Fatty acid biosynthesis
References: Mann, J (1994) Chemical aspects of Biosynthesis, Oxford Science Publications, pp. 10-18 Loudon, M. Organic chemistry Solomons, G. Organic Chemistry

Occurrence of fatty acids in Nature
• Fatty acids are seldom encountered in free form, and are usually found to be esterified with glycerol • Some plants of the Crucifer (mustard) family (which includes cabbages, broccoli, Brussel sprouts) store fatty acids in free, unesterified form. However, this is rare in Nature • Fats and oils are triacylglycerides whereby all of the three hydroxyl groups of glycerol are esterified • Membrane lipids are phospholipids in which two hydroxyl groups of glycerol are esterified with fatty acids, and one hydroxyl group is modified by phosphorylation which forms the polar (hydrophilic) portion of the molecule

The fatty acid synthase complex - Bacterial fatty acid synthases are aggregates of 6-7 enzymes while in plants and animals this synthase is a dimeric complex of two multifunctional protein which possess seven catalytic sites. Another enzyme, thioesterase which catalyses the release of the fatty - The functional groups responsible for binding to acetyl coA or malonyl CoA are thiol groups belonging either to cysteine (cys) or 4-phosphopantetheine (pant), the structure of which is similar to that of coenzyme A. Phosphopantetheine is attached to serine. -The cysteine active site is located on acyl carrier protein (ACP) while the 4-phosphopantetheine is located on ketoacyl synthase.

Simplified scheme for the reactions of fatty acid synthase (from Solomons)

Enzymes of the fatty acid synthase complex. Thioesterase which frees the synthesized fatty acid by hydrolysis is not covalently linked to the other enzymes

Dimeric structure of the complex. The thiol group is the reactive functional group on the catalytic sites

acetyl coA and malonyl coA are attached to the “cys” and “pant” active sites by a transesterification process, the reactions being catalyzed by acetyl transacyclase (“AT” and malony transacyclase (“MT”, respectively

-

β-ketoacylsynthase (“β-KS”) then catalyses a Claisen condensation between the bound acetyl coA and malonyl coA, producing a butanoyl thioester

the butanoyl thioester is reduced by β-ketoacyl reductase which uses NADPH as a hydride source, producing an R-hydroxythioester

.

The hydroxylthioester is acted upon by a dehydratase (“DH”) enzyme, producing an alkene by eliminating water, yielding an enoyl thioester

- The enoyl is in turn acted upon by enoly reductase, producing a saturated thioester - The thioester is then transferred (this is a transesterification step) to the “cys” active site of ketoacyl for another cycle of the biosynthesis process - Following this transesterification, the thiol group on the “pant” active site of the ACP is free to bind to another molecule of malonyl coA

when the thioester chain is between 14 (as in myristic acid) to 18 (as in stearic acid) carbons long, it is released by a thioesterase (“TE”) –catalysed hydrolysis
-

Balanced equation for palmitic acid biosynthesis
CH3COSCoA + 7 HOCO-CH2-COSCoA + 14 NADPH + 14 H+ → CH3(CH2)14COOH + 8CoASH + 14 NADP+ + 6 H2O A molecule of acetyl CoA is extended using 7 molecules of malonyl CoA. In the process 7 new carbonyl groups and 7 double bonds are formed, which have to be reduced, hence the need for 2 X 7 =14 molecules of NADPH

Key points
• Acetyl CoA and malonyl CoA are bound by enzymes of the fatty acid synthase complex prior joining by Claisen condensation • Claisen condensation is followed by: reduction of a carbonyl group, dehydration (the first two steps result in the removal of an oxygen atom), and reduction of a double bond • The number of repetitions of the step involving Claisen condensation determines the length of the hydrocarbon chain of the fatty acid • When the speficied length of the hydrocarbon chain is reached, the fatty acid ester is released from the synthase by hydrolysis catalyzed by the thioesterase enzyme

unsaturated fatty acids
• Double bonds in the aliphatic chains of fatty acids are generated by a free radical mechanism in some anaerobic bacteria • Double bond formation in aerobes are probably obtained through catalysis by an enzyme that utilizes a disulphide bridge in its active site. Reduction of the disulphide bridge removes two atoms of hydrogen, resulting in unsaturation of the aliphatic chain

