eLS || Fatty Acid Biosynthesis

Fatty Acid BiosynthesisBernard James Rawlings, University of Leicester, Leicester, UK

Fatty acids are produced by most organisms on this planet, mainly for use in membranes or

as a long-term store of energy. While the overall organization and arrangement of the

enzyme complexes varies, the underlying chemistry used to assemble fatty acids is virtually

identical in every organism; the crucial step being decarboxylative addition of malonate

thioesters to elongate the growing acyl chain by two carbons, followed by reduction,

dehydration and reduction of the resulting 3-oxo functionality to 3-CH2, prior to further

chain elongation.

Overview of Fatty Acid BiosynthesisPathway

Most organisms biosynthesize their own fatty acids;exceptions are the archaebacteria, which can use terpene-based compounds in their membranes, and a few bacteriasuch as the mycoplasmas that obtain their fatty acids fromthe external environment. Fatty acids rarely occur innature in their free carboxylic acid form but are usuallyfound esterified to glycerol (propan-1,2,3-triol) as thediesters (diglycerides, e.g. phosphodiglycerides) or triesters(triglycerides). The process of converting acetyl-CoA intofatty acidswould appear to be related to the reverse processof converting fatty acids into acetyl-CoA (fatty acidoxidation). However, nature has to ensure that thesemutually reverse processes donot interferewith eachother.In higher organisms the oxidation occurs in the energy-producingorganelles, such as themitochondria,while fattyacid synthesis occurs in the cytosol, a different ‘compart-ment’. In oxidation all the intermediates are as freecoenzyme A (CoA) thioesters, while in synthesis theintermediates are always covalently attached to a protein(e.g. acyl carrier protein ACP); the oxidants in oxidationare FAD and NAD1 , while in synthesis the reductant isNADPH; and fatty acid synthesis involves carboxylationof acetyl-CoA to malonyl-CoA, prior to C–C bondformation that is coupled with decarboxylation, whilefatty acid oxidation involves no such carboxylated species.Bacteria and plants can assemble amonounsaturated fattyacid through the aerobic or anaerobic pathway, whileanimals are dependent upon the aerobic pathway that usesdesaturases. Animals have only a limited range of furtherdesaturation, requiring dietary sources of some polyunsa-turated fatty acids. Bacteria and plants can then use thesedioxygen-dependent membrane-associated desaturases toproduce polyunsaturated fatty acids found in many seedoils, plants and fish (obtained via dietary sources). Allproducer organisms produce fatty acids using the samebasic mechanistic pathway; the main difference is that theenzymes in most bacteria are each on a separate peptide

(Type II), while in fungi and vertebrates they are onmultienzyme polypeptides (Type I) (Rawlings, 1998, 1997)To assemble a typical fatty acid such as hexadecanoic

acid (palmitic acid), eight units of acetyl-coenzyme A(acetyl-CoA) are sequentially combined, with reduction,dehydration and reduction in each cycle. The first step isthe acetyl-CoA carboxylase (ACC)-mediated carboxyla-tion of acetyl-CoA to malonyl-CoA. One unit of acetyl-CoA and one of malonyl-CoA are then transthioesterifiedon to protein thiols. Simultaneous decarboxylation andClaisen condensation between these two moieties gives 3-oxobutanoyl thioester, which is then reduced, dehydratedand further reduced to the corresponding butanoylthioester. This cycle of two-carbon extension reactions,keto reduction, dehydration and enoyl reduction followedby the next two-carbon extension is then repeated until thedesired length of fatty acid is obtained, when thethioesterase (TE) liberates the product as the free acid oras CoA thioesters that can then be esterified directly on toglycerol. Subsequent modifications can occur, such asfurther elongation or cyclopropanation; these aremediated by membrane-bound enzymes.

Acetyl-CoA Carboxylase (ACC)

Acetyl-CoA is produced by glycolysis in themitochondria,while the first committed step of fatty acid biosynthesis,acetyl-CoA carboxylation to malonyl-CoA, occurs in thecytosol (chloroplast in plants), separated by the mitochon-drial membrane impervious to acetyl-CoA. Acetyl-CoA inthe mitochondrion is condensed with 2-oxobutan-1,4-dioate (oxaloacetate) to give 3-carboxylato-3-hydroxy-pentan-1,5-dioate (citrate), which can diffuse through themembrane into the cytosol, where it is converted back intoacetyl-CoA and oxaloacetate by ATP-dependent citratelyase. The oxaloacetate is reduced (NADH) by malatedehydrogenase to 2-hydroxybutan-1,4-dioate (malate),which is oxidatively decarboxylated to 2-oxopropanoate

Article Contents

Secondary article

. Overview of Fatty Acid Biosynthesis Pathway

. Acetyl-CoA Carboxylase (ACC)

. Fatty Acid Synthase (FAS)

. Regulation

. Fatty Acid Elongation

. Fatty Acid Desaturation

. Variation Between Classes of Organism

. Acylglycerol Synthesis

1ENCYCLOPEDIA OF LIFE SCIENCES © 2002, John Wiley & Sons, Ltd. www.els.net

(pyruvate), which is able to diffuse across the mitochon-drial membrane back into the mitochondria for recyclingto oxaloacetate. The net result of this cycle is that oneNADPH is generated for every acetyl-CoA moleculetransferred into the cytosol.

