Download Current understanding of fatty acid biosynthesis and the acyl carrier

Biochem. J. (2010) 430, 1–19 (Printed in Great Britain)
1
doi:10.1042/BJ20100462
REVIEW ARTICLE
Current understanding of fatty acid biosynthesis and the acyl carrier protein
David I. CHAN and Hans J. VOGEL1
Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4
FA (fatty acid) synthesis represents a central, conserved process
by which acyl chains are produced for utilization in a number
of end-products such as biological membranes. Central to FA
synthesis, the ACP (acyl carrier protein) represents the cofactor
protein that covalently binds all fatty acyl intermediates via a
phosphopantetheine linker during the synthesis process. FASs
(FA synthases) can be divided into two classes, type I and II,
which are primarily present in eukaryotes and bacteria/plants
respectively. They are characterized by being composed of either
large multifunctional polypeptides in the case of type I or
consisting of discretely expressed mono-functional proteins in the
type II system. Owing to this difference in architecture, the FAS
system has been thought to be a good target for the discovery of
novel antibacterial agents, as exemplified by the antituberculosis
drug isoniazid. There have been considerable advances in this
field in recent years, including the first high-resolution structural
insights into the type I mega-synthases and their dynamic
behaviour. Furthermore, the structural and dynamic properties of
an increasing number of acyl-ACPs have been described, leading
to an improved comprehension of this central carrier protein. In
the present review we discuss the state of the understanding of FA
synthesis with a focus on ACP. In particular, developments made
over the past few years are highlighted.
INTRODUCTION
Aside from supplying FAs for biomembrane synthesis, the FAS
system also provides acyl chains for a host of other processes.
ACP (acyl carrier protein), the central cofactor protein for
FA synthesis, supplies acyl chains for lipid A and lipoic acid
synthesis, as well as quorum sensing, bioluminescence and toxin
activation [14–18]. Furthermore, ACPs or PCPs (peptidyl carrier
proteins) are also utilized in polyketide and non-ribosomal peptide
synthesis, which produce important secondary metabolites such
as the lipopeptide antibiotic daptomycin and the iron-carrying
siderophore enterobactin [19,20]. In the present review, we
provide an overview of the FA synthesis processes in bacteria and
higher organisms. In particular, recent discoveries in this field will
be highlighted with a special focus on the universal carrier protein
ACP.
De novo FA (fatty acid) synthesis represents a crucial pathway
in all living organisms. Its products are mainly utilized in
membrane biosynthesis, but are also needed in a number of
other processes such as bacterial quorum sensing and protein
post-translational modification. Owing to its ubiquitous nature
and importance, FAS (FA synthase) systems have long been a
subject for investigation and significant progress has been made
[1]. For example, a virtually complete catalogue of high-resolution
structures is available for each of the proteins involved in bacterial
FA synthesis [2]. Nevertheless, there have been many important
contributions to this field in recent years. Structures of the megasynthases from mammalian and fungal sources have become
available and researchers also continue to make strides in our
understanding of FA synthesis regulation [3–7]. Interest in the FA
synthesis pathways has remained high since it is believed that its
machinery represents a promising target for the development of
novel antibiotic agents [8]. Compounds such as triclosan and the
drug isoniazid used against tuberculosis have fuelled the search for
further medicinal agents that target the FAS systems. In addition
to these commercially available compounds, a large number of
natural products, such as platensimycin and cerulenin, have been
noted to target FA synthesis [9,10], although these have yet to be
developed to the point of being actively used pharmaceuticals. In
addition, a recent study suggests that Gram-positive organisms
may be able to completely circumvent de novo FA synthesis by
acquiring FAs from their surroundings, illustrating the need for
a better understanding of FASs [11]. Inhibition of FASs has also
been implicated as an avenue for treatment and prevention of
cancers as well as obesity, further raising interest in the study
of FA metabolism [12,13].
Key words: acyl carrier protein, antibiotic, fatty acid biosynthesis,
lipid, protein complex.
FATTY ACID SYNTHESIS
De novo fatty acid synthesis
FAS systems are grouped into two major classes, type I and II.
The main difference between the two is the organization of the
genes and the proteins needed for FA biosynthesis. In dissociated
type II FAS systems, the proteins are all expressed as individual
polypeptides from a series of separate genes [2]. These systems
are found mostly in bacteria but also in specialized eukaryotic
organelles such as mitochondria and plastids in plants. In contrast,
type I FAS systems express large multienzyme complexes that
carry all the proteins necessary for FA biosynthesis on one or two
large polypeptide chains [21]. Type I FAS systems are divided into
two sub-groups, type Ia and Ib. Type Ia systems are found in fungi
as well as a few bacterial species and produce either α6β6 or α6
Abbreviations used: ACC, acetyl-CoA carboxylase; ACP, acyl carrier protein; ACPS, ACP synthase; EM, electron microscopy; FA, fatty acid; FAS, FA
synthase; LPA, lysophosphatidic acid; MD, molecular dynamics; NRPS, non-ribosomal peptide synthetase; PA, phospatidic acid; PCP, peptidyl carrier
protein; ppGpp, guanosine tetraphosphate; PKS, polyketide synthase; RMSD, root mean square deviation; SFA, saturated FA; UFA, unsaturated FA.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2010 Biochemical Society
2
Table 1
D. I. Chan and H. J. Vogel
E. coli proteins involved in FA biosynthesis and their abbreviations
Gene
Protein name
Abbreviation
acpP
acpS
accABCD
fabD
fabH
fabB
fabF
fabG
fabA
fabZ
fabI
Acyl carrier protein
ACP synthase
Acetyl-CoA carboxylase
Malonyl-CoA–ACP transacylase
β-Oxoacyl synthase III
β-Oxoacyl synthase I
β-Oxoacyl synthase II
β-Oxoacyl reductase
β-Hydroxydecanoyl dehydratase
β-Hydroxyacyl dehydratase
Enoyl reductase
ACP
ACPS
ACC
FabD
FabH
FabB
FabF
FabG
FabA
FabZ
FabI
Figure 2
Overview of the bacterial FA synthesis mechanism
After initiation of the acyl chain synthesis (reaction 3), the synthetic cycle is repeated multiple
times until saturated C16 or C18 acyl-ACP is diverted for utilization in membrane biosynthesis.
For the description of the reactions please refer to the text.
Figure 1
Conversion of apo- into holo-ACP by ACPS
CoA donates the phosphopantetheine arm, measuring approx. 18 Å, that is attached to the
hydroxy group of the conserved serine residue in a Asp-Ser-Leu motif of ACP. The sulfhydryl in
the prosthetic group is used to bind acyl chains via a thioester bond.
multimeric proteins respectively. The second sub-group, type Ib,
is found in mammals and produce α2 homodimers, which each
contain the entire suite of proteins needed for FA synthesis [22].
De novo biosynthesis of FAs occurs via a conserved set of
reactions and, although the architecture of the FAS system can
vary greatly between various organisms, the individual enzymatic
steps are essentially the same. In fact, the FA biosynthetic
machinery that is present in humans evolved from the bacterial
systems, and the reactions carried out during the elongation cycle
are identical [23]. To this end, FA synthesis is described as it
occurs in Escherichia coli, the most studied system to date, and
differences to the type I systems are noted later in the present
review. Saturated FA synthesis in E. coli involves more than ten
genes and proteins (Table 1). Central to this process is the ACP, a
cofactor protein that covalently binds all fatty acyl intermediates.
Before ACP can accept acyl chains, it must be activated by ACPS
(ACP synthase), which attaches a phosphopantetheine group from
CoA on to a serine residue of ACP (Ser36 in E. coli) in a conserved
Asp-Ser-Leu motif (Figure 1). The resulting terminal sulfhydryl
group of the phosphopantetheine arm is then used to bind all the
FAs in a covalent high-energy thioester bond. The counterpart
to ACPS is ACPH, which is a phosphodiesterase that cleaves
acylated phosphopantetheine groups off of ACP [24]. This protein
is only found in Gram-negative bacteria and the physiological
purpose of this enzyme has not yet been established [25].
c The Authors Journal compilation c 2010 Biochemical Society
The FA biosynthesis process begins with the conversion
of acetyl-CoA into malonyl-CoA using ACC (acetyl-CoA
carboxylase), which adds an HCO3 − group to the substrate
(Figure 2; step 1). ACC consists of four proteins encoded for
by accABCD and requires a biotin cofactor as well as ATP [26].