Biosynthesis of odd-numbered fatty acids
• Nearly all naturally-occurring fatty acids have an even number of carbon atoms, because the primer molecule (acetyl coA) and the extender (malonyl coA) contribute 2 carbon atoms to the aliphatic chain of the amino acid • Acetyl coA + n malonyl coA → fatty acid with even numbers of C atoms

• Rarely, fatty acids with odd numbers of carbon atoms in their aliphatic chain occur. These are usually obtained by using propionyl CoA (which contributes 3 carbon atoms to the aliphatic chain) with malonyl CoA as extenders
O
SCoA

+ n malonyl CoA

fatty acids with odd numbers of C atoms

(propionyl CoA)

Branched fatty acids
• Branched, usually methyl branched, fatty acids occur rarely • A well-known example is the fatty acids in mutton which confers it with its unique flavour and aroma • Branching is usually facilitated by methylbranched primers (e.g. isobutyryl coA) or extenders (e.g. methyl malonyl coA)

Iso-branched fatty acids

O

OH O

OH

isobutyrl CoA as primer

O

OH

acetyl CoA as primer + malonyl CoA as extender + methyl malonyl CoA + 6 malonyl CoA O
-

OOC

CoA

methyl malonyl CoA

Prostaglandins and leukotrienes • Prostaglandins are are modified oxygenated C20 caboxylic acids found in animal tissues. These were first isolated from human semen, and found to cause contraction of smooth muscles. • Prostaglandins and leukotrienes have been found in be present in many organs and tissues, and they control a wide range of physiological responses, including immunity

O

COOH

O OOH PGG2

• Cyclooxgenase (COX) or prostaglandin synthase is the enzyme responsible for the conversion of arachidonic acid to prostaglandin • COX is an inducible enzyme, produced by macrophages at sites of inflammation • Biosynthesis begins with the action of cyclooxygenase on an unsaturated C20 fatty acid, arachidonic acid. The formation of the peroxide bridge takes place by a free radical mechanism

• The active site of the enzyme is a tyrosine residue at position 385, where the a phenoxide radical abstracts a labile hydrogen from C13 of arachidonic acid • This H is especially labile as it is located at a position that is alpha to two double bonds • The abstraction of a H radical from C13 results in the formation of a radical from arachidonic acid, which undergoes a series of electron movement which results in the binding of a molecule of oxygen,the formation of a peroxide bridge, the formation of a cyclopentane ring (a C-C bond is formed) and the binding of another molecule of oxygen

Physiological roles of prostanoids
• Prostacyclin (PGI2) and PGD2 inhibit the aggregation of blood platelets • PGE2 and PGF2 control the conctraction of bronchioles (small airways in the lungs) • PGE2 promotes the formation of the protective layer of mucus of intestinal walls, thus reducing possible damage by gastric juice

Leukotrienes
• Leukotrienes are chemical messengers produced during allergic reactions • Their biosynthesis also starts from arachidonic acid, which is acted upon by the enzyme 5-lipoxygense to produce a hydroperoxide which rearranges to form an epoxide

• The enzyme first acts by abstracting an H radical from C7 of arachidonic acid, resulting in the formation of a radical • This generates a radical which binds oxygen at C5 (hence the name of the enzyme, 5-lipooxygenase) • The reaction with oxygen results in the formation of a hydroperoxide, which reacts with a double bond forming an epoxide

Key points
• The biosyntheses of prostaglandin and leukotrienes involves free radical attack on different labile hydrogen atoms • The C-C bond in the 5-membered ring of prostaglandin is unique in biochemistry as it is one of the very few instances where such a bond is formed by a free radical mechanism • The biosynthesis of leukotrienes involves formation of an epoxide. This is not so for prostaglandin