ACC catalyses the biotin-dependent carboxylation ofacetyl-CoA to malonyl-CoA, and is related to pyruvatecarboxylase. The reaction has been shown to occur withoverall retention of stereochemistry, as illustrated by thereaction of isotopically labelled chiral acetate (eqn[I],D=deuterium, T=tritium). The C–H bond, beinglonger and weaker than either the C–D or C–T bond, ispreferentially removed by the enzyme, to give stereospe-cifically labelled product.

SCoA

O

H

D T

Acetyl-CoA carboxylase

BiotinHydrogencarbonate

SCoA

O

D T

−O

O

[I]

Most prokaryotic acetyl-CoAcarboxylases consist of threereadily dissociable proteins – biotin carboxylase, biotincarboxyl carrier protein and transcarboxylase (carboxyltransferase). The first step is the biotin carboxylase-mediated magnesium-(or manganese-) dependent activa-tion of hydrogencarbonate (bicarbonate) by ATP to givecarboxyphosphate (carbamoylphosphate) and ADP. Themagnesium, hydrogencarbonate and ATP are presumablyall bound on the protein, and the magnesium stabilizes theputative product, which has not been isolated or chemi-cally synthesized (eqn [II]).

O

HO O−P

O

−OP

OO

O

PO

O

O− O− O−

Ad

O

HO OP O−

O

O−

+ ADP

Mg2+ Mg2+

[II]

The next step is transfer of the activated carboxylmoiety tobiotin. Biotin is attached to the e-amino group of a lysineresidue in the biotin carboxyl carrier protein (BCCP) (eqn[III]).

The carboxylated biotin, still covalently attached to theBCCP via a long flexible arm, is then transferred to theactive site of the third component, the transcarboxylase,

where the carboxyl group is reversibly transferred toacetyl-CoA, forming malonyl-CoA. A base on the car-boxyl transferase removes a proton (pKa� 20) from theacetyl-CoA (the proton being removed would at the timebe perpendicular to the planar carbonyl group). A possiblemechanism is the resulting planar enolate then attackingthe carboxyl biotin, the new C–C bond being formed withoverall retention of configuration, to give the tetrahedralintermediate. This breaks down to give malonyl-CoA(as its oxyanion) and biotin. The reaction is illustrated inScheme 1 with ‘chiral acetate’, [3H,2H,1H]acetyl-CoA.

Fatty Acid Synthase (FAS)

The fatty acid synthase is a collection of seven enzymes anda protein (acyl carrier protein ACP) responsible fortypically converting one unit of acetyl-CoA and sevenunits of malonyl-CoA into hexadecanoyl-ACP thioester,which is then hydrolysed to hexadecanoic acid (palmiticacid) or transthioesterified to the corresponding CoAthioester, or esterified on to glycerol. The process involvesthe sequential formation of C–C bonds, followed byNADPH-mediated carbonyl reduction, dehydration, andNAD(P)H-mediated enoyl reduction. Thewhole process isrepeateduntil the desired chain length is achieved (eqn [IV],for example).

Acetyl-CoA + 7 malonyl-CoA + 14 NADPH + 14 H+ hexadecanoic

acid + 14 NADP+ + 8 CoASH + 7 H2O [IV]

The FAS is a huge (250 kDa) multienzyme complex,containing all seven enzyme ‘activities’ and a protein(ACP) required to assemble one unit of acetyl-CoA andtypically seven units ofmalonyl-CoA into a saturated fattyacid, these activities being either noncovalently associated(as found in most bacteria) or covalently attached (as infungi and animals). On the complex are all the functionalgroups and active sites necessary for each transformation,including two different thiol groups. One (the thiol of a

cysteine) is represented in the schemes below by a shortstraight line; the ACP thiol is attached to a long, flexibleswinging arm and is represented by a wavy line. There are

NHHN

S

O

HN

O

C

N

HHH

Lysine residueBiotin

HO OP

OMg2+

NHN

S

O

HH

HO

O

[III]

Fatty Acid Biosynthesis

2

also one or two serine hydroxyl groups that act asacyltransferases, two NAD(P)H binding sites for thereductases, and acid–base functionality for the dehydraseactivity.

Acyl transferase (AT)

The first step is the loading on to the complex of one unit ofacetyl-CoA and one unit of malonyl-CoA. One or moreacyl transferase domains first bind the required substratesand then covalently attach them to the complex by formingan O-ester linkage to serine hydroxyl groups. The acetylgroup is then thioesterified on to the ‘short’ cysteine thiol,and the malonyl group on to the ‘long’ ACP thiol(Scheme 2).

Acyl carrier protein (ACP)

ACP is a small protein (10 kDa) with an active-site serinethat has a phosphopantetheinyl group attached to thehydroxyl group. This prosthetic group acts as a 2 nm-longflexible swinging armending in a thiol towhich the growingacyl chain is attached during the cycle of reactionsdiscussed below, andwhich delivers the growing acyl chainto various active sites on the FAS complex.

