Lipid Biosynthesis
chapter
21
1. Pathway of Carbon in Fatty Acid Synthesis Using your knowledge of fatty acid biosynthesis, pro-
vide an explanation for the following experimental observations:
(a) Addition of uniformly labeled [
14
C]acetyl-CoA to a soluble liver fraction yields palmitate uni-
formly labeled with
14
C.
(b) However, addition of a trace of uniformly labeled [
14
C]acetyl-CoA in the presence of an excess of
unlabeled malonyl-CoA to a soluble liver fraction yields palmitate labeled with
14
C only in C-15
and C-16.
2. Synthesis of Fatty Acids from Glucose After a person has ingested large amounts of sucrose, the
glucose and fructose that exceed caloric requirements are transformed to fatty acids for triacylglycerol
synthesis. This fatty acid synthesis consumes acetyl-CoA, ATP, and NADPH. How are these substances
produced from glucose?
3. Net Equation of Fatty Acid Synthesis Write the net equation for the biosynthesis of palmitate in
rat liver, starting from mitochondrial acetyl-CoA and cytosolic NADPH, ATP, and CO
2
.
c21LipidBiosynthesis.qxd 11/30/12 6:45 PM Page S-247
S-248 Chapter 21 Lipid Biosynthesis
4. Pathway of Hydrogen in Fatty Acid Synthesis Consider a preparation that contains all the en-
zymes and cofactors necessary for fatty acid biosynthesis from added acetyl-CoA and malonyl-CoA.
(a) If [2-
2
H]acetyl-CoA (labeled with deuterium, the heavy isotope of hydrogen)
C
OOC
2
H
2
H
C
O
S-Co
A
and an excess of unlabeled malonyl-CoA are added as substrates, how many deuterium atoms are
incorporated into every molecule of palmitate? What are their locations? Explain.
(b) If unlabeled acetyl-CoA and [2-
2
H]malonyl-CoA
C
OOC
2
H
2
H
C
O
S-Co
A
are added as substrates, how many deuterium atoms are incorporated into every molecule of
palmitate? What are their locations? Explain.
Answer
5. Energetics of b-Ketoacyl-ACP Synthase In the condensation reaction catalyzed by b-ketoacyl-ACP
synthase (see Fig. 21–6), a four-carbon unit is synthesized by the combination of a two-carbon unit
and a three-carbon unit, with the release of CO
2
. What is the thermodynamic advantage of this process
over one that simply combines two two-carbon units?
Chapter 21 Lipid Biosynthesis S-249
6. Modulation of Acetyl-CoA Carboxylase Acetyl-CoA carboxylase is the principal regulation point in
the biosynthesis of fatty acids. Some of the properties of the enzyme are described below.
(a) Addition of citrate or isocitrate raises the V
max
of the enzyme as much as 10-fold.
(b) The enzyme exists in two interconvertible forms that differ markedly in their activities:
Protomer (inactive) 34 filamentous polymer (active)
Citrate and isocitrate bind preferentially to the filamentous form, and palmitoyl-CoA binds pref-
erentially to the protomer.
Explain how these properties are consistent with the regulatory role of acetyl-CoA carboxylase in
the biosynthesis of fatty acids.
7. Shuttling of Acetyl Groups across the Mitochondrial Inner Membrane The acetyl group of
acetyl-CoA, produced by the oxidative decarboxylation of pyruvate in the mitochondrion, is
transferred to the cytosol by the acetyl group shuttle outlined in Figure 21–10.
(a) Write the overall equation for the transfer of one acetyl group from the mitochondrion to the
cytosol.
(b) What is the cost of this process in ATPs per acetyl group?
(c) In Chapter 17 we encountered an acyl group shuttle in the transfer of fatty acyl–CoA from the
cytosol to the mitochondrion in preparation for boxidation (see Fig. 17–6). One result of that
shuttle was separation of the mitochondrial and cytosolic pools of CoA. Does the acetyl group
shuttle also accomplish this? Explain.
Answer
c21LipidBiosynthesis.qxd 11/30/12 6:45 PM Page S-249
S-250 Chapter 21 Lipid Biosynthesis
8. Oxygen Requirement for Desaturases The biosynthesis of palmitoleate (see Fig. 21–12), a com-
mon unsaturated fatty acid with a cis double bond in the
9
position, uses palmitate as a precursor.
Can this be carried out under strictly anaerobic conditions? Explain.
9. Energy Cost of Triacylglycerol Synthesis Use a net equation for the biosynthesis of tripalmitoyl-
glycerol (tripalmitin) from glycerol and palmitate to show how many ATPs are required per molecule
of tripalmitin formed.