Malonyl-CoA-ACP transacylase (FabD) next transfers malonylCoA to ACP (Figure 2; step 2). The malonyl-ACP then combines
with an acetyl-CoA group in the first condensation reaction, which
is catalysed by β-oxoacyl synthase III (FabH) and forms βoxobutyryl-ACP (Figure 2; step 3). This intermediate represents
the first compound in the FA synthesis cycle, which performs the
same four reactions in a cyclic manner to extend the saturated
fatty acyl chain by two carbon units during each cycle. In this
first instance, the acetyl-CoA unit (C2 ) is expanded to a butyryl
group (C4 ). The following cycles give rise to a hexanoyl (C6 ) and
then an octanoyl (C8 ) group, which would be continued until a
final length of 16 or 18 carbons is reached. In recent years, an
alternative to the FabD reaction has been described. Holo-ACP
has been shown to be capable of self-acylating and trans-acylating
other holo-ACP proteins [27,28]. This function of ACP was first
described in PKS (polyketide synthase) systems (see below) and
subsequently FAS proteins have been demonstrated to possess one
or both of these activities as well [27–30]. It is unclear at this time
what the physiological function of these reactions is; however, it
has been shown that bacteria with a temperature-sensitive version
of FabD may be rescued if the ACP that is present possesses both
self- and trans-acylation activities [28].
The first reaction in the FA synthesis cycle is a NADPHdependent reduction of the β-oxoacyl-ACP into β-hydroxyacylACP, which is catalysed by β-oxoacyl reductase (FabG) (Figure 2;
step 4). The hydroxy group is removed by one of two βhydroxyacyl dehydratases (FabA or FabZ), which converts the
chain into a trans-2-enoyl group (Figure 2; step 5). The double
bond is then reduced in an NADH-dependent reaction by an enoylreductase (FabI). This yields acyl-ACP bound by a saturated acyl
chain extended by two carbon units compared with the start of the
Fatty acid biosynthesis and the acyl carrier protein
Figure 3
3
Extension of fatty acyl chains
Fatty acyl chain extension occurs via the condensation reaction, in which ACP transfers its acyl chain on to one of the condensing enzymes FabB or FabF (step 1). The holo-ACP from this step is
lost (step 2) and a malonyl-ACP enters, carrying the acyl chain extender unit. The malonyl group first loses a carbon dioxide (blue) and the growing acyl chain (red) is subsequently added onto the
remaining two carbon groups attached to ACP (step 3). The resulting β-oxoacyl-ACP represents the first intermediate of a new fatty acyl elongation cycle. ‘R’ represents a saturated acyl chain of
length 3 + 2n carbons. FabH catalyses only the very first condensation reaction that forms β-oxobutyryl-ACP. In that case, the acetyl starter group from acetyl-CoA is bound by FabH and condensed
with malonyl-ACP.
cycle (Figure 2; step 6). In the first round just described, acetylCoA represents the start of the cycle. At this point the cycle begins
again through the condensation reaction of acyl-ACP with another
malonyl-ACP group, generated as described above by ACC. The
condensation reactions other than the first step involving acetylCoA are catalysed by either β-oxoacyl synthases I or II (FabB or
FabF) (Figure 2; step 7). Notably, during each of the condensation
reactions, the acyl chain is detached from ACP and bound in a
thioester bond by a cysteine residue in the active sites of FabH,
FabB or FabF (Figure 3) [2]. An extender malonyl-ACP then
enters the active site and the acyl chain is added to the tip of the
malonyl unit, which loses a CO2 group in the process. Therefore
the acyl chain is constructed from the ‘inside out’ as the additional
carbon groups are added to the base of the acyl chain rather than
the tip (Figure 3). The cycle is repeated until the acyl chain reaches
16–18 carbon groups in length, at which point the vast majority
of acyl-ACPs are utilized in membrane biosynthesis.
In bacteria, the process of membrane biosynthesis takes place
in two different ways, the most common being the PlsX/PlsY
pathway [31]. First, the peripheral membrane protein PlsX
cleaves acyl-ACP to form an acyl-phosphate. This is then
attached on to glycerol-3-phosphate to form 1-acyl-glycerol-3phosphate (LPA; lysophosphatidic acid) by the acyltransferase
membrane protein PlsY [31]. Another acyltransferase, PlsC,
that is expressed in all bacteria, then adds a second acyl
chain to the 2-position of LPA to form PA (phospatidic acid)
[32]. PA represents the fundamental building block from which
the phospholipids are derived, giving rise to phosphatidylserine,
phosphatidylethanolamine and phosphatidylglycerol [33]. In a
second, not as widely distributed pathway, the acyltransferase
PlsB utilizes acyl-ACP and transfers the acyl chain to glycerol3-phosphate directly, followed by the same reaction catalysed by
PlsC to generate PA [34]. The PlsB pathway has the advantage
that it can utilize both acyl-ACP and acyl-CoA, which is the form
of FAs from exogenous sources [34]. In E. coli, PlsC also has the
ability to function with either substrate, but this is not the case in
all organisms [31,35].
Biosynthesis of unsaturated fatty acids
The pathway described above details the production of SFAs
(saturated FAs). The reaction scheme is slightly different for
the production of UFAs (unsaturated FAs). The first step
in the production of these acyl chains is catalysed by the fabA
gene product β-hydroxyacyl dehydratase (FabA), which catalyses
both a dehydration as well as an isomerization reaction. The
isomerase function is exclusively performed on C10 substrates.
FabA converts β-hydroxydecanoyl-ACP into trans-2-decenoylACP and subsequently isomerizes this fatty acyl intermediate to
cis-3-decenoyl-ACP (Figure 4) [36,37]. Subsequently, the cis-3decenoyl group undergoes a condensation reaction with another
malonyl-ACP group, yielding cis-5-dodecenoyl-ACP, since the
two carbon groups are introduced closest to the carbonyl carbon
(Figures 3 and 4). This reaction is catalysed by the second essential
enzyme for UFA synthesis, β-oxoacyl synthase I or FabB [38].
Thus in E. coli the FabB and FabI enzymes compete for cis3- and trans-2-decenoyl-ACP respectively (Figure 4), and the
relative abundance of these two enzymes determines the ratio
of saturated to unsaturated FAs in biological membranes. In the
following acyl chain elongation cycles, the same enzymes are used
as in SFA synthesis, with the exception of FabA, as only FabZ (the
other β-hydroxyacyl dehydratase; Figure 2) is capable of utilizing
cis-acyl chains [36]. Owing to the nature of the FA synthetic
cycle, there are only a small number of specific UFAs produced in
E. coli. The inside-out carbon group additions mean the cis double
bond essentially gets ‘pushed’ away from the thioester link by two
carbon units per cycle (Figure 2). Therefore the vast majority of
UFAs are cis-9-hexadecenoic acid (palmitoleic acid) and cis-11octadecenoic acid (vaccenic acid) [39,40].
The E. coli system described above represents the most
extensively studied bacterial system. However, variations to this
UFA synthesis mechanism exist and are notably different in Grampositive bacteria. For example, Streptococcus pneumoniae does
not possess a FabA enzyme, but contains a specific cis/trans
isomerase, termed FabM, which is responsible for the conversion
of trans-2- into cis-3-decenoyl-ACP [41]. Furthermore, additional
condensation reactions are not dependent on FabB, but rather FabF
extends both the saturated and unsaturated FAs. Enterococcus
faecalis possesses a system more similar to the E. coli pathway,
where FabN replaces FabA and FabO replaces FabB [42].
Interestingly, FabN in terms of its amino acid sequence is more
closely related to FabZ, despite its functional relationship to FabA
[42]. The mechanisms in other organisms, such as Clostridium
acetobutylicium, are still being investigated and it appears a
number of enzymes involved in UFA synthesis have yet to emerge
[43].