3-Oxoacyl ACP synthase (KAS)

The next step is the crucial formation of a new C–C bondby a 3-oxoacyl ACP synthase (condensing enzyme, keto-synthase, KAS). In the laboratory, chemical formation of

NHN

S

O

HHBCCP

O

O

CoAS

OH

D

T

B-carboxyl transferase

CoAS

O

D

T

CoAS

OCOO-

D

(S)-[3H,2H]Malonyl-CoA

(S)-[3H,2H,1H]Acetyl-CoA 'Enolate' 'Carboxylated biotin'

H

NHN

S

O

HHBCCP

O

O

CoAS

O

D

H

Tetrahedral intermediate

NHN

S

O

HHBCCP

H

+

••

TT

Scheme 1

Fattyacid synthase

OH OH

(Ser) (Ser)

SH

SH

(Cys)

ACPFattyacid synthase

OH OH

(Ser) (Ser)

S

S

(Cys)

ACPFattyacid synthase

O O

(Ser) (Ser)

SH

SH

(Cys)

ACP

Me SCoA

O

SCoA

O−O2C

(acetyl-CoA)(malonyl-CoA)

(AT)Me

OO−O2C

Step 1Acetyl-CoA and malonyl-CoAapproach acyl transferase (AT) bindingpockets, ready for covalent attachment

Step 2Both acyl transferase serinehydroxyl groups loaded withacetate and malonate

Step 3Thioesterification:acetate on to cysteine thiolmalonate on to ACP thiol

H+

Me

O

O

O−

O

Scheme 2


3

C–C bonds is a difficult process, often with mediocreyields. The reaction of ethyl ethanoate with the basesodium ethoxide gives a low yield of the desired product,ethyl 3-oxobutanoate, and several byproducts. The reac-tion involves removal of a proton adjacent to the estercarbonyl (pKa 25), reaction of this carbanion with thepositively polarized carbonyl carbon, followed by break-down of the tetrahedral intermediate with ethoxide asleaving group (Scheme 3).

Nature exploits the chemical properties of thioesters:owing to poor overlap of the large and diffuse sulfur 3p or3d orbitals with the empty carbonyl p* orbitals, thereactivity of nucleophiles (such as a carbanion) with athioester carbonyl carbon is much greater than that for anester carbonyl; that is the d1 is much greater on a thioestercarbon than on an ester carbon. In addition, the thiolate isa much better leaving group that an alkoxide anion, and itis much easier to form a carbanion adjacent to a thioester(pKa 20) than adjacent to an ester (pKa 25). Thus thereactivity of a thioester is like that of a ‘ketone with a goodleaving group’. In addition, the entropically favourablecoupling of the reaction to the simultaneous loss of carbondioxide helps to drive the overall reaction to completion.The reaction occurs with overall inversion of configurationat the C2 carbon, as illustrated in Scheme 4 using chiral (R)-[2H1]malonate as substrate. The new C–C bond is formedopposite the original C–CO2

2 bond to give (S)-[2H1]3-oxobutanoyl thioester (acetoacetyl thioester).

It should be noted that while the scheme shows the twosubstrates in ‘mid-air’, they would both be intimately

associated with the surface (or on an inner cavity) of theFAS complex as the reaction proceeds, with sources ofprotons, etc., required supplied by amino acid residues inthe complex. The carbon dioxide is the same carbondioxide (via hydrogencarbonate) thatwas used to carboxy-late acetyl-CoA in the first place. When radioactive 14C-labelled CO2 is added to a FAS in vivo, no radioactivity isincorporated into fat, even though CO2 is needed for thesynthesis to proceed.Some organisms, such as E. coli, have more than one

KAS; KAS3 to form 3-oxobutanoyl-ACP, and two others(KAS1 or KAS2) for all the later C–C bond formingsteps.

3-Oxoacyl ACP reductase (KR)

The next step is reduction of the 3-oxobutanoyl thioesterby a 3-oxoacyl ACP reductase or ‘ketoreductase’ or ‘KR’activity (Scheme 5).The swinging arm of the ACP ‘delivers’ or positions the

3-oxo (carbonyl/keto) group to be reduced between thesource of H2 and quenching H1 on surface of the FAScomplex. Hydride is delivered exclusively to only one face(the ‘si’ face) of the planar ketone functionality, resulting inonly the (R) enantiomer of 3-hydroxybutanoate thioester.The source of hydride is NADPH bound noncovalently tothe surface of FAS. TheFAS enzyme complex also deliversa proton on to the opposite face of the carbonyl group.

