Chapter 20 Lipid Biosynthesis

Chapter 21
Lipid Biosynthesis
1. Fatty acids;
2. Eicosanoids;
3. Triacylglycerols;
4. Membrane phospholipids;
5. Cholesterol, steroids, and
1. Fatty acid synthesis takes a different
pathway from its degradation
 Occurs in the cytosol (chloroplasts in plants).
 Acetyl-CoA provides the first two carbons,
which is elongated by sequential addition of
two-carbon units donated from malonyl-CoA.
 Intermediates are attached to the -SH groups
of an acyl carrier protein (ACP).
 NADPH is the reductant.
 The enzymes are associated as a multi-enzyme
complex or even being in one polypeptide
chain in higher organisms (fatty acid synthase).
 Elongation
by the fatty acid synthase
complex stops upon formation of palmitate
(C16), further elongation and desaturation are
carried out by other enzyme systems.
2. Malonyl-CoA is formed from acetylCoA and bicarbonate
discovered that HCO3- is required
for fatty acid synthesis.
 Acetyl-CoA carboxylase (being trimeric in
bacteria, monomeric in animals and both in
plants) catalyzes this carboxylation reaction.
 The enzyme has three functional parts: a biotin
carrier protein; an ATP-dependent biotin
carboxylase; and a transcarboxylase.
 The enzyme exemplifies a ping-pong reaction
 Salih Wakil
 This
irreversible reaction commits acetyl-CoA
to fatty acid synthesis.
Acetyl-CoA carboxylase
catalyzes the two-step
carboxylation reaction
of acetyl-CoA in two
active sites.
3. The acetyl and malony groups are first
transferred to two –SH groups of the
fatty acid synthase complex
The acetyl group of acetyl-CoA is first transferred to
the –SH group of a Cys residue on the b-ketoacylACP synthase (KS) in a reaction catalyzed by
acetyl-CoA-ACP transacetylase (AT).
 The malonyl group is transferred from malonyl-CoA
to the –SH group of the 4`-phosphopantetheine
covalently attached to a Ser residue of the acyl
carrier protein (ACP).
The acyl carrier protein (ACP) is very similar to
CoA (thus can be regarded as “macro CoA”)
4. Fatty acids are synthesized by a
repeating four-step reaction sequence
 In
the condensation reaction (step 1),
catalyzed by b-ketoacyl-ACP synthase, the
methylene group of malonyl-CoA (linked to
ACP) undergoes a nucleophilic attack on the
carbonyl carbon of the acetyl group linked to
KS, forming the b-ketobutyryl-ACP with
simultaneous elimination of CO2.
 the b-ketobutyryl-ACP is then reduced to D-bhydroxybutyryl-ACP (step 2), using NADPH
and the b-ketobutyryl-ACP reductase (KR).
molecule is then removed from the bhydroxybutyryl-ACP to produce trans-2butenoyl-ACP in a reaction catalyzed by bhydroxybutyryl-ACP dehydratase (step 3).
 A further reduction (step 4), also using
NADPH, of the carbon-carbon double in
trans-2-butenoyl-ACP, catalyzed by enoylACP reductase produces a saturated acyl on
ACP (butyryl-ACP).
 The butyryl group is then transferred to the
Cys –SH group of b-ketoacyl-ACP synthase
for another round of four reactions, which will
extend the chain by two more carbons.
 A water
 Seven
rounds of the four-step lengthening
reactions produces palmitoyl-ACP, which will
be hydrolyzed to release a free palmitate.
 The flexible 4`-phosphopantetheine group
covalently attached to ACP is believed to act
as a switch arm to move the intermediates
from one active site to the next on the enzyme
complex (i.e., the substrates are channeled).
 A total of 7 ATP and 14 NADPH will be
consumed for making one palmitate molecule.
5. The seven activities of fatty acid
synthesis from different organisms have
different level of integration
 Each activity resides in a separate polypeptide
chain in bacteria and higher plants.
 The seven activities reside in two separate
polypeptide chains, with the synthase present
as dodecamers (a6b 6).
