Chapter Review in Chapter 21 Lipid Biosynthesis

Chapter Review

KEY TERMS

Terms in bold are defined in the glossary.

Problems

1. Pathway of Carbon in Fatty Acid Synthesis Using your knowledge of fatty acid biosynthesis, provide an explanation for the two experimental observations.
1. A biochemist adds uniformly labeled $[^{14} C]$ $left-bracket Superscript 14 Baseline zero width space zero width space upper C right-bracket$ acetyl-CoA to a soluble liver fraction, which yields palmitate uniformly labeled with $^{14} C$ $Superscript 14 Baseline zero width space zero width space upper C$ .
2. In a second experiment, the biochemist adds a trace of uniformly labeled $[^{14} C]$ $left-bracket Superscript 14 Baseline zero width space zero width space upper C right-bracket$ acetyl-CoA in the presence of an excess of unlabeled malonyl-CoA to a soluble liver fraction, which yields palmitate labeled with $^{14} C$ $Superscript 14 Baseline zero width space zero width space upper C$ only in C-15 and C-16.
2. Synthesis of Fatty Acids from Glucose After a person has ingested large amounts of sucrose, the body transforms the glucose and fructose that exceed caloric requirements to fatty acids for triacylglycerol synthesis. This fatty acid synthesis consumes acetyl-CoA, ATP, and NADPH. How do cells produce acetyl-CoA, ATP, and NADPH 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 ${CO}_{2}$ $CO Subscript 2$ .
4. Pathway of Hydrogen in Fatty Acid Synthesis A researcher has prepared a solution that contains all the enzymes and cofactors necessary for fatty acid biosynthesis from added acetyl-CoA and malonyl-CoA.
1. She then adds $[2 -^{2} H]$ $left-bracket 2 hyphen squared upper H right-bracket$ acetyl-CoA (labeled with deuterium, the heavy isotope of hydrogen) and an excess of unlabeled malonyl-CoA as substrates.
  
  How many deuterium atoms incorporate into every molecule of palmitate? What are their locations? Explain.
2. In a separate experiment, the researcher adds unlabeled acetyl-CoA and $[2 -^{2} H]$ $left-bracket 2 hyphen squared upper H right-bracket$ malonyl-CoA as substrates.
  
  How many deuterium atoms incorporate into every molecule of palmitate? What are their locations? Explain.
5. Energetics of β-Ketoacyl-ACP Synthase The condensation reaction catalyzed by β-ketoacyl-ACP synthase (see Fig. 21-6) synthesizes a four-carbon unit by combining a two-carbon unit and a three-carbon unit, with the release of ${CO}_{2}$ $CO Subscript 2$ . What is the thermodynamic rationale for this process relative to one that simply combines two two-carbon units?
6. Modulation of Acetyl-CoA Carboxylase Acetyl-CoA carboxylase is the principal regulation point in the biosynthesis of fatty acids. Following are some of the properties of the enzyme:
1. Addition of citrate or isocitrate raises the $V_{max}$ $upper V Subscript max$ of the enzyme as much as 10-fold.
2. The enzyme exists in two interconvertible forms that differ markedly in their activities:
  $Protomer (inactive) ⇌ filamentous polymer (active)$ $Protomer left-parenthesis inactive right-parenthesis right harpoon over left harpoon filamentous polymer left-parenthesis active right-parenthesis$
  
