LIPID MAPS Lipid Metabolomics Tutorial

Fatty Acid Biosynthesis

Professor Edward A. Dennis

Department of Chemistry and Biochemistry
Department of Pharmacology, School of Medicine
University of California, San Diego

Copyright/attribution notice: You are free to copy, distribute, adapt and transmit this tutorial or
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Attribution: Edward A. Dennis (2010) “LIPID MAPS Lipid Metabolomics Tutorial” www.lipidmaps.org

E.A. DENNIS 2010 ©

Metabolism and Energy Overview
• Many biomolecules are
Carbo- degraded to Acetyl CoA
Proteins Lipids
hydrates • Acetyl CoA provides
biologic energy
• Excess acetyl CoA is
Amino Simple Fatty stored as Fatty Acids
(FA’s)
Acids Sugars Acids
• FA’s are assembled into
more complex lipids like
triglycerides (TG’s)
Pyruvate

Energy
Acetyl CoA (CO2, H2O)

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 What is a “Fatty Acid”?

Palmitic acid
Fatty acid: a carboxylic acid with a long hydrocarbon chain. Usually,
they have an even number of carbons. Reactive and toxic.

Ester group
Fatty acid ester: a fatty acid in which the carboxylic acid
group has reacted with the alcohol group of another molecule
(often glycerol) to form a stable, less reactive ester bond.

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 What is a “Triglyceride”?

Glycerol: common name for

1,2,3-trihydroxy-propane.
Glycerol

Triglyceride: a glycerol
molecule with three esterfied
fatty acid side chains. Also
known more correctly as a
“triacylglycerol”. Stable, non-
polar, hydrophobic. Triacylglycerol

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Common Saturated Fatty Acids
Saturated FA’s have no double bonds

16 Carbons = Palmitic Acid (Palmitate)

18 Carbons = Stearic Acid (Stearate)

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Common Unsaturated Fatty Acids
Unsaturated FA’s have at least one double bond,
usually in the Z (cis) conformation

18 Carbons, 1 double bond at c9 = Oleic Acid (Oleate)

18 9 1

18 Carbons, 2 double bonds at c9 and c12 = Linoleic Acid (Linoleate)

18 12 9 1

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More Unsaturated Fatty Acids
18 Carbons, 3 cis double bonds at 9, 12 & 15 =
a-Linolenic Acid (a-Linolenate)

18 15 12 9 1

20 Carbons, 4 cis double bonds at 5,8,11 & 14

Arachidonic Acid (Arachidonate)

(5Z,8Z,11Z,14Z-Eicosatetraenoic Acid)
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What are Essential Fatty Acids?
• Two “Essential” FA’s
Diet cannot be synthesized
by humans
– Linoleic acid
Linoleic Linolenic – Linolenic acid
acid acid • Used in the
biosynthesis of
polyunsaturated fatty
acid
Arachidonic • Must come from diet
EPA
acid

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Key Enzyme: Acetyl-CoA Carboxylase
• Acetyl-CoA Carboxylase is
a key enzyme
• Converts acetyl-CoA into
malonyl-CoA
– The CO2 is released
later
– Biotin is a cofactor
• It is the “committed step”
in FA synthesis
• It is the regulated, rate-
limiting enzyme in FA
synthesis
“E” above is the enzyme acetyl-CoA
carboxylase, which is conjugated to biotin.

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FA Synthesis: Step 1
Step 1: Set up acetyl-ACP

Acetyl-CoA-ACP
Transacylase

ACP is “acyl carrier protein” and is

a part of a large enzyme complex.
It holds the reactants in place
while other enzymes catalyze the
subsequent reaction steps.

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 FA Synthesis: Step 2
Step 2: Set up malonyl-ACP
Acetyl-CoA-ACP
Transacylase

HCO3-
Acetyl-CoA
Carboxylase

Malonyl-CoA-ACP
Transacylase

This step will iterate many times, adding carbons to the growing FA backbone.

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 FA Synthesis: Step 3
Step 3: Condense them, giving Acetoacetyl-ACP and CO2

condensing enzyme

CO2 ACP

The condensing enzyme is also known as b-ketoacyl-ACP

synthase. It is part of the FA synthase complex
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 FA Synthesis: Step 4
Step 4: Use NADPH to reduce the b-carbonyl to a hydroxyl group

H+ + NADPH
b-ketoacyl-ACP
reductase
NADP+

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 FA Synthesis: Step 5
Step 5: Remove the hydroxyl group as H 2O leaving a double bond

b-hydroxylacyl-ACP
dehydratase

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 FA Synthesis: Step 6
Step 6: Use another NADPH to reduce the double bond

H+ + NADPH
Enoyl-ACP
reductase

NADP +

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 FA Synthesis: Repeat Cycle
Repeat from step 2 using the new 4-carbon butyryl-ACP in place of acetyl-ACP

• 6 iterations
makes
Palmitoyl-ACP.
recycle reactions 2-6 • Finally, the
six more times enzyme
thioesterase
cleaves the ACP
from palmitoyl-
ACP
thioesterase • Palmitate is
released.