 The seven activities reside in one large
polypeptide chain in vertebrates, with the
synthase present as dimers.
The seven activities
of fatty acid synthase
are integrated to
different levels in
different organisms.
6. Fatty acid synthesis occurs in cellular
compartments having a high
and NADP have selected for functioning
as electron carriers in oxidative catablism and
reductive anabolism respectively.
 In the hepatocytes and adipocytes, NADPH is
mainly produced in the cytosol via the pentose
phosphate pathway and by the malic enzyme.
 In photosynthetic plants, fatty acid synthesis
occur in the chloroplast stroma, using NADPH
made from photophosphorylation.
Pentose phosphate
NADPH in the cytosol of animal cells
is largely produced by the oxidative
decarboxylation of malate and the
pentose phosphate pathway
7. The acetyl groups of the
mitochondrion are transported into the
cytosol in the form of citrate
 The acetyl-CoA molecules are made from
glucose and amino acids in mitochondria.
 The are shuttled into the cytosol in the form of
citrate via the citrate transporter of the inner
 Acetyl-CoA is regenerated by the action of
ATP-dependent citrate lyase in the cytosol.
 Oxaloacetate is shuttled back into the
mitochondria as malate or pyruvate.
8. The rate of fatty acid biosynthesis is
controlled by acetyl-CoA carboxylase
 Excess
fuel is generally converted to fatty
acids/triacylglycerol for longer term storage.
 Acetyl-CoA carboxylase, catalyzing the
committing and rate-limiting step of fatty acid
synthesis, is allosterically inhibited by palmitoylCoA and activated by citrate.
 Glucagon and epinephrine triggers the
phosphorylation and disassociation of the
polymeric enzyme subunits, which inactivates
the enzyme.
 Citrate
partially activate the phosphorylated
acetyl-CoA carboxylase (similar to how AMP
partially active the dephosphorylated glycogen
 In plants, acetyl-CoA carboxylase is activated by
a increase of Mg 2+ concentration and decrease of
H+ concentration that accompany illumination.
 (Malonyl-CoA inhibits carnitine acyltransferase I)
Acetyl-CoA carboxylase
is regulated by allosteric
effectors and reversible
Citrate partially activate the
phosphorylated acetyl-CoA carboxylase
9. Palmitate can be further elongated and
desaturated in smooth ER
 Palmitoyl-CoA can
be further elongated by the
fatty acid elongation system present mainly in
the smooth endoplasmic reticulum, with twocarbon units also donated by malonyl-CoA.
 Palmitoyl-CoA and Stearoyl-CoA can be
desaturated between C-9 and C-10 to produce
palmitoleate, 16:1(9), and oleate, 18:1(9)
 The
double bonds are introduced by the
catalysis of fatty acyl-CoA desaturase (a
mixed-function oxidase), where both the fatty
acyl group and NADPH are oxidized by O2.
 The electrons of NADPH are transferred to O2
via Cyt b5 reductase and cytochrome b5.
 Further desaturation of oleate occur on
phosphatidylcholine and is catalyzed by
another desaturase, which is present only in
plant cells.
 Linoleate and linolenate, needed to make other
polyunsaturated fatty acids like arachidonate
are essential fatty acids for mammals.
Palmitate is the
Precursor for the
biosynthesis of
other fatty acids
Fatty acyl-CoA is desaturated (oxidized)
by O2 and NADPH.
Oleate can be
desaturated on
(often attaching to C-2)
to form linoleate
and linolenate
10. Eicosanoids are derived from
arachidonate, 20:4 (5,8,11,14)
 The arachidonate (花生四烯酸) is first cleaved
off from membrane phospholipids by
phospolipase A2, in response to hormonal or
other stimuli.
 Arachidonate is then converted to PGH2 by the
catalysis of the bifunctional cyclooxygenase
(COX): the cyclooxygenase activity converts
arachidonate to PGG2; the peroxidase activity
then converts PGG2 to PGH2.
 PGH2 is the immediate precursor of other
prostaglandins and thromboxanes.