  Citrate and isocitrate bind preferentially to the filamentous form, and palmitoyl-CoA binds preferentially 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 Inner Mitochondrial Membrane The acetyl group shuttle transfers acetyl-CoA, produced by oxidative decarboxylation of pyruvate in the mitochondrion, to the cytosol as outlined in Figure 21-10.
1. Write the overall equation for the transfer of one acetyl group from the mitochondrion to the cytosol.
2. What is the cost of this process in ATPs per acetyl group?
3. 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 β oxidation (see Fig. 17-6). One result of that shuttle is separation of the mitochondrial and cytosolic pools of CoA. Does the acetyl group shuttle also accomplish this? Explain.
8. Oxygen Requirement for Desaturases The biosynthesis of palmitoleate (see Fig. 21-12), a common unsaturated fatty acid with a cis double bond in the $Δ^{9}$ $upper Delta Superscript 9$ position, uses palmitate as a precursor. Can palmitoleate synthesis be carried out under strictly anaerobic conditions? Explain.
9. Energy Cost of Triacylglycerol Synthesis Use a net equation for the biosynthesis of tripalmitoylglycerol (tripalmitin) from glycerol and palmitate to show how many ATPs are required per molecule of tripalmitin formed.
10. Turnover of Triacylglycerols in Adipose Tissue A researcher adds $[^{14} C]$ $left-bracket Superscript 14 Baseline zero width space zero width space upper C right-bracket$ glucose to the balanced diet of adult rats. She finds no increase in the total amount of stored triacylglycerols, but the triacylglycerols become labeled with $^{14} C$ $Superscript 14 Baseline zero width space zero width space upper C$ . 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, dihydroxyacetone 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. Energetics of Acetyl-CoA Condensation to Form Acetoacetyl-CoA The formation of a thioester of acetoacetate is catalyzed by fatty acid synthase during fatty acid synthesis, and by acetyl-CoA acetyltransferase in the first step of cholesterol biosynthesis. Both are Claisen condensations. However, in fatty acid synthesis, malonyl-CoA forms in an earlier step so that decarboxylation facilitates the condensation. In the cholesterol biosynthesis pathway, the condensation occurs between two acetyl-CoA molecules, and no decarboxylation occurs to facilitate the reaction. Suggest a reason why the thermodynamic augmentation of decarboxylation is needed in fatty acid synthesis, but not in the first steps of cholesterol biosynthesis.
14. Synthesis of Isopentenyl Pyrophosphate A researcher adds $[2 -^{14} C]$ $left-bracket 2 hyphen Superscript 14 Baseline zero width space zero width space upper C right-bracket$ acetyl-CoA to a rat liver homogenate that is synthesizing cholesterol. Where will the $^{14} C$ $Superscript 14 Baseline zero width space zero width space upper C$ label appear in $Δ^{3}$ $upper Delta Superscript 3$ -isopentenyl pyrophosphate, the activated form of an isoprene unit?
15. 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:
1. phosphate;
2. d-glucosyl;
3. phosphoethanolamine;
4. d-galactosyl;
5. fatty acyl;
6. methyl;
7. the two-carbon group in fatty acid biosynthesis;
8. $Δ^{3}$ $upper Delta Superscript 3$ -isopentenyl.
16. Importance of Fats in the Diet When young rats are placed on a completely fat-free diet, they grow poorly, develop a scaly dermatitis, lose hair, and soon die. These symptoms 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?
17. 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?
18. Lowering Serum Cholesterol Levels with Statins Patients treated with a statin drug generally exhibit a dramatic lowering of serum cholesterol. However, the amount of the enzyme HMG-CoA reductase present in cells can increase substantially. Suggest a simple explanation for this effect.
19. Roles of Thiol Esters in Cholesterol Biosynthesis Draw a mechanism for each of the three reactions shown in Figure 21-34, detailing the pathway for the synthesis of mevalonate from acetyl-CoA.
20. Potential Side Effects of Treatment with Statins Although the benefits of taking statins have become clear, side effects have yet to be documented in detail. Some physicians have suggested that patients being treated with statins should also take a supplement of coenzyme Q. Suggest a rationale for this recommendation.

DATA ANALYSIS PROBLEM

21. Engineering E. coli to Produce Large Quantities of an Isoprenoid There are more than 20,000 naturally occurring isoprenoids, some of which are medically or commercially important and produced industrially. The production methods include in vitro enzymatic synthesis, an expensive and low-yield process. In 1999, Wang, Oh, and Liao reported on their experiments to engineer the easily grown bacterium E. coli to produce large amounts of astaxanthin, a commercially important isoprenoid. 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 $C_{40} H_{52} O_{4}$ $upper C Subscript 40 Baseline upper H Subscript 52 Baseline upper O Subscript 4$ .

A figure shows the structure of astaxanthin.

