Lecture 10: Fatty Acid Biosynthesis

Importance of Lipids:
  1. Major form of Energy storage in mammals
  2. Component of cell membranes
  3. pigment (retinal)
  4. cofactors (vitamin K)
  5. Detergents (bile salts)
  6. Transporters (dolichol)
  7. Hormones (Vitamin D and sex hormones

Overview of FA synthesis: "Reversal" of beta-oxidation

LOCATION of FA Synthesis = cell cytoplasm
- Inner mitochondrial membrane is impermeable to Acetyl-CoA;
Tricarboxylate transporter translocates it out of mitochondria as citrate
Tricarboxylate transporter summary:
- Brings acetyl-CoA from mitochondrial matrix to cytosol as citrate
- Facilitates directional transport
- Citrate synthase catalyzes formation of citrate within the mitochondrial matrix
- ATP-citrate lyase catalyzes reverse rxn in the cytosol to release Acetyl-CoA

Starting Substrates for FA Synthesis: Acetyl-CoA and Malonyl-CoA (donor of 2-carbons)
How to form Malonyl-CoA:
USE Acetyl-CoA Carboxylase (ACC) which is multi-domain/multi-functional and has biotin carboxylase & carboxyltransferase activity
Functional domains:
i. biotin carboxylating domain (BC)
ii. biotin binding domain (BB)
iii. Carboxyltransferase domain (CT)

STEP 1: Bicarbonate ion loaded on the biotin prosthetic grp attached to ACC
MECHANISM: Carboxylation driven by ATP hydrolysis
ENZYME: Biotin Carxoylase activity of ACC

STEP 2: Transfer carboxyl group from prosthetic to acetyl-CoA
MECHANISM: transcarboxylation
ENZYME: Transcarboxylase activity of ACC

STEP 1:      STEP 2:

NOTE: Need to convert Malonyl- CoA to ACP (immediate substrates for FA synthesis are attached to ACP not CoA)

Synthesis, Let's Go!
Enzyme responsible for catalysis = Fatty acid synthase (FAS)
  1. Converts Acetyl-CoA to Acetyl-ACP and malonyl-CoA to Malonyl-ACP
  2. Combine Acetyl-ACP and Malonyl-ACP into acetoacyl-ACP
  3. A bunch of rxns produce butyryl-ACP
Repeat 2 and 3 for an extra malonyl-CoA and keep adding on malonyl-CoA

FAS: Megasynthase = 272 kDA (HUGE ENZYME) with six major domains and one ACP; folds into seven domains with ACP right in the middle of everything

Loading Malonyl-CoA
STEP 1a: Convert acetyl-CoA into acetyl-ACP
START: acetyl-CoA
ENZYME: malonyl/acetyl CoA-ACP transacylase (MAT)
END: acetyl-ACP

STEP 1b: Load  Acetyl-ACP onto FAS 
ENZYME: beta-ketoacyl-ACP synthase

STEP 2: Convert malonyl-CoA into malonyl-ACP
START: malonyl-CoA
ENZYME: malonyl/acetyl CoA-ACP transacylase (MAT)
END: malonyl-ACP

*Top shows step 1a and 2, bottom shows the loading

6 Reactions in each round of FA synthesis.

STEP 1: Decarboxylation condensation to form C-C bond
ENZYME: KS subunit of FAS
END: beta-ketobutyryl-ACP

STEP 2: beta-carbon modification to reduce beta-keto group in beta-ketobutyryl-ACP
ENZYME: Ketoacyl-ACP reductase (KR)
END: beta-hydroxybutyryl-ACP

STEP 3: beta-carbon modification to dehydrate beta-hydroxybutyryl-ACP
ENZYME: Hydroacyl-ACP Dehydrase (DH)
END: trans-deltasquared- Butenoyl-ACP

STEP 4: beta-carbon modification: reduction of double bond
ENZYME: Enoyl-ACP reductase (ER)
END: Butyryl-ACP