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Fatty Acid Synthesis Summary
One iteration:
Step 1: Set up acetyl-ACP

Step 2: Set up malonyl-ACP

Step 3: Condense them, giving Acetoacetyl ACP

Step 4: Use NADPH to reduce distal carbonyl to a hydroxyl group

Step 5: Remove the hydroxyl group as H 2O leaving a double bond

Step 6: Use another NADPH to reduce the double bond

REPEAT: From step 2 using the new 4-carbon butyryl-ACP in

place of acetyl-ACP in step 3

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 [2a]
Acetyl-CoA
Acetyl-CoA enters cycle Carboxylase

Acetyl-CoA Malonyl-CoA

[1] Acetyl-CoA-ACP transacylase Malonyl-CoA-ACP transacylase [2b]

Initiation
Acetyl-ACP Malonyl-ACP

Fatty acid [3]

synthase cycle b-ketoacyl-
ACP synthase

Release from
FA synthase complex
Acetoacetyl-ACP

b-ketoacyl-ACP reductase [4]

Elongation b-hydroxybutyryl-
ACP
Palmitoyl-ACP Butyryl-ACP
thioesterase
[5]
[6] enoyl-ACP reductase b-hydroxyacyl-ACP dehydratase

Palmitate
2-trans-butenoyl-ACP E.A. DENNIS 2010 ©
 The FA Synthase Enzyme
• FA synthase is an enzyme
complex
• It includes all the FA synthesis
enzymes except for acetyl-CoA
carboxylase
• Actually exists as a dimer of two
complete, anti-parallel complexes
-- like Ying and Yang
• Cytosolic
Figure: Voet, D, Voet JG, Pratt CW (2002),
Fundamentals of Biochemistry: Lif e at the
Molecular Level, 2nd ed. Reprinted with
permission of John Wiley & Sons, Inc.

Figures: Nelson DL, Cox MM (2005), Lehninger Principles of

Biochemistry, 4th ed. W.H. Freeman & Co. E.A. DENNIS 2010 ©
 Tuberculosis
Reported Tuberculosis in the US 1982-2008 • Incidence: one of the leading
Tuberculosis cases (thousands)

AIDS increase
causes of death due to infectious
50% decrease
diseases
– Often follows HIV infection
• Symptoms: pulmonary infection
– cough, sputum, pleural effusions
• see picture below left
– urogenital & brain affects also seen
• Mechanism: Mycobacterium
tuberculosis infection
Year • Treatments:
– First-line combination:
• Pyrazinamide and Isoniazid
– stop mycobacterial FA synthase!
• Rifampin (RNA transcription inhibitor)
– Various second-line agents
– If needed, HIV treatment

Normal lung Tuberculosis infection

Source: CDC
 Treating Tuberculosis
• Mycobacteria
– make their outer membrane with
mycolic acids using FA synthases
• FAS-1 is a single, eukaryote-like
enzyme with multiple actions
• FAS-2 is a multi-unit, prokaryote-like
enzyme with multiple actions
• Pyrazinamide (“peer-ah-ZIN-a-mide”)
– Inhibits FAS-I
– Relatively specific for M. tuberculosis
Figure: Draper, Nat. Med. 6, 977-8 (2000).
– Arrests synthesis of both fatty acids
and mycolic acids
Mycolic acid; • Isoniazid (“eye-so-NYE-a-zid”)
R1 and R2 are
– Inhibits FAS-II
long-chain
aliphatic – Stops synthesis of mycolic acids
hydrocarbons

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 Bacteria vs. Mammals

FA Synthesis in Bacteria FA Synthesis in Mammals

• Acyl carrier protein (ACP) • ACP is part of the FA
is a separate molecule, as synthase complex, which
are each of the six is one large protein
enzymes. present as a dimer.

• “Acetyl CoA carboxylase” • Acetyl-CoA carboxylase is

is two separate enzymes, one enzyme conjugated to
plus a biotin cofactor a biotin cofactor.
joined to a third enzyme.

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 Regulation of FA Synthesis
Regulation occurs primarily at acetyl-
CoA carboxylase, the rate limiting step

Feedback Mechanisms Hormonal Mechanisms

• Citrate, which builds up • Insulin, which signals a resting,
when acetyl-CoA is energy rich state, dephosphoryl-
ates and accelerates the enzyme.
plentiful, accelerates FA
synthesis. • Glucagon, epinephrine and
norepinephrine, which signal
• Palmitoyl-CoA weakly immediate energy needs, phos-
inhibits FA synthesis. phorylate and slow the enzyme
[via AMP -dependent protein
kinase and also via CMP-
dependent PKA].

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 Acknowledgement
This tutorial is based on an evolving subset of lectures and
accompanying slides presented to medical students in the Cell
Biology and Biochemistry course at the School of Medicine of the
University of California, San Diego.

I wish to thank Dr. Bridget Quinn and Dr. Keith Cross for aid in
developing many of the original slides, Dr. Eoin Fahy for advice in
applying the LIPID MAPS nomenclature and structural drawing
conventions [Fahy et al (2005) J Lipid Res, 46, 839-61; Fahy et al
(2009) J Lipid Res, 50, S9-14] and Masada Disenhouse for help in
adopting to the tutorial format.
Edward A. Dennis
September, 2010
La Jolla, California

E.A. DENNIS 2010 ©