Astaxanthin has a six-carbon ring on the left side with a double bond on its right side, a lower right vertex bonded to C H 3, a bottom vertex double bonded to O, a lower left vertex bonded to O H, a top vertex bonded to 2 C H 3, and a right side vertex bonded to C H double bonded to C H bonded to C bonded to C H 3 above double bonded to C H bonded to C H double bonded to C H bonded to C bonded to C H 3 above double bonded to C H bonded to C H double bonded to C H bonded to C H double bonded to C bonded to C H 3 below bonded to C H double bonded to C H bonded to C H double bonded to C bonded to C H 3 below bonded to C H double bonded to C H bonded to the lower left vertex of a six-membered ring with a double bond on its left side, an upper left vertex bonded to C H 3, a top vertex double bonded to O, an upper right vertex bonded to O H, and a bottom vertex bonded to 2 C H 3.

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 next page, starting with $Δ^{3}$ $upper Delta Superscript 3$ -isopentenyl pyrophosphate (IPP). Steps and are shown in Figure 21-36, and the reaction catalyzed by IPP isomerase is shown in Figure 21-35.
In step of the pathway, two molecules of geranylgeranyl pyrophosphate are linked to form phytoene. Is this a head-to-head joining or is it a head-to-tail joining? (See Figure 21-36 for details.)
Briefly describe the chemical transformation in step .
The synthesis of cholesterol (Fig. 21-37) includes a cyclization (ring closure) that requires a net oxidation by $O_{2}$ $upper O Subscript 2$ . Does the cyclization in step of 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, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate. Wang and colleagues cloned several of the E.coli genes that encode enzymes needed for astaxanthin synthesis, in plasmids that allowed their overexpression. These genes included idi, which encodes IPP isomerase, and ispA, which encodes a prenyl transferase that catalyzes steps and .

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 ; and crtB, crtI, crtY, crtZ, and crtW from Agrobacterium aurantiacum, which encode enzymes for steps , , , , and , respectively.

Arrow 1 points down from delta superscript 3 end superscript-isopentenyl pyrophosphate (I P P) to geranyl pyrophosphate (C subscript 10 end subscript) plus P P subscript i end subscript and is accompanied by a curved arrow showing the addition of dimethylallyl pyrophosphate (D M A P P). Diagonal forward and reverse arrows labeled I P P isomerase indicate a reversible reaction between delta superscript 3 end superscript-isopentenyl pyrophosphate and dimethylallyl pyrophosphate. Arrow 2 points from geranyl pyrophosphate (C subscript 10 end subscript) plus P P subscript i end subscript to farnesyl pyrophosphate (C subscript 15 end subscript) plus P P subscript i end subscript and is accompanied by a curved arrow showing the addition of I P P. Farnesyl pyrophosphate is a twelve-carbon zigzag chain with C 1 on the right bonded to O bonded to a circle labeled P bonded to a circle labeled P; with double bonds between C 2 and C 3, C 6 and C 7, and C 10 and C 11; and with methyl groups bonded to C 3, C 7, and C 11. Arrow 3 points down to geranylgeranyl pyrophosphate (C subscript 20 end subscript) plus P P subscript i end subscript and is accompanied by a curved arrow showing the addition of I P P. Geranylgeranyl pyrophosphate is a sixteen-carbon zigzag chain with C 1 on the right bonded to O bonded to a circle labeled P bonded to a circle labeled P; with double bonds between C 2 and C 3, C 6 and C 7, C 10 and C 11, and C 14 and C 15; and with methyl groups bonded to C 3, C 7, C 11, and C 15. Arrow 4 points down to phytoene (C subscript 40 end subscript) plus 2 P P subscript i end subscript and is accompanied by an arrow showing the addition of geranylgeranyl pyrophosphate. Phytoene is a thirty-two carbon zigzag chain with a peak in the center at a cis double bond from which it bends down to the left and right. From left to right, it has double bonds between C 2 and C 3, C 6 and C 7, C 10 and C 11, C 14 and C 15, C 16 and C 17, C 18 and C 19, C 22 and C 23, C 26 and C 27, and C 30 and C 31 and methyl groups bonded to C 2, C6, C 10, C 14, C 16, C 19, C 23, C 27, and C 31. Arrow 5 points down to lycopene (C subscript 40 end subscript), which is horizontal. From left to right, it has double bonds at C 2 to C 3, C 6 to C 7, C 8 to C 9, C 10 to C 11, C 12 to C 13, C 14 to C 15, C 16 to C 17, C 18 to C 19, C 20 to C 21, C 22 to C 23, C 24 to C 25, C 26 to C 27, and 30 to C 31 and methyl groups at C 2, C 6, C 10, C 14, C 19, C 23, C 27, and C 31. Arrow 6 points down to beta-carotene (C subscript 40 end subscript). Beta-carotene has a six-membered ring on the left with a double bond at its right side, a lower right vertex bonded to C H 3, a top vertex bonded to 2 C H 3, and an upper right vertex bonded to C H double bonded to C H bonded to C bonded to C H 3 above and double bonded to C H bonded to C H double bonded to C H bonded to C bonded to C H 3 above and double bonded to C H bonded to C H double bonded to C H bonded to C H double bonded to C bonded to C H 3 below bonded to C H double bonded to C H bonded to C H double bonded to C bonded to C H 3 below bonded to C H double bonded to C H bonded to the lower right vertex of a six-membered ring with a double bond at the left side, an upper right vertex bonded to C H 3, and a bottom vertex bonded to 2 C H 3. Arrow 7 points down to arrow 8, which points down to astaxanthin (C subscript 40 end subscript).

