Biosynthesis of saturated fatty acids

The content describes the various steps of fatty acid biosynthesis

1. Biosynthesis of saturated fatty acids Dr Renjana P. K . Assistant professor, Department of Botany, Govt Arts & Science College, Kozhikode

2. Two pathways Intramitochondrial Reversal of β -oxidation Synthesis of long chain FAs & elongation of already existing ones Anaerobic Extramitochondrial Located in the ER FA synthesis from acetyl Co-A Production of malonyl Co-A from acetyl Co-A End product is palmitate

3. Extramitochondrial FA synthesis Acetyl Co-A as a primer Precursor is malonyl Co-A formed from Acetyl Co-A Principal compund is Palmitic acid Fatty acid synthase complex Cofactors- NADPH, HCO 3 - , ATP, Mn ++ , Mg ++ Cyclic process

4. Enzymes & Co-factors Two main enzymes- 1. Acetyl Co-A carboxylase 2. Fatty acid synthase Co- fcators - 1. Biotin 2. NADH 3. Mg ++ 4. Mn ++ 5. ATP 6. HCO 3 -

5. Stages of Fatty acid biosynthesis Transport of Acetyl-Co A and NADPH to the cytoplasm Conversion of Acetyl-Co A to Malonyl Co-A Reactions of Fatty acid synthase complex

6. Transport of Acetyl Co-A Acetyl Co-A condenses with oxaloacetate in mitochondria to form citrate Citrate is transported to cytoplasm In the cytosol it is cleaved by ATP-citrate lyase to liberate Acetyl Co-A and oxaloacetate This transport is coupled with the production of NADPH & CO 2 in the cytoplasm (needed for FA biosynthesis)

7. Pyruvate Fatty acids Amino acids Acetyl Co-A Production & Transport of Acetyl Co-A Formed in the mitochondrion Transported to cytoplasm Mitochondrial membrane is impermeable to Acetyl Co-A 3 major routes (shuttle mechanisms )

8. Transportation of Acetyl co A

9. Transfer of Acetyl Co-A from mitochondria to cytosol Glucose Pyruvate Malate Oxaloacetate Citrate Fatty acid HMP Shunt NADPH+H + NADP + CO 2 NADH+H + NAD + Acetyl Co-A CoASH ADP+Pi ATP Pyruvate CYTOSOL MITOCHONDRIAL MATRIX Acetyl Co-A Oxaloacetate Citrate Pyruvate carboxylase PDH Complex Citrate Synthase Amino acids FA Citrate lyase MDH Malic Enzyme

10. Addition of 2-carbon acetyl units to carboxyl end of a growing chain Obtained from first two C-atoms of 3-carbon compound malonyl Co-A Major steps of FA synthesis Production of malonyl Co-A from acetyl Co-A Fatty acid synthesis from malonyl Co-A Malonyl Co-A

11. Formation of malonyl Co-A Acetyl Co-A carboxylase (rate limiting enzyme) Acetyl Co-A + HCO 3 - Malonyl Co-A Acetyl Co-A carboxylase Biotin ATP ADP

12. Fatty acid synthesis from malonyl Co-A Several steps Multienzyme system (Fatty acid synthetase system) 7 enzymes and Acyl Carrier protein (ACP)- centre Prosthetic group of ACP is 4-phospho-pantetheine – Sulfhydryl (-SH) group Another –SH group in the peripheral part -SH groups act as acceptors of acyl groups

13. Once malonyl-CoA is synthesized, long carbon FA chains may be assembled in a repeating four-step sequence. With each passage through the cycle the fatty acyl chain is extended by two carbons. When the chain reaches 16 carbons, the product palmitate (16:0) leaves the cycle . Assembly of a long chain fatty acid

14. Each segment of the disk represents one of the six enzymatic activities of the complex ( Thioesterase not shown) At the center is the ACP – acyl carrier protein - with its phosphopantethein -e arm ending in –SH. Fatty acid synthase complex

15. Fatty acid synthesis- Steps The 2 imp prosthetic groups of FA synthase complex : - thiol (-SH)of phosphopantetheine , covalently bound prosthetic group of Acyl carrier protein - thiol (-SH)of the side-chain of a cysteine residue of keto acyl synthase enzyme(condensing enzyme-3 rd enzyme) After the formation of malonyl Co-A, all the remaining steps are catalyzed by Fatty acid synthase complex

16. A ctivation of the acetyl group The acetyl group from acetyl-CoA is transferred to the Cys-SH group of the b - ketoacyl ACP synthase This reaction is catalyzed by Acetyl transacylase .