 Aspirin
(acetylsalicylate) irreversibly inhibits
the cyclooxygenase by acetylating an active
site Ser, thus blocking the synthesis of
prostglandins and thromboxanes; Ibuprofen
also inhibit the same enzyme.
 Arachidonate can also be modified by adding
hydroperoxy groups at various positions to
form various hydroperoxyeicosatetraenoates
(HPETEs) in reactions catalyzed by various
lipooxygenases with the incorporation of O2.
 The HPETEs will be further converted to
leukotrienes (白细胞三烯).
Prostaglandins and
thromboxanes are
synthesized from
activity of COX
activity of COX
Tyr385, a key
residue for the
Heme for the peroxidase
active site
The dimeric bifunctional cyclooxygenase
11. Newly synthesized fatty acids have
mainly two alternative fates in cells
 Fate
I: be incorporated into triacylglycerols as
a form to store metabolic energy in long terms.
 Fate II: be incorporated into membrane
phospholipids (during rapid growth).
12. Phosphatidic acid is the common
precursor for the syntheses of both
triacylglycerols and
Phosphatidic acid (or diacylglycerol 3-phosphate) is
made by transferring two acyl groups from two acylCoAs to L-glycerol 3-phosphate, which is derived
from either glycerol or dihydroxyacetone phosphate.
 A phosphatidic acid is converted to a triacylglycerol
via a dephosphorylation reaction (catalyzed by
phosphatidic acid phosphatase) and a acyl
transferring reaction.
acid is derived
from L-glycerol 3phosphate and two
Often saturated
Often unsaturated
Phosphatidic acid
is the common
precursor for both
triacylglycerols and
Phosphatidic acid
13. Insulin stimulates conversion of
dietary carbohydrates/proteins into fat
Diabetes patients due to lack of insulin would
neither be able to use glucose properly, nor to
synthesize fatty acids from carbohydrates and amino
 They show increased rates of fatty acid oxidation
and ketone body formation, thus losing weight.
14. Two strategies are taken for
converting phosphatidic acid to
 Eugene Kennedy revealed in 1960s that either
the –OH group of the diacylglycerol (strategy
1) or that of the polar head (strategy II) is first
activated by attaching to cytidine nucleotide.
 The CMP moiety is displaced by the other –
OH group in a nucleophilic attack reaction to
synthesize a glycerophospholipid.
 Both strategies are used in eukaryotic cells,
but only strategy I is use in bacterial cells.
Eukaryotic cells
use both strategies
(occurring on sER and
inner membrane of
Bacteria mainly use
this strategy
Phospholipid synthesis
in E. coli employs
15. Acidic (anionic) phospholipids in
eukaryotic cells are synthesized using
 These
include phosphatidylglycerol,
cardiolipin, phosphatidylinositol,
 eukaryotic cardiolipin is synthesized from one
phosphatidylglycerol and one CDPdiacylglycerol (from two
phosphatidylglycerols in bacteria).
16. Phosphatidyl choline (PC) and
phosphatidyl ethanolamine (PE) are
often made from the salvage (reuse)
pathway in mammals
 Diet choline and ethanolamine are first
converted to CDP-choline and CDPethanolamine after an initial phosphorylation
 The CMP moiety is then replaced by a
diacylglycerol, forming PC and PE.
 Phosphatidylserine (PS) is often made from
PE by a head exchange reaction (reversible).
 PC
can be made from PE by three methylation
reactions using S-adenosylmethionine
(adoMet) in the liver cells.
 PS
can also be converted to PE by a
decarboxylation reaction.
PC and PE are
made from the
salvage pathway
in mammals
The synthesis of PE,
PC, PS in eukaryotic
17. The synthesis of ether lipids involves
a displacement of fatty acyl by fatty
alcohol step and a desaturation step
 Both
plasmalogen (缩醛磷脂) and plateletactivating factor are made using this pathway.
 The acyl group on 1-acyldihydroxyacetone 3phosphate is replaced by a long chain alcohol
group to form the ether linkage.
