Fatty Acid Synthesis

Fatty acid synthesis (also termed de novo lipogenesis) is the anabolic metabolic pathway that converts two-carbon acetyl-CoA precursors derived from excess dietary carbohydrates and amino acids into long-chain saturated fatty acids—principally the 16-carbon saturated fatty acid palmitate (hexadecanoate, C16:0)—for esterification into storage triacylglycerols and membrane phospholipids. In vertebrates, de novo lipogenesis takes place inside the cytosol of hepatocytes (liver), adipocytes (white and brown adipose tissue), the lactating mammary gland, and rapidly proliferating cells.

Whereas fatty acid catabolism (β-oxidation) occurs inside the mitochondrial matrix via discrete soluble enzymes using FAD and NAD⁺, mammalian fatty acid synthesis is spatially segregated to the cytosol and catalyzed by two sequential enzyme systems: acetyl-CoA carboxylase (ACC), which uses ATP and bicarbonate (HCO₃⁻) to activate acetyl-CoA into three-carbon malonyl-CoA, and Type I Fatty Acid Synthase (FAS), a 540-kDa homodimeric multi-catalytic megasynthase complex that uses NADPH (supplied by the pentose phosphate pathway and malic enzyme) to execute seven iterative cycles of condensation, reduction, dehydration, and saturation.

Because the pyruvate dehydrogenase complex (PDH) oxidizes glycolytic pyruvate into acetyl-CoA exclusively inside the mitochondrial matrix—and the inner mitochondrial membrane is impermeable to acetyl-CoA—cells employ the citrate–malate–pyruvate shuttle (tricarboxylate transport system) to export two-carbon units into the cytosol:

1. Citrate Export: Inside the mitochondrion, citrate synthase condenses acetyl-CoA with oxaloacetate to form citrate (the first intermediate of the citric acid cycle). When cellular energy charge is high, ATP and NADH inhibit mitochondrial isocitrate dehydrogenase, causing citrate to accumulate and exit across the inner mitochondrial membrane via the tricarboxylate carrier (SLC25A1) in exchange for malate.
2. ATP-Citrate Lyase (ACLY): In the cytosol, ATP-citrate lyase cleaves citrate using CoA-SH and the hydrolysis of 1 ATP → ADP + P_i to regenerate cytosolic acetyl-CoA and oxaloacetate.
3. NADPH Generation via Malic Enzyme: Cytosolic malate dehydrogenase reduces oxaloacetate to L-malate (oxidizing cytosolic NADH to NAD⁺). Cytosolic NADP⁺-dependent malic enzyme (ME1) then oxidatively decarboxylates L-malate into pyruvate + CO₂ while reducing NADP⁺ to NADPH. Pyruvate re-enters the mitochondrion to complete the cycle. For every 8 acetyl-CoA molecules shuttled to the cytosol to build one palmitate molecule, malic enzyme generates up to 8 of the 14 required NADPH molecules, while the oxidative branch of the pentose phosphate pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) supplies the remaining 6 NADPH molecules.

The irreversible, rate-limiting committed step of fatty acid synthesis is the carboxylation of acetyl-CoA (C2) to malonyl-CoA (C3) catalyzed by acetyl-CoA carboxylase (ACC):

Acetyl-CoA + HCO₃⁻ + ATP → Malonyl-CoA + ADP + P_i (Δ*G*°' = −1.2 kJ/mol)

Like pyruvate carboxylase, ACC utilizes a covalently linked biotin prosthetic group (tethered to a lysine residue on its biotin carboxyl carrier protein [BCCP] domain) as a flexible swinging arm. In the first half-reaction at the biotin carboxylase (BC) site, ATP phosphorylates bicarbonate to form a transient carboxyphosphate intermediate that carboxylates the N1 nitrogen of biotin. The 1.4-nm biotinyl-lysine arm then swings to the carboxyltransferase (CT) active site, transferring the activated –COO⁻ group onto the α-carbon of acetyl-CoA to yield malonyl-CoA.

Mammals express two isoforms: ACC1 (cytosolic, enriched in lipogenic tissues like liver and adipose tissue to supply malonyl-CoA for FAS) and ACC2 (tethered to the outer mitochondrial membrane in oxidative tissues like skeletal and cardiac muscle, where local malonyl-CoA potently inhibits carnitine palmitoyltransferase I [CPT-I], preventing newly synthesized or circulating fatty acids from entering mitochondria for β-oxidation and thus preventing a futile cycle).

Acyl Carrier Protein (ACP) and Priming

Mammalian Type I Fatty Acid Synthase (FAS) is an X-shaped homodimer of two identical 2,511-amino-acid (~270-kDa) polypeptides, each containing seven distinct functional domains. Growing acyl chains remain covalently bound throughout synthesis to the thiol (–SH) terminus of a flexible 2.0-nm 4'-phosphopantetheine prosthetic group (derived from vitamin B₅, pantothenic acid) attached to a serine residue of the Acyl Carrier Protein (ACP) domain.

Before the first elongation cycle, the malonyl/acetyltransferase (MAT) domain primes the complex by transferring an acetyl group from acetyl-CoA onto the ACP thiol, which is immediately translocated onto a conserved active-site cysteine thiol (–SH) of the β-ketoacyl-ACP synthase (KS) domain. MAT then loads a three-carbon malonyl group from malonyl-CoA onto the freed ACP phosphopantetheine thiol.