17. A ctivation of the malonyl group Transfer of the malonyl group to the –SH group of the ACP is catalyzed by Malonyl transacylase The charged acetyl and malonyl groups are now in close proximity to each other

18. After activation, the processes involved are- 1. Condensation 2. Reduction 3. Dehydration 4. Reduction These steps are repeated till a fatty acid with 16 carbon atoms is synthesized Series of Reactions

19. To initiate FA biosynthesis, malonyl and acetyl groups are activated on to the enzyme fatty acid synthase . Initially, a priming molecule of acetyl- CoA combines with a cysteine —SH group catalyzed by acetyl transacylase Malonyl-CoA combines with the adjacent —SH on the 4'-phosphopantetheine of ACP of the other monomer, catalyzed by malonyl transacylase (to form acyl-malonyl enzyme. The first round of FA biosynthesis-

20. Step-1(Condensation) The acetyl group attacks the methylene group of the malonyl residue, catalyzed by β -ketoacyl synthase , and liberates CO 2 , forming Acetoacetyl enzyme, freeing the cysteine —SH group.

21. Condensation – Condensation of the activated acetyl and malonyl groups takes place to form Acetoacetyl-ACP Enzyme β - ketoacyl -ACP synthase.

22. Step-2 (Reduction) Reduction - The Acetoacetyl-ACP is reduced to b- hydroxybutyryl -ACP, catalyzed by β - ketoacyl -ACP reductase NADPH + H + are required for reduction

23. Step-3 (Dehydration) Dehydration – Dehydration yields a double bond in the product, Crotonyl -S-ACP Reaction is catalyzed by β - hydroxybutyryl -ACP dehydratase .

24. Step-4 (Reduction) Reduction Reduction of the double bond takes place to form butyryl -S-ACP, Reaction is catalyzed by enoyl-reductase. Another NADPH dependent reaction.

25. This reaction makes way for the next incoming malonyl group. The enzyme involved is acetyl- transacetylase The growing chain is transferred from the acyl carrier protein

26. Beginning of the second round of the FA synthesis cycle The butyryl group is on the Cys -SH group The incoming malonyl group is first attached to ACP. In the condensation step, the entire butyryl group is exchanged for the carboxyl group on the malonyl residue

27. Repetition of these four steps leads to fatty acid synthesis The β - ketoacyl group is reduced, dehydrated, and reduced again (reactions 2, 3, 4) to form the corresponding saturated acyl -S-enzyme. A new malonyl-CoA molecule combines with the —SH of 4'-phosphopantetheine, displacing the saturated acyl residue onto the free cysteine —SH group. The sequence of reactions are repeated until a saturated 16-carbon acyl radical ( Palmityl ) has been assembled. It is liberated from the enzyme complex by the activity of a seventh enzyme in the complex, Thioesterase ( deacylase ).

28. Repetition of these four steps leads to fatty acid synthesis

29. Sequence of reactions Acyl transfer Malonyl transfer Condensation Reduction Dehydration Reduction Acyl transfer Reactions of 2-6 are repeated 6 times (7 cycles of reactions) End product is palmitate 2C – Acetyl Co-A directly 14C- Malonyl Co-A

30. 8 Acetyl Co-A = 1 Acetyl Co-A + 7 Malonyl Co-A 7 cycles of fatty acid synthesis 14 ATP 14 ADP 14 NADPH + H + 14 NADP + Palmitic acid + 7CO 2 + 8Co-A-SH + 6H 2 O Summary of Palmitic acid synthesis

31. 1. Acetyl Co-A + ACP-SH Acetyl S-ACP + Co-A 2. Malonyl Co-A + ACP-SH 4. Malonyl –S-ACP + Acetyl S- Cys -ENZ (3) Malonyl -S-ACP + Co-A Acetoacetyl -S-ACP+ 3. Acetyl S-ACP + ENZ (3)- Cys - SH Acetyl S- Cys -ENZ (3) 5. Acetoacetyl S-ACP + NADH + H + β - hydroxy butyryl -S-ACP + NADP + Reduction Condensation Acetyl transacylase Malonyl transacylase β - ketoacyl synthetase β - ketoacyl reductase ENZ(3)+CO 2

32. 6. β - hydroxy butyryl -S-ACP Crotonyl -S-ACP + H 2 7. Crotonyl -S-ACP + NADPH + H + Butyryl -S-ACP + NADP + 8. Butyryl -S-ACP + ENZ- Cys -SH Butyryl -S- Cys -ENZ(3)+ ACP Butyryl -S- Cys -ENZ(3) + Malonyl -S-ACP Continues Reduction Dehydration β - ketoacyl dehydratase Enoyl reductase Enzyme bound palmitic acid Pamlitic Acid + Free enzyme Thioesterace / Deacylase Cleavage

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