Some bacteria also possess desaturases that can introduce
unsaturated bonds into acyl chains after FA synthesis.
c The Authors Journal compilation c 2010 Biochemical Society
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Figure 4
D. I. Chan and H. J. Vogel
UFA synthesis pathways in bacteria
(A) Two UFA synthesis pathways are shown for E. coli (left) and S. pneumoniae (right). E. coli utilizes FabA, which has a dual dehydratase and isomerase activity, whereas S. pneumoniae has a
dedicated cis /trans isomerase (FabM), both of which are specific for C10 acyl chains. (B) The structure of the resulting cis -3-decenoyl-ACP.
For example, Bacillus subtilis and Pseudomonas aeruginosa
both express a membrane-bound enzyme known as DesA
that introduces cis double bonds at either the 5- or 9position respectively [44,45]. Ps. aeruginosa expresses a
second desaturase known as DesB, which also introduces
cis double bonds at the 9-position. However, DesB is active
on acyl-CoA taken up from exogenous sources rather than
phospholipids that are part of the membrane [45]. Unsaturated
cis-acyl chains may be modified further in the membrane via
other mechanisms, including cis/trans isomerization and the
introduction of cyclopropane FAs [46,47]. These adjustments
help membranes adapt to environmental factors such as acid
stress in the case of cyclopropane FAs [48]. In B. subtilis,
a two-component system regulates the expression of DesA
via the membrane-bound thermosensor DesK and the response
regulator DesR [44]. At temperatures lower than 30 ◦C,
DesK phosphorylates DesR, which activates the transcription of
DesA and leads to lipid desaturation. Recently, the structure
of the catalytic portion of DesK was determined in a number of
conformations, showing major rearrangements of its four helix
bundle [49]. This explains how DesK alternates between its
different states in response to changes in membrane fluidity.
Type I fatty acid synthase systems
The type I FASs are believed to have evolved from a dissociated
system, much like the type II systems found in bacteria and plants
[23]. Various evolutionary processes such as gene duplication and
gene fusion probably led to the emergence of the multifunctional
systems now found in modern type I FASs [23]. Despite
obvious differences in the overall organization of the two FAS
c The Authors Journal compilation c 2010 Biochemical Society
systems, the enzymatic reactions and mechanism of de novo FA
synthesis are essentially identical [22]. For example, exactly the
same intermediates and reactions are present in the elongation
cycle. However, some differences do occur in the initiation
and termination processes. The mammalian type Ib systems
contain a malonyl-/acetyl-CoA ACP transacylase (FabD) that
loads both the initiator and extender units on to ACP. This
contrasts the bacterial systems, which utilize FabH and FabD
for these processes (Figure 2). Fungal type Ia systems have a
discrete acetyl transferase that is specific for acetyl-CoA in the
transfer to ACP. The malonyl-CoA extender unit is transferred on
to ACP via a malonyl/palmitoyl transferase which is also involved
in the FA synthesis termination process, as it transfers the mature
palmitoyl group from ACP back on to CoA for use in various
cellular processes. On the other hand, type Ib systems utilize
a dedicated thioesterase enzyme that cleaves acyl chains off of
ACP and releases free FAs. Type II systems instead use enzymes,
such as the aforementioned PlsB and PlsX, to transfer acyl chains
directly from acyl-ACP for integration into the membrane [31].
Mitochondrial fatty acid synthase
In addition to the cytosolic FAS systems that are present in
eukaryotic organisms, mitochondria harbour a further set for
de novo FA synthesis. The system found in mitochondria differs
from the cytosolic FAS as it is of the type II dissociated organization and many of the proteins are highly homologous with
their bacterial counterparts [50]. Human and yeast mitochondrial
genes equivalent to the bacterial type II proteins continue to
be discovered and the structures of a number of mitochondrial
proteins have been solved [51–54]. Although both systems
Fatty acid biosynthesis and the acyl carrier protein
Table 2
Summary of bacterial FAS regulation
Effector
Target
Ligand
Consequence
Acyl-ACP
Acyl-ACP
Acyl-ACP
PpGpp
SpoT
ACC
FabH
FabI
PlsB
ppGpp synthesis
Acyl-ACP
FAS inhibition
FAS inhibition
FAS inhibition
PlsB inhibition
FAS inhibition
FabT
FapR
FadR
FabR
FabR
Transcription level regulation
FAS genes
Acyl-ACP
FAS genes
Malonyl-CoA or- ACP
fabA , fabB
Acyl-CoA
fabA , fabB
Saturated acyl-CoA or -ACP
fabA , fabB
Unsaturated acyl-CoA or -ACP
FAS inhibition
FAS activation
Activation
Activation
Repression
are present in eukaryotic cells, the vast majority of the FAs
synthesized are produced by the cytosolic machinery. The
mitochondrial FAS synthesizes octanoic acid, which serves
as the precursor for lipoic acid [55]. There is also evidence
that the mitochondrial system can produce longer FAs, however,
it is presently unclear what these acyl chains are used for
[55,56]. The exception is β-hydroxytetradecanoyl-ACP, which is
known to be part of the respiratory complex I in mammalian
mitochondria [57]. Although the role of the mitochondrial
FAS system is not entirely clear, it has been shown that
an impaired system leads to respiratory incompetence in
yeast [58], whereas in Trypanosoma brucei malfunctioning
mitochondrial FAS leads to abnormal morphology [59], and
in mammalian tissues to cell death [60]. Recently, evidence
has been presented that this FAS system is implicated in
monitoring the metabolic condition of the cell [61]. Decreasing
the amount of pyruvate available in mitochondria will limit the
acetyl-CoA pool for FAS synthesis and thereby lower the rate
of lipoic acid formation. Since pyruvate dehydrogenase, the
committing enzyme for TCA (tricarboxylic acid) cycle activity,
is strictly dependent on lipoic acid, this presents a positivefeedback loop involving FAS in mitochondria. Furthermore,
it has been suggested that a mitochondrial FAS product is
implicated in the RNase P maturation process, which is required for tRNA processing [61]. As such, inhibition
of the FAS will lead to decreased protein synthesis in
mitochondria. A link between RNase P and 3-hydroxyacyl dehydratase was also found in humans, as they are expressed
in a bicistronic fashion [62]. That these two proteins, which
are seemingly only distantly related in function, are transcribed
together further substantiates the notion that mitochondrial FAS
plays a role in sensing the metabolic state of a cell.
REGULATION OF FATTY ACID SYNTHESIS
Regulation through product inhibition
A great deal of energy in the form of ATP is committed to
de novo synthesis of fatty acyl chains and it is therefore of
paramount importance to regulate this process. The primary
pathway for the regulation of FA synthesis in E. coli is through
feedback inhibition by long-chain acyl-ACPs. This was initially
demonstrated through the overexpression of thioesterases that
cleave acyl-ACPs, leading to excessive FA synthesis [63]. Longchain acyl-ACPs affect the pathway at three discrete steps by
inhibiting ACC, FabH and FabI (Table 2) [64]. Inhibition of ACC
prevents the creation of malonyl-CoA, which is the extender unit
needed for the elongation of the growing acyl chains (Figure 2;
step 1) [65]. FabH catalyses the formation of the starter unit
5
β-oxobutyryl-ACP and thus its inhibition prevents the initiation of
further acyl chain formation cycles (Figure 2; step 3) [66]. Finally,
FabI catalyses the reduction of enoyl-ACP, which is critical for the
completion of the acyl chain elongation cycle due to the reversible
nature of the preceding dehydration reaction (Figure 2; step 6)
[67].
Since the vast majority of the synthesized FAs are utilized in
biomembranes, the acyl-ACP concentration is highly dependent
on the level of acyl chain integration into membranes, which
is catalysed in part by the enzyme PlsB. PlsB activity is in turn
inhibited by guanosine tetraphosphate (ppGpp), which is a general
stress response metabolite in E. coli and related bacteria as well
as in plants [68–70]. When the bacterium undergoes a ppGppmediated stress response, PlsB activity is decreased, which leads
to a build-up of long-chain acyl-ACPs and consequently to the
attenuation of FA synthesis (Table 2). On the other hand, it has
been shown that ACP interacts and alters the activity of the enzyme
SpoT, which is responsible for both the synthesis and breakdown of ppGpp [71,72]. This means that ACP itself can act as
a sensor of disrupted FA metabolism, leading to a build-up of
ppGpp and consequently to a stress response in E. coli [71].