H3C OEt

O

Ethyl ethanoate

H2C OEt

ONaOEt

(low concentration)

H3C OEt

O

H3C

O

OEt

O

OEtH3C

O

OEt

O

Ethyl 3-oxobutanoate(+ other byproducts)

δ+

δ−

Scheme 3

Fattyacid synthase

S

S

(Cys)

ACP

Decarboxylative C–Cbond formation withinversion of configuration

Me

O

O

O−

O

1H 2HFattyacid synthase

SH

S

(Cys)

ACP

Me

O

O

1H

2H

+CO2

Fattyacid synthase

SH

S

(Cys)

ACP Me

OO

1H2H

+CO2

Products:(S)-[2H1]-3-oxobutanoyl thioesterplus CO2

Redrawn to highlight inversionof configuration

(R)-[2H1]malonate

OR

Scheme 4


4

(3R)-Hydroxyacyl ACP dehydrase (DH)

The next step is dehydration by (3R)-hydroxyacyl-ACPdehydrase (DH) to give the (2E)-butenoyl (crotonyl)thioester. Again, the substrate attached to theACPflexiblearm will be brought intimately into contact with the DHactivity or domain of the FAS complex. Here a basicresidue (B) will stereospecifically remove one of the twodiastereotopic C2 hydrogens (the pro-S or Hs), and aprotonated residue (AH+) will be strategically placed toprotonate the hydroxyl group forming water and the E(trans) double bond (Scheme 6).

Enoylacyl-ACP reductase (ER)

All the above steps occur with identical stereochemistryin all organisms thus far examined, from bacteria, to

fungi, plants and mammals. The next step is reductionof the (2E) double bond to the saturated butanoylthioester by the NADPH-or NADH-dependent enoyla-cyl-ACP reductases (ER). However, the stereochemistryof this reduction depends upon the organism in whichit is occurring. There are two faces (re and si) that a hydride(H#2 ) can be added to C3 and two faces (re and si)that a proton (H@+) can be added to C2 in the secondstep, giving either syn or anti overall addition. Synaddition is where both hydride and proton are addedto same face of the double bond. All four possiblecombinations have now been observed in nature. Themechanism is analogous to a ‘Michael’ addition of anNADPH-derived hydride (H#) to C3 to give an enolateintermediate, followed by quenching with a solvent oramino acid residue-derived proton (H@) (Scheme 7). InE. coli there are two ERs, with differing specificity for

SH

S

FAS Me

O H

H

H#SH

S

FAS Me

O H

H

H#

Enolate intermediatetwo possiblestereochemistries at C-3protonation above orbelow plane of doublebond

Attack by NADH hydride on C-3either above or below plane of double bond

H@

SH

S

FAS Me

O H

H

H#

H@

Four possibleproductstereochemistries

Scheme 7

SH(Cys)

ACP

NAD

H*

H

FAS

SMe

OO

SH(Cys)

ACP

NAD+

SMe

OHO H*

NADH*

H+

SH

S

(Cys)

ACP

O

Me

OH

NAD+

*Hwhich is

KRdomain (3R)-Hydroxybutanoyl thioester

FAS

FAS

Scheme 5

SH

S

FAS Me

O OH*H

HRHSB

AH+ SH

S

FAS Me

O *H

HRBHS

A H2O

(2E)-butenoyl thioester+

Scheme 6


5

different length or unsaturation of substrate; one usesNADH, one NADPH.

The four different overall stereochemistries possible, andan example of where they occur, are listed in Table 1. Thus,for yeast, the observed stereochemistry is as shown inScheme 8.

We now have a two-carbon extended homologue ofethanoate – butanoate – on the swinging arm thiol. This istransferred to the cysteine thiol by transthioesterification(Scheme 9).

A new molecule of malonyl-CoA is now bound andO-esterified on to an AT serine hydroxyl as before, andthen transferred on to the ACP thiol. The saturated acyl

chain is now ready for another cycle, i.e. C–C bondformation (KAS), ketoreduction (KR), dehydration (DH)and enoyl reduction (ER), with exactly the same mechan-istic stereochemistry as before, to form hexanoyl ACPthioesters (Scheme 10).The product saturated acyl chain is now transesterified

to the cysteine thiol, another unit of malonate is loadedon to the ACP thiol, and the whole process repeatsitself until an acyl chain of the desired length (C16/C18)is obtained, when a thioesterase (TE, part of the FAScomplex) uses an active-site serine hydroxyl groupto liberate the acyl chain as a CoA thioester, as the freeacid or as an ester of propane-1,2,3-triol (glycerol)(Scheme 11).Thus, the first C2 unit (at the methyl terminus) of a fatty

acid is derived directly from acetyl-CoA (referred to as the‘starter unit’). All other C2 units are also derived fromacetyl-CoA, but via malonyl-CoA – these are ‘extenderunits’ (Figure 1).The overall FAS complex could be represented as in

Figure 2, a three-dimensional complex with the ACPswinging arm delivering the growing acyl chain to each ofthe required functionalities in turn.

SH

S

FAS Me

O H

H

SH

S Me

O H

H

H#

H@

NADPH#

H@2O

Yeast enoyl reductase

FAS

Scheme 8

SH

S

FAS Me

O Transthioesterification

S

SH

Me

O

Butanoyl-ACP thioester Butanoyl-cysteinyl thioester

FAS

Scheme 9

S

S

FAS

Me

O

O

O O

SH

S

FAS

O O

Me

3-Oxohexanoyl thioester

SH

S

FAS

O

Me

Hexanoyl thioester

KR DH ERKAS

Malonate

butanoate

Scheme 10

Table 1

Source of FAS C-2 C-3

Rat liver (mammalian) syn si reE. coli (bacteria) syn re siYeast (fungi) anti si siFlour beetle anti re re