 The double bond in plasmalogen is introduced
at the end by the catalysis of a mixed-funciton
Synthesis of
the ether lipids
1-alkylglycerol 3-phosphate
18. The sphingosine backbone of
spingolipids is derived from palmitoylCoA and Ser
 Palmitoyl-CoA condenses with serine (PLP is
needed for decarboxylate serine) to form bketosphinganine, which is then reduced to
sphinganine (二氢鞘氨醇).
 Sphinganine is then acylated and desaturated
to form ceramide (containing sphingosine).
 Addition of sugar(s) or phosphocholine heads
leads to the synthesis of cerebroside,
gangliosides, or sphingomyelin.
 The
ways for the membrane lipids
(glycerolphospholipids and spingolipids)
synthesized at smooth endoplasmic
reticulum or inner membrane of
Mitochondria to be transported to specific
cellular locations are not well understood
Spingolipid synthesis begins
with the condensation between
palmitoyl-CoA and Ser.
A glycolipid, not a
(not CDP-choline!)
19. Radioisotope tracer experiments
revealed that all the 27 carbons of
cholesterol is derived from acetyl-CoA
 The origin of the carbon atoms of cholesterol
was deduced from tracer experiments where
either with the methyl carbon or the carboxyl
carbon in acetate is labeled with 14C (1940s).
 The pattern of labeling provided the blueprint
for revealing the detail enzymatic steps for
cholesterol biosynthesis occurring in mammals.
 The 30-carbon squalene (of six isoprene units)
and later on mevalonate were found to be
intermediates of cholesterol biosynthesis.
 The
biosynthetic pathway of cholesterol, being
the most complex known, was elucidated
mainly by Konrad Bloch and Feodor Lynen in
the 1950s.
The carbon origins of cholesterol as revealed by
radioisotope labeling studies.
20. The cholesterol biosynthesis
pathway can be divided into four stages
 Stage
I: three acetyl-CoA molecules condense
to form the 6-carbon mevalonate (甲羟戊酸).
 Stage II: mevalonate is converted to activated
5-carbon isoprene (异戊二烯) units.
 Stage III: Six isoprene units condense to form
the linear 30-carbon squalene(鲨烯).
 Stage IV: The linear squalene is cyclized to
form a four-ring structure, which is eventually
converted to the 27-carbon cholesterol through
a series of complicated reactions.
Reactions assembling
cholesterol from 18
molecules of acetyl-CoA
can be divided into four
21. Mevalonate commits the acetyl
groups for cholesterol synthesis
molecule of b-hydroxy-b-methylglutarylCoA (HMG-CoA) is formed from three acetylCoA molecules in the cytosol via the same
reactions as occurring in mitochondria for ketone
body formation.
 HMG-CoA reductase (an integrated membrane
protein in the smooth ER) catalyzes the
irreversible reduction of HMG-CoA (using two
molecules of NADPH) to form mevalonate:
committing the acetyl groups for cholesterol
synthesis (thus being a major regulation step).
 One
One mevalonate is
synthesized from
three acetyl-CoA
HMG-CoA lyase
in mitochondria
Acetyl-CoA + acetoacetate
In cytosol
The irreversible
committing step
for cholesterol
22. Two activated isoprenes are formed
from mavelonate after going through
three phosphorylation steps
 Three
phosphate groups are transferred from
three ATP molecules to mevalonate to form 3phospho-5-pyrophosphomevalonate.
 The leaving of both the carboxyl and the 3phosphate groups leads to the formation of 3isopentenyl pyrophosphate.
 3-Isopentenyl pyrophosphate is isomerized to
form the second activated isoprene:
dimethylallyl pyrophosphate.
Two activated
isoprenes are
formed from
23. The 30-carbon linear squalene is
formed from the condensation of six
activated isoprene units
 A dimethylallylpyrophosphate
is joined to an
isopentenylpyrophosphate (head-to-tail) to form
the 10-carbon geranyl pyrophosphate.