10. Turnover of Triacylglycerols in Adipose Tissue When [
14
C]glucose is added to the balanced diet
of adult rats, there is no increase in the total amount of stored triacylglycerols, but the triacylglycerols
become labeled with
14
C. Explain.
11. Energy Cost of Phosphatidylcholine Synthesis Write the sequence of steps and the net reaction
for the biosynthesis of phosphatidylcholine by the salvage pathway from oleate, palmitate, dihydroxy-
acetone phosphate, and choline. Starting from these precursors, what is the cost (in number of ATPs)
of the synthesis of phosphatidylcholine by the salvage pathway?
12. Salvage Pathway for Synthesis of Phosphatidylcholine A young rat maintained on a diet deficient
in methionine fails to thrive unless choline is included in the diet. Explain.
13. Synthesis of Isopentenyl Pyrophosphate If 2-[
14
C]acetyl-CoA is added to a rat liver homogenate
that is synthesizing cholesterol, where will the
14
C label appear in
3
-isopentenyl pyrophosphate, the
activated form of an isoprene unit?
Chapter 21 Lipid Biosynthesis S-251
14. Activated Donors in Lipid Synthesis In the biosynthesis of complex lipids, components are assembled
by transfer of the appropriate group from an activated donor. For example, the activated donor of acetyl
groups is acetyl-CoA. For each of the following groups, give the form of the activated donor: (a) phosphate;
(b)
D
-glucosyl; (c) phosphoethanolamine; (d)
D
-galactosyl; (e) fatty acyl; (f) methyl; (g) the two-
carbon group in fatty acid biosynthesis; (h)
3
-isopentenyl.
c21LipidBiosynthesis.qxd 11/30/12 6:45 PM Page S-251
S-252 Chapter 21 Lipid Biosynthesis
15. Importance of Fats in the Diet When young rats are placed on a totally fat-free diet, they grow
poorly, develop a scaly dermatitis, lose hair, and soon die—symptoms that can be prevented if
linoleate or plant material is included in the diet. What makes linoleate an essential fatty acid? Why
can plant material be substituted?
16. Regulation of Cholesterol Biosynthesis Cholesterol in humans can be obtained from the diet or
synthesized de novo. An adult human on a low-cholesterol diet typically synthesizes 600 mg of cholesterol
per day in the liver. If the amount of cholesterol in the diet is large, de novo synthesis of cholesterol is
drastically reduced. How is this regulation brought about?
17. Lowering Serum Cholesterol Levels with Statins Patients treated with a statin drug generally ex-
hibit a dramatic lowering of serum cholesterol. However, the amount of the enzyme HMG-CoA reductase
present in cells can increase substantially. Suggest an explanation for this effect.
18. Roles of Thiol Esters in Cholesterol Biosynthesis Draw a mechanism for each of the three reac-
tions shown in Figure 21–34, detailing the pathway for the synthesis of mevalonate from acetyl-CoA.
19. Potential Side Effects of Treatment with Statins Although clinical trials have not yet been carried
out to document benefits or side effects, some physicians have suggested that patients being treated
with statins also take a supplement of coenzyme Q. Suggest a rationale for this recommendation.
Chapter 21 Lipid Biosynthesis S-253
c21LipidBiosynthesis.qxd 11/30/12 6:46 PM Page S-253
S-254 Chapter 21 Lipid Biosynthesis
(a) Circle the eight isoprene units in the astaxanthin molecule. Hint: Use the projecting methyl
groups as a guide.
Astaxanthin is synthesized by the pathway shown on the following page, starting with
3
-isopentenyl pyrophosphate (IPP). Steps 1and 2are shown in Figure 21–36, and the reaction
catalyzed by IPP isomerase is shown in Figure 21–35.
(b) In step 4of the pathway, two molecules of geranylgeranyl pyrophosphate are linked to form
phytoene. Is this a head-to-head or a head-to-tail joining? (See Figure 21–36 for details.)
(c) Briefly describe the chemical transformation in step 5.
(d) The synthesis of cholesterol (Fig. 21–37) includes a cyclization (ring closure) that involves a net
oxidation by O
2
. Does the cyclization in step 6of the astaxanthin synthetic pathway require a
net oxidation of the substrate (lycopene)? Explain your reasoning.