6

Regulation

Any cell needs to carefully regulate, control and direct theflow of carbon atoms from acetyl-CoA either into the citricacid cycle (for short-term production of energy, hydrideand metabolites) or into fatty acid synthesis (for the

production of diglycerides for new membranes, andtriglycerides as a long-term store of energy). The firstcommitted step in the assembly of fatty acids is acetyl-CoAcarboxylation, and regulation of the activity of acetyl-CoAcarboxylase (ACC) primarily moderates the rate of flow ofacetyl-CoA into this pathway.In E. coli, a shortage of available amino acids results in

the production of the unusual nucleotides guanosine 5’-diphosphate 3’-diphosphate (ppGpp) and the correspond-ing triphosphate pppGppp; both these nucleotides bind toa common site on the transcarboxylase domain, inhibitingthe ACC. Thus, in bacteria the rate of fatty acidbiosynthesis, and the resultant level of new membraneformation, is linked to the level of available nutrients andthe rate of production of other cellular macromolecules(Magnuson et al., 1993; Rock and Cronan, 1996). Productinhibition can also occur: long-chain acyl-ACPs caninhibit enzymes such as KAS3 (which forms 3-oxobuta-noyl-ACP) and enoyl reductases, downregulating fattyacid production.Animals accumulate triglycerides (fat) in their cells, and

need long-term (adaptive) control of production levels aswell as short-term regulation tomoderate sugar levels aftera meal. Adaptive control can be achieved by varying theexpression of enzymes and the level of the enzymes present– high-carbohydrate low-fat diets result in high levels ofACC. There is a particularly rapid formation anddegradation of FAS enzymes in brain and nervous systemtissue, and the level of enzymes may determine FASactivity in these tissues. In the shorter term, vertebratesneed tomaintain a steady flow of acetyl-CoA into the citricacid cycle to produce energy, and any excess acetyl-CoAneeds to be channelled into fatty acid synthesis and thelong-term storage of triglycerides. Thus, ACC needs to beactivated and deactivated in a rapid response/short-term

FAS

SH

S

SH

S

SS

S

R

O O

KR DH ER

R

O

R

O

O

O

R

O

Acetyl CoA+

Malonyl CoA+

FASR = Me

KAS−CO2

transthioesterification

AT

malonyl -CoA

KAS CO2

R

GlycerolGlycerides

CoA thioester

H2OCarboxylic acid

CoASH

SH

O

FAS

FAS

FAS

R+C2

Scheme 11

Acetyl-CoA Malonyl-CoA

Me OH

O

CO2

Hexadecanoic acid (palmitic acid)

Figure 1 Incorporation of acetyl-CoA into both the ‘starter unit’ and‘extender units’ and incorporation of malonyl-CoA into extender unitsonly.

SH

:B

+HA

+HA

H+HA

SH

(swinging arm)

OH

OH

OH

H

(phosphopantetheine)KR

TE

KASDH

ATER

ACP

NADP

NADSer

Ser

Ser

Cys

Figure 2 Fatty acid synthase complex showing all necessary enzymaticactivities, cofactors and ‘swinging arm’ thiol.


7

manner. ACC is product-inhibited: high levels of hexade-canoyl-CoA inhibit ACC, the inhibition being competitivewith citrate inhibition. Vertebrate ACC is inactive as themonomer (protomer), but highly active when in thefilamentous oligomeric form. A high level of acetyl-CoAand ATP is reflected by a high concentration of citrate andother tricarboxylic acids that cause polymerization andactivation of ACC. ACC is also thought to be deactivatedby phosphorylation and reactivated by dephosphorylation– high levels of hormonally produced cyclic AMPdeactivate ACC. Low blood sugar levels trigger release ofhormones that cause formation of cyclic AMP, whichactivates enzymatic phosphorylation ofACC, deactivatingit; while high insulin (a result of high blood sugar levels)levels causes activation.

Fatty Acid Elongation

Many organisms possess separate membrane-bound sys-tems to further chain-elongate the FAS product (as CoAthioester) and, though poorly characterized, these areusually a complex that adds a single unit of malonyl-CoA,with KR, DH and ER liberating the two-carbon extendedCoA thioesters (eqn [V]).

The mycobacteria are unusual in having a multifunc-tional type I FAS that can produce a C24 fatty acid, and incontaining a wide variety of very long and functionalizedfatty acids. The C24 FAS product is chain-extended byelongases, two carbons at a time, with S-adenosylmethio-nine mediated cyclopropanation of Z (cis) double bonds.The antimycobacterial drug isoniazid is thought to inhibitthe elongase enoyl reductase, preventing formation ofthese very long fatty acids. A Claisen reaction with asecond C24 fatty acid (or docosylmalonyl-CoA) and a 3-oxo reduction results in the mycolic acid [VI], which is anessential component of theMycobacterium tuberculosis cellwall and whose cyclopropyl groups are essential for itspathogenicity.