 A geranyl pyrophosphate is joined to another 3isopentenyl pyrophosphate (head-to-tail) to form
the 15-carbon farnesyl pyrophosphate(法呢基焦
 Two farnesyl pyrophosphate join (head-to-head)
to form the 30-carbon squalene.
is formed from
three activated
isoprene units
Squalene is formed
from the condensation
of two farnesyl
24. The rings of cholesterol are formed
via a concerted reaction across four
double bonds of the linear squalene
epoxide intermediate
 Squalene
2,3-epoxide, is first formed in a
reaction catalyzed by squalene monooxygenase
using O2 and NADPH.
 Concerted movement of electrons through four
double bonds and the migration of two methyl
groups generates lanosterol (羊毛固醇).
 Lanosterol is converted to cholesterol via about
20 enzymatic reactions including many double
bond reduction and demethylations.
Oxygenation induced
ring closing converts
the linear squalene
to lanosterol of four
rings, which is
converted to
after going through
another 20 or so
~ 20
25. Cholesterols made in vertebrate
livers can be converted to bile acids and
cholesterol esters before exporting
Cholesterol can be converted to bile acids and bile
salts, which will be secreted to the intestine for
emulsifying lipids.
 Cholesterol can also be converted to the more
hydrophobic cholesterol esters, which will be
stored in the liver or transported to other tissues
after being incorporated into lipoprotein particles.
Cholesterol can be converted
to bile acids (salts) :
glycocholate and taurocholate
acyl transferase
(ACAT) catalyzes the
addition of an acyl
group to the hydroxyl
group of cholesterol
26. Lipids (including cholesterols) are
transported in the vertebrate plasmia as
various lipoprotein particles
 The different lipoprotein particles, having
different combinations of lipids and
apolipoproteins, can be separated by
untracentrifugation due to different densities
and sizes.
 The human plasma lipoproteins include
chylomicrons (which transports lipids from
intestine to various tissues), VLDL (very low
density lipoproteins), LDL(Low density
lipoproteins), HDL (high density lipoproteins).
 At
least nine apolipoproteins (named as apo A,
B, C, D, E) have been revealed in human,
which act as signals to target the lipoprotein
particles to various tissues or activating
enzymes that will act on the lipoproteins.
 Endogenous lipids made in liver are
transported to other tissues as VLDL particles
(~ 87% lipids and 12% proteins).
 The apoC-II protein in VLDL activates the
lipoprotein lipase in muscle and adipocyte
tissues, thus releasing free fatty acids there.
 Some VLDL remnants
is then converted to
LDL (with 23% proteins and 75% lipids) and
with the others being absorbed by the liver
cells via receptor-mediated endocytosis.
 LDL delivers cholesterols to extrahepatic
tissues, where its apoB-100 protein (4636
residues) is recognized by specific LDL
receptors and LDL is endocytosed.
 HDL, with its precursors formed in the liver or
intestine cells, collects the cholesterols in the
plasma and deliver them to the liver cells.
 (A negative correlation between blood HDL
level and arterial diseases has been observed.)
Lipids are transported
as various lipoprotein
particles in vertebrate
LDL is uptaken by cells
via the LDL receptors,
LDL receptors
are recycled
to the cell surfaces
27. The de novo synthesis of
cholesterol is regulated to complement
dietary intake
 HMG-CoA reductase, catalyzing the ratelimiting step of the de novo cholesterol
synthesis, has an activity variable over 100 fold!
 An yet characterized sterol promotes proteolytic
degradation of HMG-CoA reductase and
inhibits the transcription of the genes of HMGCoA reductase and LDL receptor.
 Hormones (insulin and glucagon) regulate the
activity of the HMG-CoA reductase via
reversible phosphorylation.
 Genetic
defect of the LDL receptor was found
to cause the familial hypercholesterolemia and
atherosclerosis: the LDL cholesterols can not
enter the cells, while de novo synthesis
continues despite the high cholesterol level in
the blood.
 Mevalonate analogs (e.g., compactin and
lovastatin) can be used to treat patients with
familial hypercholesterolemia.
