fatty acid synthase inhibitors may be useful
  drugs
• Antitumor
• Antiobesity

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Different structures of fatty acid synthases
from different organisms
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• Those from bacteria and higher plants the seven
  activities reside in seven separate polypeptide
  chains
• Those from yeast the seven activities reside in
  two separate polypeptide chains, with the
  synthase present as dodecamers (?6?6)
• Those from vertebrates the seven activities
  reside in one large polypeptide chain, with the
  synthase present as dimers.

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Maier, T., Jenni, S. Ban, N. (2006)
Architecture of mammalian fatty acid synthase at
4.5 A resolution. Science 311, 1258-1262
Jenni, S., Leibundgut, M., Maier, T., Ban, N.
(2006) Architecture of a fungal fatty acid
synthase at 5 A resolution. Science 311,
1263-1267.
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Science 311, 1258-1262
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Regulation of fatty acid synthesis
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Regulation of Fatty Acid Metabolism short-term
regulation (substrate availability allosteric
effectors and/or enzyme modification) e.g.
Acetyl-CoA carboxylase (ACC), Carnitine
acyltransferase I (inhibited by
malonyl-CoA) long-term regulation (regulation of
the rate of enzyme synthesis and turn-over) e.g.
regulated by hormones (insulin, glucagon)
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Regulation of acetyl-CoA carboxylase (allosteric
regulation hormone-dependent covalent
modification)
Palmitoyl CoA also inhibits the translocase that
transports citrate from mitochondria to the
cytosol, as well as glucose 6-phosphate
dehydrogenase, which generates NADPH in the
pentose phosphate pathway.
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Acetyl-CoA carboxylase is regulated by covalent
modification
Citrate activates the inactive phosphorylated
enzyme rather than removing the phosphate
AMP-activated protein kinase AMP (), ATP(-)
Protein Phosphatase 2A
Acetyl-CoA carboxylase from plants and bacteria
is not regulated by citrate or by a
phosphorylation dephosphorylation cycle.
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The active (dephosphorylated) form of acetyl-CoA
carboxylase forms filaments (under electron
microscope)
Citrate partially activate the phosphorylated
acetyl-CoA carboxylase (similar to how AMP
partially activate the dephosphorylated glycogen
phosphorylase) .
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• Acetyl-CoA carboxylase (key regulatory step)
• It is inhibited by palmitoyl-CoA and fatty
  acyl-CoAs, and activated by citrate (allosteric
  regulation, local regulation)
• Glucagon and epinepherine inactivate the enzyme
  by triggering its phosphorylation insulin has
  the opposite effect (global regulation)
• The active (dephosphorylated) form of the enzyme
  forms filaments, while its inactive form
  dissociate into monomers or oligomers
• (Insulin also stimulates the activities of
  citrate lyase and pyruvate dehydrogenase complex
  by phosphorylation).

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Response to diet

• Fatty acid synthesis and degradation are
  reciprocally
• regulated so that both are not simultaneously
  active
• In starvation, the level of free fatty acids
  rises because hormones
• such as epinephrine and glucagon stimulate
  lipase. Insulin, in contrast,
• inhibits lipolysis.
• Fatty acid oxidation is blocked during
  synthesis
• Malonyl-CoA effectively inhibits carnitine
  acyltransferase I, thus
• blocking the ?-oxidation of fatty acids in
  mitochondria
• NADH inhibits 3-hydroxyacyl-CoA dehydrogenase
  acetyl-CoA inhibits
• thiolase
• Adaptive control Animals that have fasted and
  are then fed
• high-carbohydrate, low-fat diets show marked
  increases in their
• amounts of acetyl CoA carboxylase and fatty
  acid synthase within
• a few days.

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• In plant
• Fatty acid synthesis occurs in chloroplast
• (stroma) in plants
• The plant acetyl-CoA carboxylase is not
  regulated
• by phosphorylation It is activated by
  increase
• in stroma pH and magnesium

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Insulin
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Fatty acid elongation and desaturation
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Long-chain saturated FAs are synthesized from
palmitate
Formation of polyunsaturated FAs
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Elongation of fatty acids
Two systems In smooth ER membranes similar
to the last cycle of FA synthesis (CoA
replaces ACP as acyl carrier ) In
mitochondria reverse reaction of FA
?-oxidation Enoyl-CoA reductase use NADPH
Acyl-CoA dehydrogenase use FAD.
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Elongation of FAs in
mitochondria
thiolase
3-L-hydroxyacyl-CoA dehydrogenase
Enoyl-CoA hydratase
Enoyl-CoA reductase
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Desaturation of fatty acids

• The double bonds of palmitoleate and oleate are
  introduced in vertebrates by fatty acyl-CoA
  desaturase, together with cytochrome b5 and
  cytochrome b5 reductase
• Two substrates, fatty acyl-CoA and NADPH, are
  oxidized simultaneously by O2 . The desaturase is
  thus a mixed-function oxidase.