Transcriptional level regulation of fatty acid synthesis
E. coli as well as other Gram-negative organisms also regulate FA
synthesis on a transcriptional level via the FabR and FadR proteins
(Table 2). Both proteins are involved in the regulation of the fabA
and fabB genes, which code for the essential enzymes required
for UFA synthesis. FadR is a transcriptional activator protein that
is bound at position −40 to the fabA and fabB genes, whereas
the protein FabR is a repressor that can bind in the promoter
region. FadR binds to both saturated and unsaturated acyl-CoA
groups, which causes the protein to release from the DNA and
thereby inhibit fabA and fabB transcription [73]. FadR actually
plays a dual role in the modulation of FA levels, as it also acts as a
repressor of β-oxidation [74]. FabR, on the other hand, responds
to both acyl-CoA and acyl-ACP, but unsaturated and saturated
ligands elicit different responses [7]. SFAs decrease the DNA
affinity of FabR, which leads to its release from the promoter and
subsequent transcription of fabA and fabB, whereas unsaturated
groups increase the affinity resulting in the repression of these
genes [7].
Although this is the only known transcription-level regulation
of FA synthesis in E. coli, there are additional mechanisms in
Gram-positive species (Table 2). For example, in Staphyloccocus
aureus and B. subtilis, the protein FapR is a transcription factor
that represses all FAS genes [75]. FapR is regulated by malonylCoA, which leads to the release of the inhibitor and transcription
of the FAS genes [76]. Moreover, malonyl-ACP was recently
shown to play a similar role as malonyl-CoA in regulating FapR in
B. subtilis [77]. Interestingly, this process does not seem to involve
protein–protein interactions but requires the exposed malonylphosphopantetheine group.
The protein FabT was identified to regulate the entire FAS gene
cluster with the exception of FabM in S. pneumoniae, which is
involved in UFA synthesis (see above) [78]. It was subsequently
discovered that FabT is a repressor that responds to acyl-ACP
levels [6]. It was shown to be most sensitive to vaccenyl-ACP but
responds to other long chain acyl-ACPs as well in the presence of
DNA [6].
Additional means of regulation of FA biosynthesis may yet be
uncovered. Recently, malonyl-ACP has been found to copurify
and crystallize in a complex with the cytoplasmic STAS domain
of YchM, an E. coli inner membrane protein that is involved in the
transport of bicarbonate. This observation directly links regulation
c The Authors Journal compilation c 2010 Biochemical Society
6
D. I. Chan and H. J. Vogel
of the transport of the substrate bicarbonate to the activity of
acetyl-CoA carboxylase (T. Moraes and R. Reithmeier, personal
communication). Clearly, the role of this protein–protein complex
in regulating FA biosynthesis requires further study.
ACYL CARRIER PROTEINS
Structure
ACP is the carrier protein that represents the central player
responsible for attachment and presentation of all acyl chain
intermediates to the enzymes of FA biosynthesis. In E. coli, ACP
is highly abundant, comprising approx. 0.25 % of all soluble
proteins and it represents one of four major protein–protein
interaction hubs, the others being DNA and RNA polymerases
as well as ribosome-associated proteins [79,80]. Bacterial ACP
is the protein encoded by the acpP gene. It is expressed as apoACP and requires activation to holo-ACP by ACPS through the
attachment of a phosphopantetheine moiety from CoA (Figure 1).
The prosthetic arm is attached at a specific serine residue in a
conserved Asp-Ser-Leu motif. Bacterial ACPs are small, acidic
proteins, the prototypical version being E. coli ACP, which has
a molecular mass of 9 kDa and a pI of 4.1 [79]. It has a very
high concentration of anionic residues and only few cationic
residues, which number 20 and 6 respectively, out of a total of 77
amino acids for the E. coli protein. The high density of anionic
residues is responsible for the unusual migration pattern of ACP
on SDS/PAGE systems, where it appears as a 20 kDa protein. In
addition, ACP only has 22 hydrophobic amino acids, giving it a
high ratio of charged to hydrophobic residues. This amino acid
distribution is highly reminiscent of natively unfolded proteins
and ACP is known to be of rather marginal stability [81,82].
E. coli ACP displays a large hydrophobic expansion at an elevated
pH, which can be visualized by a decreased electrophoretic
mobility in native PAGEs run at alkaline pH [83]. Although
E. coli ACP is folded at low ionic strength and pH 7, Vibrio
harveyi and Helicobacter pylori ACPs are unfolded or partially
folded respectively, under these conditions [84,85]. ACP is also a
highly dynamic protein and E. coli, Plasmodium falciparum and
spinach ACPs have even been noted to exist in a conformational
equilibrium between two states [86–88]. Deuterium exchange
and 15 N-heteronuclear NMR dynamics studies reveal that loop I
is among the most flexible regions of ACP along with helices
II and III [85,89,90]. Recent 15 N relaxation dispersion NMR
studies reveal that spinach ACP displays μs and ms timescale motions, further complicating the nature of ACP dynamics
[91]. Conformational flexibility is thought to be important for
ACP’s role in substrate delivery, since the acyl chain must be
extracted from the protein and presented to the enzymes of FA
synthesis. Although the flexibility of ACP is believed to aid in acyl
chain insertion and extrusion and is also important for enzyme
interactions, unfolding of the protein is not required for these
processes. This was demonstrated utilizing a functional, cyclized
version of ACP, which stabilizes the folded conformation and
thereby prevents complete unfolding [92].
Researchers began describing the structure of ACP over
40 years ago and the high-resolution solution structure of the
E. coli protein was first published in the late 1980s by the Prestegard group [1,93,94]. Since then, a number of additional ACP
structures have been published from various organisms (Table 3,
Figure 5). Each of these structures falls in line with a consensus
fold consisting of a four α-helix bundle. Three of these helices
run approximately parallel with each other, whereas helix III is
much shorter and lies almost perpendicular to the axes of the
major helices (Figure 5). In E. coli ACP, helices I–IV run from
c The Authors Journal compilation c 2010 Biochemical Society
Table 3
High-resolution structures of FAS ACPs solved to date
Organism
Acylation state
PDB ID
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
S. oleracea
S. oleracea
P. falciparum
P. falciparum
M. tuberculosis
B. subtilis
B. subtilis
B. subtilis
Type II Proteins
Apo
Holo
Butyryl
Hexanoyl
Heptanoyl
Decanoyl
trans -2-Dodecenoyl
Decanoyl
Octadecanoyl
Holo
Holo
Holo
Apo/Holo
Holo
Malonyl
1ACP, 1T8K, 2K92
2K93
1L0I, 1L0H, 2K94
2FAC
2FAD
2FAE
2FHS
2FVF, 2FVE
2FVA, 2AVA
2FQ0, 2FQ2
3GZL, 3GZM
1KLP
1HY8
1F80
2X2B
Rat
Human
Yeast
Type I proteins
Apo
Apo
Holo
1N8L, 2PNG
2CG5
2UV8, 2PFF, 2VKZ
Comments
In complex with FabI
Disulfide-linked ACP
In complex with ACPS
In complex with ACPS
Part of mega-synthase
residues 3–15, 36–49, 56–59 and 65–74 respectively, and helix
I is antiparallel with helices II and IV [95]. The helices form a
scaffold around a hydrophobic pocket in the centre of ACP that
has long been thought to harbour the growing acyl chain during
FA biosynthesis [96,97]. This was confirmed when structures of
acyl-ACP from spinach and E. coli ACP became available and it
was shown that the opening to the pocket lies in the gap between
helices II and III (Figure 5) [88,95,98]. The electrostatic surface of
E. coli ACP is highly anionic and helix II in particular possesses a
segment of conserved negatively charged amino acids (Figure 6)
[99]. The structure of ACP is remarkably conserved throughout
many organisms even at low sequence identity. For example, E.
coli and rat ACPs only share 22.5 % sequence identity, but the
overall global fold is still largely the same [100,101]. Furthermore,
when expressed in E. coli, rat ACP is partially capable of replacing
the endogenous protein where it can be utilized by the host FAS
system [102].
Current understanding of acyl-ACPs
High-resolution acyl-ACP structures and MD (molecular
dynamics) simulations have greatly increased our understanding
of how various acyl chains are bound and accommodated by ACP.