The de novo synthesis of
cholesterol is regulated
to complement the
dietary uptake
(For storage)
The mevalonate analogs
are used to treat
28. Pregnenolone, the common
precursor of all steroid hormones, is
derived from cholesterol
 The tail chain of cholesterol is first
hydroxylated at C20 and C22, and then cleaved
between these two carbons to remove a 6carbon unit, forming pregnenolone(孕烯醇酮).
 The hydroxylation is catalyzed by cytochrome
P450 monooxygenases (a mitochondrial
enzyme) that utilize NADPH and O2.
 Cytochrome P450 is a large family of enzymes
with different substrate specificity, hydroxylates
many hydrocarbon chains.
Prognenolone, the
common precursor
of steroid hormones,
is synthesized from
cytochrome P450
29. All steroid hormones are derived
from cholesterol
 Progesterone
(孕酮) is synthesized from
pregnenolone by oxidizing the 3-OH group and
the isomerization of the double bond (from 5
to 4 position).
 Cortisol (a major glucocorticoid) is synthesized
from progesterone by hydroxylation at C-17, C21, and C-11.
 Aldosterone (a mineralocorticoid) is
synthesized from progesterone by hydroxylation
at C-21, C-11, and oxidation of C-18 to an
 Testosterone
(an androgen, or male hormone)
is synthesized from progesterone by the
removal of 2-carbon unit and hydroxylation at
 Estradiol ( an estrogen, or female hormone) is
synthesized from testosterone by the removal
of C-19 and formation of the aromatic A ring.
Progesterone is synthesized from pregnenolone
by oxidizing the 3-OH group and the
isomerization of the double bond from 5 to 4
Cortisol and aldosterone
are synthesized from
progesterone by several
oxygenation reactions
(forming hydroxyl and
aldehyde groups)
Testosterone is synthesized from
progesterone by the removal of a 2-carbon
unit and hydroxylation at C-17
Estradiol is synthesized
from testosterone by
the removal of C-19 and
formation of the
aromatic A ring
30. A hugh array of biomolecules, all
called isoprenoids are synthesized using
activated isoprenes
These include many pigments (carotenoids, phytol
chain of chlorophylls), fragrant principles, vitamines
(A, D, E, K), rubber, dolichols, quinones
(ubiquinone, plastoquinone), juvenile hormones of
 Prenylation of proteins (attaching of geranylgeranyl
and farnesyl groups) leads to membrane association.
Some plant pigments are isoprenoids
Some fragrant molecules are isoprenoids
(called terpenes)
Natural rubber is cis-polyisoprene
Isoprenoid tails
function to anchor
proteins to membranes
“Perfumes, colors and
sounds echo one
Charles Baudelaire
 Fatty
acid biosynthesis takes a different
pathway from the reverse of its degradation
and takes place in different cellular
 The aceytl-CoA units are transported out of
mitochondrial matrix as citrate.
 Acetyl-CoA carboxylase catalyzes the ratelimiting step of fatty acid synthesis and is
highly regulated by allosteric and covalent
 Palmitate,
the usual final product of fatty acid
synthesis, can be further elongated and
desaturated in sER.
 Eicosanoids are derived from arachidonate by
the action of cyclooxygenases and peroxidases.
 Phosphatidic acid (diacylglycerol 3-phosphate)
is the common precursor of both
triacylglycerol and glycerophospholipids.
 Glycerophospholipids are made using two
alternative strategies of CDP modification.
 The backbone of sphingolipids are made from
palmitoyl-CoA and Ser.
 Radioisotope
tracer experiments revealed that
all the 27 carbons of cholesterol are from
 The biosynthesis of cholesterol takes a long
pathway, with the reaction catalyzed by HMGCoA reductase being the rate-limiting step for
de novo synthesis of cholesterol.
 Activated isoprene untis, mevalonate, squalene
were found to be important intermediates of
cholesterol biosynthesis.
 The
lipids are transported as lipoprotein
particles (including chylomicrons, VLDL,
LDL, and HDL).
 The de novo biosynthesis of cholesterol is
regulated to complement the dietary uptake.
 All streroid hormones are derived from
 A huge arrays of isoprenoids are made using
activated isoprene units.