In yeast there are systems associated with the endoplas-mic reticulum that elongate 12:0CoAand 14:0CoA to 16:0

CoA and 18:0 CoA in discrete stages; and systemsassociatedwith themitochondrialmembrane that elongate18:0 CoA to 24:0 CoA in several steps.In animals there are membrane-bound elongases and

desaturases in the endoplasmic reticulum that extenddietary polyunsaturated fat such as linoleic acid (18:2,9Z,12Z) into arachidonic acid (20:4,5Z,8Z,11Z,14Z), essen-tial for transformation into prostaglandins, thromboxanesand leucotrienes. Alternatively, these C20 fatty acids can befurther extended with desaturation up to 22:5 and 22:6 forincorporation into membrane phospholipids.

Fatty Acid Desaturation

Anaerobic desaturation

Bacteria such as E. coli mainly produce (9Z)-hexadec-9-enoyl-ACP via an anaerobic pathway. (3R)-Hydroxyde-canoyl-ACP is formed from acetyl-CoAandmalonyl-CoAin the usual manner using FAS. This substrate can then bedehydrated by either the FAS dehydrase as normal, or byan additional enzyme (3R)-hydroxydecanoyl-ACP dehy-drase (HDDH), to give (2E)-dec-2-enoyl-ACP (trans-decenoyl-ACP). The dehydrase has a rate minimum for

dehydrating (3R)-hydroxydecanoyl-ACP; in contrast,HDDH had a preference for dehydrating (3R)-hydroxy-decanoyl-ACP. The same enzyme, HDDH, can thenisomerize the (2E)-dec-2-enoyl-ACP to (3Z)-dec-3-enoyl-ACP (cis-dec-3-enoyl-ACP), which is then chain-extendedin the usual manner (but only using KAS1, not KAS2) bythe FAS complex to give (9Z)-hexadec-9-enoyl ACP or(11Z)-octadec-2-enoyl ACP. HDDH contains a singleactive site that is responsible for both the dehydration andthe isomerization (Scheme 12) (Leesong et al., 1996; Heathand Rock, 1996).

Aerobic desaturation

A second system, which directly uses molecular dioxygento convert a saturated portion of a hydrocarbon to aZ (cis)

Me SCoA

O

Hexadecanoyl-CoA, 16:0-CoA(Palmitoyl-CoA)

SCoA

O

Octdecanoyl-CoA, 18:0-CoA(Stearoyl-CoA)

Elongase

malonyl-CoA

NAD(P)H

Me

16

18

[V]


8

double bond, is used widely (eqn [VII]). Plants use non-membrane-bound desaturases to form their monounsatu-rated fatty acids and membrane-bound desaturases toconvert these into polyunsaturated fatty acids, while alldesaturations inmammals and fungi usemembrane-bounddesaturases. The soluble plant desaturases are now well-characterized, but the membrane-bound desaturases re-main poorly characterized.

These desaturases are all thought to have non-haemm-oxo-bridged diiron centres with a gap for dioxygenbinding above one iron, and a bent hydrophobic groovefor binding the fatty acid. The reaction of the iron centrewith dioxygen forms an Fe–O–O� radical that stereo-specifically abstracts a hydrogen from the hydrocarbonchain strategically placed nearby (Lindqvist et al., 1996).

By varying the size and length of the bent hydrophobicpocket, different enzymes (e.g. D9-desaturase) can be usedto first insert, for example, a (9Z)-double bond to give18:1(9Z); then a D12-desaturase forms linoleic acid18:2,9Z,12Z and a D15-desaturase forms linolenic acid18:3,9Z,12Z,15Z.In plants, saturated and monounsaturated fatty acids

are first formed in the chloroplast while attached to an

ACP by a type II FAS. After conversion to the free acid bya thioesterase, they are transferred to the cytoplasm andconverted intoCoAthioesters; enzymes in the endoplasmicreticulum then effect further desaturation, elongation andattachment to glycerol.Many lower plants, such as mosses and liverworts, also

contain fatty acids containing triple (ethynic/acetylenic)

MeCOOH

OH

Me

1

1′22′

3

213352

2-Docosyl-21,22:33,34-dimethano-3-hydroxydipentacontanoic acid (a mycolic acid)

[VI]

Acetyl-CoA

+

Malonyl-CoA

FASMe S-ACP

OOH

(3R)-Hydroxydecanoyl-ACP

S-ACP

O

(2E )-Dec-2-enoyl-ACP

Saturated fatty acidsFAS

DHHDDH(dehydration)

(using KAS1 or KAS2)MeS-ACP

O

(2E )-Dec-2-enoyl-ACP

S-ACP

O

(3Z )-Dec-3-enoyl-ACP

Me

HDDH

Monounsaturated fatty acidsFAS

(isomerization)

(using only KAS1)

Me

Scheme 12

Me SCoA

O

Hexadecanoyl-CoA, 16:0-CoA(Palmitoyl-CoA)

SCoA

O

Me∆9-Desaturase

O2

(9Z)-Hexadecenoyl-CoA 16:1,9Z-CoA(Palmitoleoyl-CoA)

1

916

[VII]


9

bonds. The triple bonds are thought to be formed from thecorresponding double bond by similar diiron-based aero-bic desaturases. In some plants, notably compositae suchas daisies, these triple bonds are derivatized to furan orthiophen rings.