The Four-Step Elongation Cycle and Thioesterase Release

Each of the seven elongation cycles adds a two-carbon unit through a four-step reaction sequence that is the chemical converse of β-oxidation:

1. Condensation (KS — β-Ketoacyl-ACP Synthase): Decarboxylation of malonyl-ACP (releasing the CO₂ originally added by ACC) generates a nucleophilic carbanion that attacks the electrophilic thioester carbonyl of the acyl group on the KS cysteine, forming four-carbon acetoacetyl-ACP (a β-ketoacyl-ACP). The exergonic loss of CO₂ drives carbon–carbon bond formation toward completion.
2. First Reduction (KR — β-Ketoacyl-ACP Reductase): The β-keto group at C3 is stereospecifically reduced by NADPH + H⁺ to form the D-isomer D-3-hydroxybutyryl-ACP (contrast with L-3-hydroxyacyl-CoA in β-oxidation).
3. Dehydration (DH — β-Hydroxyacyl-ACP Dehydratase): Removal of H₂O across C2 and C3 yields the trans-Δ² unsaturated intermediate crotonyl-ACP (trans-Δ²-enoyl-ACP).
4. Second Reduction (ER — Enoyl-ACP Reductase): A second molecule of NADPH + H⁺ reduces the trans-Δ² double bond to form saturated four-carbon butyryl-ACP (C4:0).

The saturated C4 butyryl chain is then translocated from ACP back onto the KS cysteine thiol, ACP is reloaded with another malonyl-CoA, and the four-step cycle repeats six additional times (7 cycles total). Once the 16-carbon saturated palmitoyl-ACP chain is assembled, the steric bulk of C16 triggers the C-terminal thioesterase (TE) domain to hydrolyze the thioester linkage, releasing free palmitate.

Overall Stoichiometry for Palmitate

Synthesizing 7 malonyl-CoA from 7 acetyl-CoA via ACC requires: 7 Acetyl-CoA + 7 HCO₃⁻ + 7 ATP → 7 Malonyl-CoA + 7 ADP + 7 P_i

Combining 1 priming acetyl-CoA with 7 malonyl-CoA across 7 cycles on FAS (consuming 2 NADPH per cycle) yields the net overall stoichiometry:

8 Acetyl-CoA + 7 ATP + 14 NADPH + 14 H⁺ → Palmitate (C16:0) + 8 CoA-SH + 7 ADP + 7 P_i + 14 NADP⁺ + 6 H₂O

Allosteric and Hormonal Regulation of ACC

• Allosteric Control: Cytosolic citrate (high when mitochondrial acetyl-CoA and ATP are abundant) binds to inactive ACC dimers and promotes their polymerization into catalytically active filamentous polymers (V_max increases ~10-fold). Conversely, the end product palmitoyl-CoA triggers depolymerization of ACC filaments back into inactive protomers.
• Covalent Phosphorylation: When cellular energy drops (elevated [AMP]/[ATP]) or during fasting/stress, AMP-activated protein kinase (AMPK) and glucagon/epinephrine-activated protein kinase A (PKA) phosphorylate specific serine residues (Ser79 on ACC1) to lock ACC in its inactive monomeric state. In the carbohydrate-fed state, insulin activates protein phosphatase 2A (PP2A) to dephosphorylate and activate ACC while inducing SREBP-1c and ChREBP transcription of ACACA and FASN.

How are fatty acids longer than 16 carbons or unsaturated fatty acids synthesized?

Once palmitate (C16:0) is released by FAS and activated to palmitoyl-CoA, enzyme systems on the cytosolic face of the smooth endoplasmic reticulum (ER) (the ELOVL elongase family) add two-carbon units from malonyl-CoA to produce stearate (C18:0) and longer chains (C20–C24). Similarly, ER membrane stearoyl-CoA desaturase-1 (SCD1, Δ⁹-desaturase)—coupled to cytochrome b₅, NADPH, and O₂—introduces a cis-Δ⁹ double bond to form palmitoleate (16:1) and oleate (18:1).

Why are linoleic acid (ω-6) and α-linolenic acid (ω-3) essential dietary fatty acids in mammals?

Mammalian fatty acid desaturases (Δ⁹, Δ⁶, and Δ⁵ desaturases) can only introduce cis carbon–carbon double bonds between the C1 carboxyl carbon and carbon-9 (Δ⁹) of an acyl chain; they lack the Δ¹² and Δ¹⁵ desaturase enzymes present in plants and phytoplankton. Consequently, mammals cannot synthesize linoleate (18:2 cis-Δ^9,12, an ω-6 fatty acid) or α-linolenate (18:3 cis-Δ^9,12,15, an ω-3 fatty acid) de novo and must obtain them from the diet to synthesize arachidonate (20:4 ω-6) and eicosanoids.

Why is Type I FAS a drug target in oncology, while Type II FAS is targeted by antibiotics?

Whereas normal adult human tissues (outside the liver and adipose tissue) obtain most of their lipids from circulating lipoproteins and express low levels of Type I FAS, many human carcinomas (breast, prostate, and colon cancers) overexpress FASN to support rapid membrane biogenesis. Conversely, bacteria, plants, and apicoplast-bearing parasites utilize Type II FAS, in which each catalytic step is performed by a separate, dissociable monofunctional protein—allowing drugs like isoniazid (targeting mycobacterial InhA) and triclosan (targeting bacterial FabI enoyl-ACP reductase) to kill pathogens selectively without inhibiting human Type I FAS.

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