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Fatty acyl-CoA is desaturated (oxidized) using
O2 and NADPH (in smooth ER)
a mixed-function oxidase oxidize two different
substrates simultaneously
Fatty acyl- CoA desaturase
ER membrane
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Differences between Oxidase and Oxygenase
Cytochrome P450 a family of proteins belonging
to monooxygenase (mixed- function oxidase or
oxygenase here, the oxidase means oxygnease )
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Desaturation of fatty acids In vertebrates,
Palmitate(C16) Palmitoleate (C16,?9 )
Stearate(C18) Oleate (C18,?9 )
fatty acyl- CoA desaturase
Mammalian hepatocytes cannot introduce double
bonds beyond ?9. Linoleate, 182(?9,12)and
?-linolenate 183 (?9,12,15),cannot be
synthesized by mammals, but plants can synthesize
both.
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Production of polyunsaturated fatty acids in
plants

• Further desaturation of oleate (to form
  linoleate and linolenate) occur on
  phosphatidylcholine
• This is catalyzed by another desaturase, which is
  present only in plant cells, not in vertebrates
• Therefore, linoleate and linolenate are essential
  fatty acids for mammals.

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• In plants
• Oleate can be further
• desaturated (beyond
• position 9) on
• phosphatidylcholine
• (often attaching to C-2)
• to form linoleate
• and linolenate in
• plants.
• Linoleate and linolenate,
• needed to make other
• polyunsaturated fatty
• acids like arachidonate
• are essential fatty acids
• for mammals.

desaturase
desaturase
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Subcellular Localization of Lipid Metabolism
Plant cells
Animal cells
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Eicosanoids
Eicosanoids are a class of lipids that include
prostaglandins,thromboxanes and leucotrienes
Eicosanoids derive their name from their
common origin, that is, from C20 polyunsaturated
fatty acids, the eicosanoic acids, particularly
arachidonate, 204 (? 5,8,11,14),
5,8,11,14-eicosatetraenoic acid. Eicosanoids
exert specific physiological effects on target
cells, like hormones. However, eicosanoids are
distinct from most hormones in that they act
locally, near their sites of synthesis, and they
are catabolized extremely rapidly. Thus,
eicosanoids are considered to be locally acting
hormones.
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Arachidonic acid is formed as arachidonyl-CoA
from 8,11,14 eicosatrienoyl-CoA. The essential
fatty acid linoleic acid is an important
precursor of arachidonic acid.
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Plasma membrane
Stimulus
Arachidonate is derived from membrane
phospholipids
Membrane-bound phospholipids
Arachidonic acid
Tromboxanes induce constriction of blood vessels
and platelet aggregation (blood clotting)
Prostaglandins stimulate inflammation,regulate
blood flow, control ion transport and modulate
synaptic transmission.
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Cyclooxygenase (COX), also called prostaglandin
H2 synthase (bifunctional enzyme) -
cyclooxygenase activity - peroxidase
activity
cyclooxygenase activity of COX
cyclooxygenase activity of COX
peroxidase activity of COX
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Aspirin inhibits the cyclooxgenase activity of
COX by acetylating an essential Ser residue on
the enzyme.
Ibuprofen inhibits the same step, probably by
mimicking the structure of the substrate or an
intermediate in the reaction.
Aspirin and Ibuprofen belong to Non-steroidal
anti-inflammatory drugs ,NSAIDs .
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Side effect of Aspirin Aspirin inhibits both
isozymes (COX1 and COX2) equally, so a dose
sufficient to reduce inflammation also risks
stomach irritation. New developing NSAIDS should
inhibit COX-2 specifically. COX1 for the
synthesis of the prostaglandins that regulate
the secretion of gastric mucin COX2 for the
synthesis of prostaglandins that mediate
inflammation, pain and fever.
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Summary

• Fatty acid biosynthesis takes a different pathway
  from the reverse of its degradation and takes
  place in different cellular compartments.
• The aceytl-CoA units are transported out of
  mitochondrial matrix as citrate.
• Acetyl-CoA carboxylase catalyzes the
  rate-limiting step of fatty acid synthesis and is
  highly regulated by allosteric and covalent
  modifications.

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• Palmitate, the usual final product of
• fatty acid synthesis, can be further
• elongated and desaturated.
• Eicosanoids are derived from arachidonate
• by the action of cyclooxygenases
• and peroxidases