E. coli acyl-ACP structures have been published with butyryl,
hexanoyl, heptanoyl, octanoyl and decanoyl chains attached, and
decanoyl- and octadecanoyl-ACP structures from spinach have
also been reported [88,95,98]. The spinach acyl-ACP structures
were determined by NMR spectroscopy and an ambiguous
NOE (nuclear Overhauser effect) docking protocol was used
to determine the co-ordinates of the acyl chain, whereas the
E. coli structures were obtained from X-ray crystallography.
The different acyl-ACP forms from E. coli are highly similar
[Cα RMSD (root mean square deviation) ranging from 0.2 to
0.94 Å (1 Å = 0.1 nm) for decanoyl-ACP to the other forms],
whereas the spinach structures display an RMSD of 1.2 Å
from the E. coli protein (Figure 5). The structures reveal that
all the acyl chains enter the hydrophobic cavity of ACP between
helices II and III and are positioned along helix II into the
protein cavity. MD simulations of E. coli acyl-ACP captured
Fatty acid biosynthesis and the acyl carrier protein
Figure 5
7
Sample high-resolution type II acyl-ACP structures
E. coli butyryl- and decanoyl-ACPs (A and B respectively), as well as octadecanoyl-ACP from spinach (C), show the highly conserved ACP structural motif consisting of a four α-helix bundle. The
spinach ACP NMR structures are an overlay of the five lowest energy conformations and illustrate that the octadecanoyl chain is too large to fit into the hydrophobic pocket in its entirety. The acyl
chains and prosthetic groups are coloured according to atom type. A three-dimensional interactive structure of (B) is available online at http://www.BiochemJ.org/bj/430/0001/bj4300001add.htm.
the spontaneous transition process into the hydrophobic pocket
of the protein starting from a completely solvent-exposed acyl
chain conformation (see Supplementary Movie S1 at http://www.
BiochemJ.org/bj/430/bj4300001add.htm) [103]. As the acyl chain
enters the cavity, the phosphopantetheine linker arranges itself
at the cavity entrance and usually hydrogen bonds with Asp35 ,
Thr38 and Glu60 . Inside the protein, the simulations demonstrate
that the acyl chains can enter a second sub-pocket that is
directed towards helix I of the protein, which was subsequently
documented by NMR analysis as well [103,104]. The opening
of this cavity is mediated by two leucine residues which either
block or allow entry into the second sub-cavity via their χ 1
dihedral angles (see Supplementary Movie S2 at http://www.
BiochemJ.org/bj/430/bj4300001add.htm) [103]. Harbouring an
acyl chain inside the cavity of ACP expands its volume with
increasingly longer acyl chains. This has been described as an
overall swelling, as helices II and IV are observed to move further
apart, as well as helix III [88,104,105]. In spinach ACP, this
increases the cavity volume from 157 to 228 Å3 going from
decanoyl- to octadecanoyl-ACP [88]. The shorter acyl chains
attached to ACP up to octanoic or decanoic acid are buried
inside the pocket and the hydrocarbon groups are not readily
solvent-accessible [88,95,98,103]. This is in agreement with
earlier experiments such as octyl-sepharose binding studies and
native-PAGE performed at pH 9.5, which suggest that acyl chains
up to C8 are fully bound inside the hydrophobic pocket, whereas
longer acyl groups are partially solvent-exposed [79,106]. We
have also conducted MD simulations of the most prominent
E. coli UFAs, cis-palmitoleic and cis-vaccenic acids, bound to
ACP. Preliminary results indicate that these groups are more
rigid, but nonetheless utilize the same hydrophobic pocket as all
the other acyl chains (D.I. Chan, D.P. Tieleman and H.J. Vogel,
unpublished work).
Although there is now substantial evidence to understand the
behaviour of saturated acyl-ACP, only little is known on ACP
bound to the elongation cycle intermediates. It is conceivable that
the entire acyl chain does not have to be extracted from the protein
during the β-carbon processing reactions, since the reactions take
place at the β-carbon. However, high resolution structures of
FabA and FabI bound to substrate analogues consistently show
interactions between the acyl chains and the active site of the
enzymes [37,107]. Furthermore, ACP in the crystal structure
complex with FabI is positioned in a manner that the acyl chain
would have to be extruded entirely for the β-carbon to reach the
active site of FabI [108]. In that study, the authors also performed
MD simulations that position the tip of the acyl chain away from
ACP and into the active site of FabI [108]. As such, it is likely that
in the β-carbon processing reactions, the acyl chain is completely
c The Authors Journal compilation c 2010 Biochemical Society
8
Figure 6
D. I. Chan and H. J. Vogel
Electrostatic properties of ACP
Anionic and cationic residues are marked on E. coli ACP in red and blue respectively in (A) and (B). (B) The recognition helix, helix II, is highlighted, which contains a number of conserved anionic
residues as well as two hydrophobic residues (white). (C) Electrostatic surface representations of ACP, the left panel being orientated in the same way as (A) and the right panel rotated by 180◦,
displaying the highly anionic face.
extruded from the hydrophobic pocket of ACP, similar to what
takes place during the condensation reactions. A recent MD
study of β-oxoacyl-, β-hydroxyacyl- and trans-2-enoyl-ACPs
(see cycle in Figure 2) conducted by our group provides additional
information on ACP and the elongation cycle intermediates [105].
These intermediates were also observed to spontaneously enter
the hydrophobic pocket of ACP and sample both sub-pockets
within the protein (see Supplementary Movie S2). Furthermore,
it was observed that the β-oxone and β-hydroxy groups affect
the positioning of short-chain acyl groups, as these polar moieties
prefer not to reside within the hydrophobic binding pocket inside
the protein [105]. That study also managed to capture the acyl
chain extraction out of ACP in the case of the β-oxo- and βhydroxybutyryl-groups [105]. These short acyl chains possess
very little hydrophobic character and so were not constrained
inside the hydrophobic pocket as stringently. Similarly, a recent
crystal structure of B. subtilis malonyl-ACP reveals that this
moiety is not housed inside the hydrophobic pocket [77]. As the
acyl chains are extracted in the MD simulations, the structure of
ACP remains unchanged and it is essentially the reverse of the acyl
chain entry process [105]. A steered MD study of P. falciparum
β-hydroxydecanoyl-ACP suggests a similar acyl chain extrusion
pathway, however, helix III in those simulations swings away from
the protein, unlike what was observed in the unbiased simulations
[109]. Whether this reflects a real difference in the behaviour of
c The Authors Journal compilation c 2010 Biochemical Society
short- and long-chain acyl groups will have to be evaluated in
further studies.
Type I ACPs
Unlike the wide range of type II ACP structures that are currently
available, only the rat, human and Saccharomyces cerevisiae type I
ACP structures have been characterized to date (Table 3). Rat
and human apo-ACPs were cloned and expressed as discrete
proteins, separated from the large multi-enzyme complexes that
they are usually a part of. The rat ACP structure was determined by solution NMR, whereas the human protein was
crystallized in a complex with ACPS [100,101,110]. On the
other hand, the structure of yeast holo-ACP was determined
together as part of the entire 2.6 MDa FAS complex using X-ray
crystallography [5,111,112]. The structures display similar overall
folds to the type II ACPs and also possess four α-helices
(Figure 7), despite relatively low amino acid sequence identities
between the two types (rat compared with E. coli ACP: 22.5 %
identity). Intriguingly, the yeast protein possesses a second
domain in addition to the usual ACP fold, which consists of four
additional helices extending from the C-terminus, approximately
doubling the size of the protein (18 kDa) (Figure 7) [5,111]. NMR
experiments conducted on the isolated yeast ACP domain support
Fatty acid biosynthesis and the acyl carrier protein
Figure 7
9
High-resolution structures of type I ACPs
Human (A), yeast (B) and rat (C) type I ACPs. Overall, the secondary structure is conserved with type II proteins but other differences are present. ACP from yeast possesses a second domain (blue)
and the entrance to the hydrophobic pocket appears to be closed off in type I ACPs (compare red circles in B and C with Figure 5B). The opening is closed off by loop I, which is orientated more
towards helix III (thick arrow, C) and by Arg2150 (spheres, C). Ser36 is highlighted in stick representation.
the conformation observed in the X-ray mega-complex [113].