Animals do not use the anaerobic pathway; their aerobicdesaturases can introduce double bonds atD4,D5,D8 orD9,but never beyond D9, all with the Z (cis) configuration.Thus, animals are unable to convert oleic acid 18:1,9Z intolinoleic acid 18:2,9Z,12Z or a-linolenic acid 18:3,9Z,12Z,15Z, both of whichmust be obtained from dietary sources.These fatty acids are essential asmammals need to elongateand desaturate the former to arachidonic acid 20:4,5Z,8Z,11Z,14Z, which is then transformed into the icosa-noids such as the prostaglandins, thromboxanes andleucotrienes.

Variation Between Classes of Organism

Prokaryotes

Bacterial FAS produce fatty acid ACP thioesters, whichare usually directly transesterified to form glycerideswithout the intermediacy of the free carboxylic acids. Inmost bacteria, the FAS complex is presumed to be anassembly of noncovalently linked enzymes (type II FAS).Any such organization is lost upon cell breakage, and eachindividual component has been studied independently andfound to be active. A few bacteria use unusual ‘starterunits’ replacing acetyl-CoA in the above sequence. Forexample,manyStreptomyces,Bacillus,StaphylococcusandMyxococcus spp. use l-valine-derived 3-methylbutanoyl-CoA to form 13-methyltetradecanoyl-CoA [VIII]. Thethermoacidophilic Alicyclobacillus heptanicus uses cyclo-heptanecarbonyl-CoA to form 11-cycloheptanylundeca-noic acid [IX], which is used to maintain cell membranedensity in a hot (608C) environment.

Me OH

OMe

13-methyltetradecanoyl-CoA13 1

[VIII]

OH

O

1

11

11-Cycloheptanylundecanoic acid

[IX]

Several bacteria are thought to use multienzyme polypep-tides, reminiscent of animal FAS: Brevibacterium ammo-niagenes produces saturated and monounsaturated fattyacids in a type I-like process using an a6-homomultimerthat includes an HDDH domain to insert the monounsa-turation.

Eukaryotes

All eukaryotic FAS complexes are assembled from multi-enzyme peptides (Wakil and Stoops, 1983, 1984).In fungal systems (type IA), all activities are present on

two polypeptide chains: the a subunit contains the KAS,ACP and KR activity, while the b chain contains the ER,DH, AT and malonyl/hexadecanoyl transacylases. Elec-tron microscopy shows the enzymatically active complexto be an a6b6 complex with a molecular mass of 2.4MDathat resembles six discs in aplane containing thea subunits,linked by six arch-like units, three alternately above theplane and three below. A recent higher resolution studyshows the structure as barrel-like with 32 point symmetry,with 12 small funnel-shaped openings (2 nm diameter),presumably for substrate entry and product efflux for eachof six active sites, leading to a single solvent-filled cavitywith 42 active sites (Kolodziej et al., 1996). There wasconsiderable negative cooperativity between some identi-cal active sites: for instance, only six or seven of the 24acetate binding sites could be loaded, even at highconcentrations of acetyl-CoA.Many invertebrates, such as insects, produce fatty acids

that are then derivatized to form pheromones or elongatedto long-chain cuticular lipids. Several insectFAShavebeenpurified, and are reminiscent of animal FAS. Most insectsuse cuticular lipids to prevent dessication. These arecomplex mixtures of long straight-chain, methyl-branchedand unsaturated hydrocarbons. Fatty acids such as 18:0are elongated, and then either decarboxylated or reducedto the aldehyde and then decarbonylated to the hydro-carbon and carbon monoxide (Mpuru et al., 1996). Inlepidoptera, many pheromone biosyntheses involve elon-gation or shortening, reduction of the thioesters to thecorresponding alcohol, and O-acetylation (Foster, 1998).Animal FAS produces a mixture of free fatty acids,

primarily hexadecanoic acid, except in mammary glands,which form shorter chain products; or in water fowluropygial (preening) glands, which produce specializedpolymethylated fatty acids such as 2,4,6,8-tetramethylun-decanoic acid for their water-repelling properties. ChickenFAS prefers acetyl-CoA as ‘starter unit’, but rat FAS andhuman FAS prefers butanoyl-CoA or 3-oxobutanoyl-CoA, whichmay be a physiologically significant substrate.All seven FAS activities and the ACP domain are presenton a single polypeptide chain in the orderKAS, AT,DH, acentral nonfunctional core, ER, KR, ACP and TE. Thecatalytically inactive central core has a strong tendency todimerize, and is thought to be responsible for holding thetwo polypeptides together. An earlymodel showsFAS as aflat head-to-tail homodimer of two polypeptides in whichtheACP thiol fromonepolypeptide interactswith theKASthiol from the second polypeptide. Recent complementa-tion studies, in which activities from either polypeptidechain are deleted and the remaining activity is measured(examining which domains actually interact), suggest that


10

the structure involves some helical coiling of the twopolypeptides (Witkowski et al., 1996; Wakil, 1989).