E. coli does not make large quantities of many isoprenoids, and does not synthesize
astaxanthin. It is known to synthesize small amounts of IPP, DMAPP, geranyl pyrophosphate, farne-
syl pyrophosphate, and geranylgeranyl pyrophosphate. Wang and colleagues cloned several of the
E. coli genes that encode enzymes needed for astaxanthin synthesis in plasmids that allow their
overexpression. These genes included idi, which encodes IPP isomerase, and ispA, which encodes
a prenyl transferase that catalyzes steps 1and 2.
To engineer an E. coli capable of the complete astaxanthin pathway, Wang and colleagues
cloned several genes from other bacteria into plasmids that would allow their overexpression in
E. coli. These genes included crtE from Erwinia uredovora, which encodes an enzyme that
catalyzes step 3; and crtB, crtI, crtY, crtZ, and crtW from Agrobacterium aurantiacum,
which encode enzymes for steps 4,5,6,7, and 8, respectively.
The investigators also cloned the gene gps from Archaeoglobus fulgidus, overexpressed this gene
in E. coli, and extracted the gene product. When this extract was reacted with [
14
C]IPP and DMAPP,
or geranyl pyrophosphate, or farnesyl pyrophosphate, only
14
C-labeled geranylgeranyl pyrophosphate
was produced in all cases.
Data Analysis Problem
20. Engineering E. coli to Produce Large Quantities of an Isoprenoid There is a huge variety of
naturally occurring isoprenoids, some of which are medically or commercially important and produced
industrially. The production methods include in vitro enzymatic synthesis, which is an expensive and
low-yield process. In 1999, Wang, Oh, and Liao reported their experiments to engineer the easily
grown bacterium E. coli to produce large amounts of astaxanthin, a commercially important iso-
prenoid.
Astaxanthin is a red-orange carotenoid pigment (an antioxidant) produced by marine algae. Marine
animals such as shrimp, lobster, and some fish that feed on the algae get their orange color from the
ingested astaxanthin. Astaxanthin is composed of eight isoprene units; its molecular formula is
C
40
H
52
O
4
:
HO CH3
CH3CH3
H3CH3C
O
O
CH3
H3C
CH3
CH3CH3
OH
Astaxanthin
Chapter 21 Lipid Biosynthesis S-255
Geranyl pyrophosphate (C10) ⫹ PPi
Farnesyl pyrophosphate (C15) ⫹ PPi
Dimethylallyl pyrophosphate
(DMAPP)
⌬3-Isopentenyl pyrophosphate (IPP)
IPP isomerase
1
IPP
2
IPP
3
O
PP
P P
Geranylgeranyl pyrophosphate (C20) ⫹ PPi
Geranylgeranyl pyrophosphate
4
5
6
7
8
O
Phytoene (C40) ⫹ 2PPi
Lycopene (C40)
-Carotene (C40)
Astaxanthin (C40)
c21LipidBiosynthesis.qxd 11/30/12 6:46 PM Page S-255
S-256 Chapter 21 Lipid Biosynthesis
(e) Based on these data, which step(s) in the pathway are catalyzed by the enzyme encoded by
gps? Explain your reasoning.
Wang and coworkers then constructed several E. coli strains overexpressing different genes and mea-
sured the orange color of the colonies (wild-type E. coli colonies are off-white) and the amount of astaxan-
thin produced. Their results are shown below.
(f) Comparing the results for strains 1 through 4 with those for strains 5 through 8, what can you
conclude about the expression level of an enzyme capable of catalyzing step 3of the astaxan-
thin synthetic pathway in wild-type E. coli? Explain your reasoning.
(g) Based on the data above, which enzyme is rate-limiting in this pathway, IPP isomerase or the en-
zyme encoded by idi? Explain your reasoning.
(h) Would you expect a strain overexpressing crtBIZYW,gps, and crtE to produce low (), medium
(), or high () levels of astaxanthin, as measured by its orange color? Explain your rea-
soning.
Answer
Gene(s) Orange Astaxanthin yield
Strain overexpressed color (g/g dry weight)
1
crtBIZYW
ND
2
crtBIZYW, ispA
ND
3
crtBIZYW, idi
ND
4
crtBIZYW, idi, ispA
ND
5
crtBIZYW, crtE
32.8
6
crtBIZYW, crtE, ispA
35.3
7
crtBIZYW, crtE, idi
234.1
8
crtBIZYW, crtE, idi, ispA
390.3
9
crtBIZYW, gps
35.6
10
crtBIZYW, gps, idi
1,418.8
Note: ND, not determined
Chapter 21 Lipid Biosynthesis S-257
c21LipidBiosynthesis.qxd 11/30/12 6:46 PM Page S-257