The rationale for this additional domain in yeast ACP is currently
unknown. In two of the yeast FAS complex structures, ACP is
positioned in contact with the oxoacyl synthase domain and the
phosphopantetheine group is directed away from the protein into
the active site of the enzyme [5,111]. The interactions between
ACP and the oxoacyl synthase unit occur both between the
typical and the extended domains of yeast ACP. Notably, helix
II, similar to what is seen in type II complexes, is implicated
as part of the interaction interface (see below) [111,114]. Helix
II is also involved in the complex between human ACP and
ACPS [110]. However, Bunkoczi et al. [110] indicated that
the most important protein–protein interactions in this case
occur between hydrophobic regions and are based on shape
complementarity. It is also noted that human ACP is far less
negatively charged compared with the type II E. coli protein
(overall charges of −5 and −14 respectively), which supports
an increased role for hydrophobic interactions in the type I
systems.
It has been postulated that type I ACPs do not harbour their
bound acyl chain inside the hydrophobic pocket, which would
represent a major difference between the type I and II proteins
[101]. The C-terminal region of loop I in type I ACPs is observed
to rest further towards helix III compared with type II proteins,
in which this portion of the loop is directed closer to helix I
(compare Figure 5 with Figure 7). This arrangement is held in
place by a hydrophobic triad composed of Leu2144 , Leu2149 and
Val2174 (residues 29, 34 and 59 in E. coli numbering) which
is not conserved in type II ACPs [101]. This hypothesis was
derived from the observation that only very small NMR chemical
shift perturbations are seen upon conversion of holo-ACP into
hexanoyl- or hexadecanoyl-ACPs in rat [101]. Yeast type I ACP
is in apparent contrast to this hypothesis, since it does not
possess the same hydrophobic triad and a substantial hydrophobic
pocket was detected [5]. Nevertheless, the loop I arrangement
in yeast ACP is still equivalent to that seen in the rat protein,
further complicating the interpretation of these data [101]. We
conducted MD simulations of isolated type I rat ACP similar to the
simulations performed with acyl-ACP from E. coli [103]. Unlike
the E. coli protein, neither rat octanoyl-ACPs nor butyryl-ACPs
displayed a transition of the acyl chain inside the hydrophobic
pocket during 3 × 20 ns of simulations (see Supplementary Movie
c The Authors Journal compilation c 2010 Biochemical Society
10
D. I. Chan and H. J. Vogel
S3 at http://www.BiochemJ.org/bj/430/bj4300001add.htm). Interestingly, the acyl chains appear to have a propensity to associate
with loop I of ACP, similar to simulations of the bacterial
type II ACPs [105]. The opening to the pocket is observed to
narrow further during the simulations of rat ACP, and Arg2150
(Arg60 in E. coli numbering) frequently hydrogen bonds with
the phosphopantetheine linker and thereby somewhat blocks the
entrance to the cavity. This arginine residue is well conserved
among type I ACPs and represents a hydrogen bond donor group
in the binding pocket entrance, which is absent in type II ACPs.
Thus these MD simulations support the hypothesis that type I
ACPs do not protect acyl chains inside a hydrophobic pocket, and
it remains to be seen what role Arg2150 may play. Recently, data was
also presented on type I PKS ACPs (see below), indicating that
these proteins do not bind their acyl chains inside the hydrophobic
pocket either, further supporting this theory for type I FAS ACPs
[115].
Although additional data is needed to evaluate the mechanism
of acyl chain binding in type I ACPs, it is certainly conceivable
that the acyl-chain in these systems need not be shielded inside the
ACP pocket. In type II systems, ACP is expressed as an individual
protein, whereas in type I FAS, ACP is attached to much larger
540 kDa or 2.6 MDa protein complexes. If type II ACPs expose
their bound acyl chains to the solvent, it may very well be targeted
to the cell membrane, much like myristoylated or palmitoylated
proteins in mammalian cells (e.g. recoverin) [116]. However, in
type I FASs, because ACP is covalently bound to the other protein
subunits, it is unlikely that a single acyl chain would target the
whole complex to the membrane. In fact, in the fungal type I
systems, ACP is located inside the scaffold of the overall barrel
shape (see below) and therefore would not be accessible to the
cell membrane.
ACP–protein interactions
For each of the reactions that occurs in the type II FA synthesis
cycle, the substrate that is covalently bound to ACP and hidden
inside its pocket must be extracted and placed into the active
site of the partner enzyme. Because the substrate is not readily
accessible inside the hydrophobic pocket of ACP, the partner
enzyme must bind the carrier protein and then somehow access
the bound cargo. Although ACP interacts with a large number
of proteins during FA synthesis, no common ACP-binding
sequence has been noted, unlike, for example, the protein kinases.
Nevertheless, information has been gained on ACP–protein
interactions from a number of structural, mutagenesis, NMR
chemical shift perturbation and bioinformatic studies. Sequence
alignment of ACPs from various organisms indicates that many
anionic residues in helix II are conserved and are therefore
believed to be important for ACP–enzyme interactions (Figure 6)
[99]. Helix II was subsequently dubbed the ‘recognition’ helix
[99] and it has been a recurring theme in virtually all studies
of type II ACP–enzyme interactions. It has been shown that
ACPs from a broad range of organisms are capable of restoring
growth in an E. coli acpP temperature-sensitive strain, which
is at least partially related to the similarity of helix II in ACPs
from different organisms [117]. The two ACP–enzyme complex
structures that are available support the important role of helix
II (Figure 8) [108,118]. In both the ACP–ACPS and ACP–
FabI structures, helix II represents the central interaction site.
Furthermore, chemical-shift perturbation and docking studies
involving FabG and FabH respectively, heavily implicate helix
II of ACP [119,120]. Mutations of various portions of helix II
suggest that different enzymes recognize specific regions of the
c The Authors Journal compilation c 2010 Biochemical Society
Figure 8
ACP–enzyme complex structures
The structure of the B. subtilis ACPS–ACP complex shows the functionally relevant homotrimer
of ACPS (blue) that binds ACP (coloured) between the interface of two dimers. (B) An expanded
snapshot showing the electrostatic interactions between ACP (coloured from N- to C-terminal
in red to white) and ACPS (blue). Anionic residues are shown in red. The β-strands of ACPS
are transparent for clarity. (C) Structure of the docked complex between FabG and ACP (purple),
which binds between helices II and III and across the dimer interface of FabG. The β-oxoacyl
substrate (starred arrow) would have to be extracted from the internal hydrophobic pocket of ACP
and directed into the active site of FabG next to the NADPH cofactors (spherical representation).
For simplicity, only the dimer of FabG is shown instead of the tetramer.
Fatty acid biosynthesis and the acyl carrier protein
helix. For example, interactions with ACPS are highly affected
by mutations to the N-terminal region of helix II, whereas
LpxA, which is involved in lipid A biosynthesis, and acyl-ACP
synthetases were more affected by mutations to the C-terminal
region of this helix [121].
Another important feature for ACP binding appears to be a
common surface pattern on the partner enzyme, which consists
of an electropositive/hydrophobic surface patch and lies in close
proximity to the active site [119,122]. In one study of FabH, it
was shown that the mutants R249A or A253Y in the patch region,
representing cationic and hydrophobic residues respectively,
largely affect the affinity for ACP [122]. These observations again
stress the importance of helix II of ACP, since the docking model
of FabH–ACP predicts that Arg249 of FabH interacts with Glu41 ,
which resides in the recognition helix [122]. Arginine residues in
the cationic portion of the ACP-binding enzymes appear to play
a particular role in these protein–protein interactions. In each
of the complexes characterized to date (ACP and ACPS, FabD,
FabH, FabG and FabI), arginine residues were identified that,
when mutated to uncharged residues, have highly detrimental
consequences for ACP binding [108,118–120,122]. Although
some lysine residues have been implicated as well, it appears
that arginine residues may be more suitable, potentially due to
their superior hydrogen-bonding ability.
Although helix II has been heavily implicated as an interaction
feature between ACP and various enzymes, other regions of
ACP are involved as well. The docking study mentioned above
between ACP and FabH also shows that the N-terminal region of
ACP participates in binding, whereas residues close to helix III
are involved in FabG and ACPS binding [118,119,122]. Type I
FAS and PKS systems provide yet another situation. For example,
thioesterases use carbon rulers to select for appropriate substrates
and, in the case of at least one PKS thioesterase, it was
shown that protein–protein interactions make only negligible
energetic contributions to their association [123,124]. A recent
EM (electron microscopy) study of the yeast type I FASs
suggests that ACP spends different amounts of time at each
enzyme site depending on the protein–protein interactions [125].