Acylglycerol Synthesis

The glycolysis intermediate 3-hydroxypropanonyl phos-phate (dihydroxyacetone phosphate), is reduced by anNADH-dependent enzyme to (2R)-2,3-dihydroxypropylphosphate (sn-glycerol 3-phosphate). The trivial name andcommonly used diagrams assume the Fischer projection,as there is (2R) stereochemistry at the central carbon. InE.coli, a complex of inner membrane-bound enzymes formtriacyl glycerol. In mammals, these enzymes are found as atriacylglycerol synthetase complex bound to the endoplas-mic reticulum. The first, an acyl transferase, reacts theglycerol 3-phosphate with a saturated fatty acid CoAthioester to give the corresponding 1-acylglycerol 3-phosphate (lysophosphatidate) (Vanden Boon and Cro-nan, 1989). A secondmembrane-bound enzyme transfers asecond acyl group, frequently a monounsaturated fattyacid-CoA thioester, on to the remaining hydroxy group togive a phosphatidate. The phosphate group is hydrolysedto give the diacylglycerol, which is then acylated to give thetriacyl glycerol or triglycerides (Scheme 13).

References

Foster SP (1998) Sex pheromone biosynthesis in the Tortricid moth

Planotortrix excessana (Walker) involves chain shortening of palmi-

toleate and oleate. Archives of Insect Biochemistry and Physiology 37:

158–167.

Heath RJ and Rock CO (1996) Roles of FabA and FabZ b-hydroxyacylacyl carrier protein dehydratases in Escherichia coli fatty acid

biosynthesis. Journal of Biological Chemistry 271: 27795–27801.

Kolodziej SJ, PenczeK PA, Schroeter JP and Stoops JK (1996)

Structure–function relationships of the Saccharomyces cerevisiae

fatty acid synthase. Three-dimensional structure. Journal of Biological

Chemistry 271: 28422–28429.

Leesong M, Henderson BS, Gillig JR, Schwab JM and Smith JL (1996)

Structure of a dehydratase-isomerase from the bacterial pathway for

the biosynthesis of unsaturated fatty acids: two catalytic activities in

one active site. Structure 4: 253–263.

Lindqvist Y, Huang WJ, Schneider G and Shanklin J (1996) Crystal

structure of a D9 stearoyl-acyl carrier protein desaturase from castor

seed and its relationship to other di-iron proteins. EMBO Journal 15:

4081–4092.

Magnuson K, Jackowski S, Rock CO and Cronan JE Jr (1993)

Regulation of fatty acid biosynthesis in Escherichia coli. Microbiolo-

gical Reviews 57: 522–542.

Mpuru S, Reed JR, Reitz RC and Blomquist GJ (1996) Mechanism

of hydrocarbon biosynthesis from aldehyde in selected insect

species: requirement for O2 and NADPH and carbonyl group

released as CO2. Insect Biochemistry and Molecular Biology 26:

203–208.

CH2OH

CH2OPO32−

HHO

sn-Glycerol 3-phosphate(Fischer projection)

HOH2C CH2OPO32−

O

Dihydroxyacetone phosphate

NADH

Enzyme

HOH2C

OHH

(2R)-2,3-dihydroxypropyl phosphate

CH2OH

CH2OPO32−

HHO

MeCoA-S

O

CH2OPO32−

HHO

Me

O

OAcyl transferase

CH2OPO32−

HO

Me

O

OO

Me

HO

R1

O

O

R2

O

OH

HO

R1

O

O

R2

O

O R3

OR3COSCoA Phosphatase

Lysophosphatidate

Phosphatidate

TriglycerideDiacylglycerol

Stearoyl -CoA

Acyl transferase

Palmitoleoyl -CoA

CH2OPO32−

Scheme 13


11

Rawlings BJ (1997) Biosynthesis of fatty acids and related metabolites.

Natural Product Reports 14: 335–358.

Rawlings BJ (1998) Biosynthesis of fatty acids and related metabolites.

Natural Product Reports 15: 275–308.

Rock CO and Cronan JE Jr (1996) Escherichia coli as a model for the

regulation of dissociable (type II) fatty acid biosynthesis.Biochimica et

Biophysica Acta 1302: 1–16.

Vanden Boon T and Cronan JE Jr (1989) Genetics and regulation

of bacterial lipid metabolism. Annual Review of Microbiology 43:

317–343.

Wakil SJ (1989) Fatty acid synthase, a proficient multifunctional

enzyme. Biochemistry 28: 4523–4530.

Wakil SJ and Stoops JK (1983) Structure and mechanism of fatty acid

synthase. The Enzymes 16: 3–61.

Wakil SJ andStoops JK (1984)Fatty acid synthetases of eukaryotic cells.

In: Martonsi AN (ed.) The Enzymes of Biological Membranes, vol. 2,

pp. 59–109. New York: Plenum Press.

Witkowski A, Joshi A and Smith S (1996) Fatty acid synthase:

in vivo complementation of inactive mutants. Biochemistry 35:

10569–10575.

Further Reading

Abeles RH, Frey PA and Jencks WP (1992) Biochemistry. Boston, MA:

Jones and Bartlett.

Dewick PM (1997) Medicinal Natural Products. Chichester: Wiley.

O’Hagan D (1991) The Polyketide Metabolites. Chichester: Ellis

Horwood.

Mann J (1987) Secondary Metabolism, 2nd edn. Oxford: Oxford

University Press.

Stryer L (1988) Biochemistry, 3rd edn. New York: WH Freeman.


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