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 $[^{14} C] IPP$ $left-bracket Superscript 14 Baseline zero width space zero width space upper C right-bracket IPP$ and DMAPP or geranyl pyrophosphate or farnesyl pyrophosphate, only $^{14} C$ $Superscript 14 Baseline zero width space upper C$ -labeled geranylgeranyl pyrophosphate was produced in all cases.

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; they measured the orange color of the colonies (wild-type E. coli colonies are off-white) and the amount of astaxanthin produced (as measured by its orange color). Their results are shown below (ND indicates not determined).

Strain Gene(s) overexpressed Orange color Astaxanthin yield (μg/g dry weight)

1 crtBIZYW $-$ $minus$ ND

2 crtBIZYW, ispA $-$ $minus$ ND

3 crtBIZYW, idi $-$ $minus$ ND

4 crtBIZYW, idi, ispA $-$ $minus$ ND

5 crtBIZYW, crtE $+$ $plus$ 32.8

6 crtBIZYW, crtE, ispA $+$ $plus$ 35.3

7 crtBIZYW, crtE, idi $+ +$ $plus plus$ 234.1

8 crtBIZYW, crtE, idi, ispA $+ + +$ $plus plus plus$ 390.3

9 crtBIZYW, gps $+$ $plus$ 35.6

10 crtBIZYW, gps, idi $+ + +$ $plus plus plus$ 1,418.8

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 3 of the astaxanthin synthetic pathway in wild-type E. coli? Explain your reasoning.

Based on the data above, which enzyme is rate-limiting in this pathway, IPP isomerase or the enzyme encoded by idi? Explain your reasoning.

Strain	Gene(s) overexpressed	Orange color	Astaxanthin yield (μg/g dry weight)
1	crtBIZYW	$-$ $minus$	ND
2	crtBIZYW, ispA	$-$ $minus$	ND
3	crtBIZYW, idi	$-$ $minus$	ND
4	crtBIZYW, idi, ispA	$-$ $minus$	ND
5	crtBIZYW, crtE	$+$ $plus$	32.8
6	crtBIZYW, crtE, ispA	$+$ $plus$	35.3
7	crtBIZYW, crtE, idi	$+ +$ $plus plus$	234.1
8	crtBIZYW, crtE, idi, ispA	$+ + +$ $plus plus plus$	390.3
9	crtBIZYW, gps	$+$ $plus$	35.6
10	crtBIZYW, gps, idi	$+ + +$ $plus plus plus$	1,418.8

Reference

Wang, C.-W., M.-K. Oh, and J.C. Liao. 1999. Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli. Biotechnol. Bioeng. 62:235–241.