Furthermore, it was shown that the FapR of B. subtilis, involved in
FAS regulation, only recognizes the malonyl-phosphopantetheine
moiety of malonyl-ACP [77]. Therefore the picture is not as
straightforward as it might appear. Part of the problem is the
relatively low affinity (low μM range) between ACP and its
partner enzymes [119,121], which has been noted as a likely
reason for why only two ACP–protein complexes have been
crystallized to date. The highly dynamic nature of ACP itself
probably contributes to this situation as well.
NMR chemical-shift perturbations and computational docking
algorithms may present one approach to help circumvent the
difficulties of studying the structures of ACP–enzyme complexes.
This methodology has been used to characterize the ACP–FabD
complex of the FAS system in Streptomyces coelicolor [120] and
also the ACP–LpxA complex [126]. LpxA is an acyl transferase
involved in lipid A synthesis, which is a major component of
Gram-negative bacterial cell walls [127]. In addition to the NMR
chemical shift perturbations, RDCs (residual dipolar couplings)
were also utilized in the information-driven docking, which
provides additional orientational restraints for the placement
of ACP [126]. The resulting complex of ACP–LpxA is highly
consistent with the experimental data and reveals important salt
bridges, again involving helix II of ACP [126]. We used a
similar data-driven docking approach to determine the interactions
between ACP and FabG. We utilized previously published NMR
chemical-shift perturbation data as well as mutational data for
Arg129 and Arg172 of FabG [119] and employed experimental
11
information driven docking by HADDOCK [128]. HADDOCK
is one of a number of programs, such as ZDOCK [129] and
PatchDock [130], that are available and allow various forms of
data-driven docking. Since the location of the FabG active site is
known, as well as the length of the phosphopantetheine linker of
ACP, we were able to significantly limit the search space for ACP
and arrive at a model highly consistent with the experimental data
(Figure 8). This complex once more highlights the importance
of helix II of ACP in its interaction with partner enzymes, as it
is in close proximity to Arg129 and Arg172 of FabG, which have
been shown to be crucial for ACP binding [119]. Furthermore, the
prosthetic group with the attached acyl chain is well situated for
delivery of the substrate into the active site of FabG (Figure 8).
With the continued development and improvement of datadriven docking protocols, this approach may prove itself as a
feasible and effective alternative to obtaining crystal structures
of ACP–enzyme complexes. In particular, the accuracy of the
docked complex can be increased vastly if mutagenesis or
similar information is available on both the target and the ligand
proteins.
ACP within type I fatty acid mega-synthases
In type I FAS systems, ACP is part of large, multi-domain
polypeptides that also carry the other protein domains for FA
synthesis in a linear fashion (Figures 9B and 10A). These chains
arrange the mammalian and fungal systems in different multimeric
assemblies to build the FAS machinery (Figures 9 and 10).
Structures for mammalian, fungal and yeast type I FAS systems
have been determined previously by X-ray crystallography
[3–5,111,112,114,131,132]. The type Ia mega-complexes (2.6
MDa) of the yeast S. cerevisiae and the fungus Thermomyces
lanuginosus display similar overall folds, described as a barrel
shape with a central wheel in the middle that splits the complex
into two reaction chambers (Figure 10) [3,5]. Each chamber
contains three units each possessing the enzymes necessary for
performing the acyl chain elongation cycle. The mammalian FAS
structure looks significantly different and is markedly smaller with
each monomer of the homodimer measuring approx. 270 kDa in
size. In the mammalian complex, of the entire polypeptide chain,
only 9 % is used in linker regions, whereas in the much larger,
fungal type Ia protein approx. 50 % of the polypeptide is used
for scaffolding [21]. The human protein shape resembles a person
sitting with their legs and arms stretched out (Figure 9) [131].
The last two proteins on the mammalian polypeptide chain are
the ACP and thioesterase domains. Neither could be identified
in the electron density of the mammalian FAS complex, in contrast
with the yeast protein in which the structure of ACP was observed
(see above). In the mammalian system, ACP is anchored by
the oxoreductase domain on the N-terminal side, whereas the
thioesterase domain lies on its C-terminal side. In contrast, ACP
is connected via linker regions to the side wall of the fungal barrel
structure N-terminally and to the central wheel C-terminally. In
addition, the N-terminal linker region is alanine- and proline-rich,
which might make it more rigid and further restrict the motion
of ACP in fungal FAS systems [21]. It is therefore perhaps not
surprising that the electron density for ACP was not observed in
the mammalian complex.
In the crystal structure of the yeast FAS, the ACP was
locked into a fixed position with the active site of the oxoacyl
synthase enzyme (Figure 10) [5,111,112]. Hence this provided
a somewhat static picture, whereas ACP is believed to move
dynamically from enzyme active site to active site within each
chamber of the complex. Fortunately, for both the mammalian
and fungal FASs, EM studies have provided further insights into
c The Authors Journal compilation c 2010 Biochemical Society
12
Figure 9
D. I. Chan and H. J. Vogel
Mammalian type I FAS structure
(A) Crystal structure of the mammalian type I FAS complex in surface representation, coloured according to the proteins’ subunits (B). The yellow arrow denotes the location of ACP, for which there was
no electron density in the crystal structure. The broken arrows indicate the directions of FAS movement as was identified by EM (as explained in the main text). (B) The mammalian FAS is composed
of two α-chains which each contain a complete set of proteins for FA synthesis. DH, dehydratase; ER, enoyl reductase; KR, oxoacyl reductase; KS, oxoacyl synthase; MAT, malonyl-/acetyl-CoA
transacylase; TE, thioesterase.
the dynamic aspects of ACP and FAS action [125,133]. In the
mammalian system, a continuous spectrum of FAS conformations
was observed, indicating that the lower half of the X-shaped
structure can rotate around and thereby access both active site
cavities (Figure 9A, lower broken arrow) [133]. In addition to
rotation around the vertical axis of the protein, an in-plane
swinging by 25◦ is also observed, which alters the active site chambers between open and closed conformations (Figure 9A, upper
broken arrow) [133]. These data consolidate the biochemical
c The Authors Journal compilation c 2010 Biochemical Society
evidence that each ACP in the dimer can access both reaction
chambers, which was not apparent from the crystal structures
alone [134]. The yeast FAS seen in the EM studies is slightly
relaxed compared with the crystal structure, probably due to the
absence of crystal contacts [125]. Densities were identified that
correspond to ACP being located at the acetyl transferase, oxoacyl
synthase, oxoacyl reductase and the enoyl reductase active sites
[125]. ACP exposes the same anionic surface to each domain and
the enzymes all possess a cationic region, highly reminiscent of
Fatty acid biosynthesis and the acyl carrier protein
Figure 10
13
Structures of the fungal 2.6 MDa type I crystal structure
(A) The heterododecameric arrangement consisting of six α- and β-chains (lower diagram), resembling an overall barrel shape. (B) X-ray structures of the yeast protein reveal ACP (surface
representation) locked into position at the oxoacyl synthase domains. (C) EM experiments captured ACP at the active sites of a number of enzymes, revealing the dynamic manner of substrate shuttling
during FA synthesis. (C) reproduced from Gipson, P., Mills, D.J., Wouts, R., Grininger, M., Vonck, J. and Kuhlbrandt, W. (2010) Direct structural insight into the substrate-shuttling mechanism of
yeast fatty acid synthase by electron cryomucroscopy. Proc. Natl. Acad. Sci. U.S.A. 107, 9164–9169; [125]. AT, acetyl transferase; DH, dehydratase; ER, enoyl reductase; KR, oxoacyl reductase; KS,
oxoacyl synthase; PT, phosphopantetheine transferase; TE, thioesterase.
what has been described for type II FAS systems. It was also
noted that the electron density inside the reaction chamber is
highly variable, suggesting that ACP moves about stochastically
in each of the six reaction chambers [125]. This means that the
ACP position in one of the reaction chambers is not co-ordinated
with the carrier protein position in the other chambers. These
EM results clearly support the notion of ACP being able to move
around and present the correct acyl chains sequentially to the
desired enzymes, so that it can be synthesized in an efficient
assembly line fashion. Because ACP cannot leave the reaction
chambers, it does not need to hide its acyl chain inside the protein,
which could also help expedite its processing in the enzymes of
the type I systems.