STEP 5: Translocation of of butyryl group to Cysteine on beta-ketoacyl-ACP synthase (KS)
Done by KS
i.e. move from the ACP to the KS

"STEP 5.5": Recharge ACP with another malonyl group by MAT domain
Condensation via KS domain

Question: for C16-Palmitic acid, how many rounds of FA synthesis needed?
ANSWER: Since it has 16 carbons in the chain and each round produces 2 carbons, need 8 rounds
NOTE: New carbons always added on the carboxyl side, so the oldest malonyl-CoA added will be at the end of the chain

STEP 6: Hydrolysis
ENZYME: Thioesterase (TE) which cleaves the ester bond
END: A Fatty Acid chain

In mammalian cells, FA synthesis is catalyzed by the multi-domain, multi-function enzyme FAS that contains SIX enzyme activities (KS, KR, DH, ER, MAT, HS)
ACP is the prosthetic group involved in all steps of the enzymatic reactions
STARTER = acetyl-ACP, EXTENDER = malonyl-ACP (so 1 acetyl ACP : many malonyl-ACPs)

What about Elongation of very long fatty acids?

- FA synthase primarily produces palmitate (C16:0)
- In eukaryotic cells, Elongation happens in both the cytoplasm and the ER (endoplasmic reticulum)
- Microsomal FA Elongation that happens in ER involves acyl-CoA (malonyl CoA) derivatives; it's performed by separate enzymes bound to cytoplasmic face of ER membrane

In Humans...
EVOVL1-7: Elongation of very-long-chain FAs (7 genes)
KAR: beta-ketoreductase
HACD1-4: 3 hydroxyacyl-CoA dehydratase (4 genes)
TER: Trans-2,3-enoyl-CoA reductase

Fatty Acid Desaturation
Eukaryotic cells produce several ER-bound acyl-CoA desaturases that catalyze production of mono- and polyunsaturated FAs
Diff desaturases exhibit activity at bonds 9, 6 and 5
Two distinct families: 
  1. Stearoyl-CoA desaturases (SCDs)
  2. Fatty acid desaturases (FADs)
Typically, the first cis-double-bond is introduced between C9 and 10 (delta 9) and its reaction catalyzed by SCD
Additional double bonds are added by 6- and 5-desaturases (FADs)

PROBLEM: Humans unable to introduce double bonds beyond bond 9.
- SOLUTION: Fatty acids must come in from the diet:

Regulation of FA Synthesis

REGULATORS OF: Acetyl-CoA Carboxylase (ACC)
Activated by...
- citrate (allosterically)
Dephosphorylation (Insulin-activated protein phosphatase 2A or PP2A)
INActivated by...
- Palmitoyl-CoA (fatty acid-CoA) via feedback inhibition
Phosphorylation (AMP kinase, or AMPK; is an Energy sensor)

Regulators of actual FA synthesis: reciprocal regulation of beta-oxidation
- Malonyl-CoA inhib's CPT1 activity so FA synthesis inhib's beta-oxidation
- High FA concentration suppresses FA synthesis by blocking malonyl-CoA formation

diagram for regulation of ACC

In summary...

LOCATION: Mitochondria vs cytosol
COENZYMES: FAD and NAD+ vs NADPH (see the 6 steps per round of FA synthesis)
C2 UNIT: acetyl-CoA (product) vs malonyl-CoA (donor)

  1. FA Synthesis requires the activity of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS)
  2. Acetyl-CoA is transported from the mitochondria to the cytosol in the form of citrate (Tricarboxylate transport system)
  3. ACC, a biotin-dependent enzyme, catalyzes ATP- dependent carboxylation of acetyl-CoA resulting in malonyl-CoA
  4. Mammalian FAS (7 enzymatic activities plus acyl-carrier protein) catalyzes FA elongation to a maximum length of 16 carbons (palmitic acid-C16)