Although the architecture and sequence identity of the type I
FAS systems are drastically different from the type II dissociated
enzymes, many of the functional units in these complexes are
remarkably similar. For example, the oxoacyl reductase domain
(FabG) of E. coli only has 21 % sequence identity with the fungal
protein, but the RMSD for both structures measures 2.5 Å [114].
On the other hand, other domains, such as the enoyl reductase and
dehydratase enzymes, vary significantly between the type Ia, Ib
and II systems. It should be noted that human mitochondrial type II
ACP shares similar properties to E. coli ACP and is expected to
have a similar structure [135], capable of harbouring acyl chains.
ACYL CARRIER PROTEINS IN SECONDARY METABOLISM
Three major processes involve carrier proteins that bind substrates
via a phosphopantetheine prosthetic group. These are FA
c The Authors Journal compilation c 2010 Biochemical Society
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D. I. Chan and H. J. Vogel
biosynthesis, PKS systems and NRPSs (non-ribosomal peptide
synthetases). Although FAS and PKS systems exist in both type I
and type II organizational units, NRPSs mainly exist as multifunctional type I polypeptides [136]. In contrast with FAS systems
that only utilize one ACP, NRPS and PKS systems may contain
a large number of carrier proteins. For example, Streptomyces
avermitilis has been documented to have over 80 carrier proteins
to accommodate all of its PKS and NRPS assembly lines [137].
Both NRPS and PKS systems are involved in the synthesis of
small natural molecules such as the antimicrobials gramicidin S,
erythromycin, vancomycin, and iron-chelating compounds such
as enterobactin [19,136]. The key difference between the two
systems is that the former catalyses the synthesis of peptide
bonds, whereas the latter forms carbon–carbon bonds, much
like the FAS system. The organization of these multidomain
units can become quite complex and even PKS–NRPS hybrids
exist, but each system can be decomposed into minimal essential
components that are needed for function. The NRPS system
minimally requires a carrier protein, an adenylation domain and
a condensation domain [136]. Since this system is involved in
peptide synthesis, the ACP analogue here is usually termed PCP.
In the first step, the adenylation domain catalyses the initiation
of the PCP by adding an amino acid via a thioester linkage. The
condensation domain then forms a peptide bond by combining two
PCP-bound substrates. Similarly, PKS systems minimally require
ACP, oxo synthase and acyl transferase domains [136]. The acyl
transferase usually loads a malonyl group from malonyl-CoA
on to ACP. The oxo synthase domain then extends this substrate
through a condensation reaction with another loaded ACP protein.
These systems function in an assembly-line fashion and product
diversity can be varied depending on the modular units that are
present. Whereas in FAS systems each cycle is catalysed by four
separate enzymes that reduce the β-oxoacyl chain to its saturated
form, PKS and NRPS systems may omit various enzymes in this
cycle and thereby create a variety of products. Modifying the
assembly line of enzymes has been proposed as a convenient way
to create novel bioactive compounds with high stereoselectivity
[138,139]. A system of interest is the biosynthesis of daptomycin,
which is an antibacterial lipopeptide that is synthesized by an
NRPS system [140] and is in use as an antibacterial agent for
the treatment of endocarditis and skin infections. Not only are
ACP-like proteins involved in the NRPS pathway leading to
biosynthesis of daptomycin, but it has recently been shown that
the fatty acyl group attached to this lipopeptide is provided by
DptF, which is itself also an ACP [140].
Many NRPS PCP and PKS ACP structures have been determined (Table 4), and the overall folds are largely conserved with
FAS ACPs (Figure 11). They possess the same helical bundle
arranged in a highly similar manner to the FAS counterparts
(Figures 11B and 11D). However, unlike FAS ACPs, a distinct
difference between the apo and holo forms of PCPs has been
noted. For example, in the tyrocidine A synthetase system from
B. subtilis, a PCP has been observed to have three distinct
conformations. Two states are only accessed by either apo- or
holo-PCP, whereas a third conformation is shared by both PCP
forms, known as the A/H state (Figure 11) [141]. The A/H form
is very similar to the canonical ACP conformation consisting
of a four-helix bundle that resembles those seen in the FAS
carrier proteins (Figure 11B) [141]. The conformation exclusive
to apo-PCP contains shortened helices I–III and, most notably,
the region corresponding to helix III is moved further towards the
protein core. The structure only seen in holo-PCP is characterized
by an altered helix IV that lies almost perpendicular to helix I
(Figure 11C). The conformational changes of PCP allow it to alter
its interaction interface, exposing different regions of the protein,
c The Authors Journal compilation c 2010 Biochemical Society
Table 4
High-resolution PKS ACP and NRPS PCP structures available
Organism
PKS
S. coelicolor
S. rimosus
S. roseofulvus
S. erythraea
A. parasiticus
NRPS
B. brevis
E. coli
Anabaena sp.
Other
L. rhamnosus
Acylation state
End product
PDB ID
Apo
Holo
Acetyl
Malonyl
3-Oxobutyl
3,5-Dioxobutyl
Butyryla
Hexanoyla
Octanoyla
Apo
Holo
Apo
Holo
Actinorhodin
2AF8, 2K0Y
2K0X
2KG6
2KG8
2KGD
2KGE
2KG9
2KGA
2KGC
1NQ4
1OR5
2JU1, 2JU2
2KR5
Oxytetracycline
Frenolicin
6-Deoxyerythrololide Bb
Norsolorinic acid
Holo
Apo
Apo/holo
Holo
Apoc
Apod
Apo
Tyrocidine
Enterobactin
Unknown
1DNY
2GDY
2GDW
2GDX
2JGP
2FQ1
2AFD
Apoe
Lipoteichoic acid
1DV5, 1HQB
a
Non-native substrate
b
This product is a building block of erythromycin
c
Solved in a complex with a condensation domain
d
Solved in a complex with an isochorismate lyase domain
e
D-alanyl carrier protein
which selects the appropriate binding partner in the synthetic cycle
[141]. This is not observed in PKS ACPs, which behave more
similarly to the FAS counterparts (Figure 11D). In PKS systems,
differences between apo- and holo-ACP have been noted, but they
are far more subtle [142]. Interestingly, the structure of the type I
PKS ACP involved in norsolorinic acid synthesis was determined,
and although this is not the first type I PKS ACP determined [143],
it was noted in this case that, similarly to the rat ACP structure, the
acyl chain does not enter the hydrophobic pocket of the protein
[115].
ANTIBIOTICS AND THE FAS SYSTEM
Owing to obvious architectural differences between the human
type I FAS and the bacterial type II FAS and their perceived
essential function, this particular system has long been considered
a good target for the development of novel antibiotics. In addition,
there are high-resolution structures available for each enzyme in
the FAS pathway, which provides a good starting point for rational
structure-based approaches to drug design and lead optimization
[2]. Nevertheless, the presence of the type II FAS system in
mitochondria, which closely resembles the bacterial systems, puts
a high demand on the selectivity of any new compounds developed
in this manner. Moreover, whether FASs are plausible targets has
been under debate recently, as it was demonstrated that numerous
pathogenic Gram-positive bacteria can actually survive in the
absence of a functioning FAS system [11]. It was shown that
these bacteria have the ability to grow on FAs taken up from
external sources, such as human blood plasma [11]. Nevertheless,
this does not apply to all bacteria and considering that there are
already antibiotics in use that successfully target bacterial FASs, it
Fatty acid biosynthesis and the acyl carrier protein
Figure 11
15
Carrier proteins from NRPS and PKS systems
The PCP from the tyrocidine NRPS system undergoes changes depending on whether apo- or holo-PCP is present (A–C). The A form shown in (A) is only used by apo-PCP, whereas the A/H
form (B) is shared by the apo- and holo-proteins. The H form (C) is exclusive to the holo state. The structures are coloured in a continuous spectrum from red (N-terminus) to blue (C-terminus).
(D) The 3-oxobutyl-ACP intermediate from the S. coelicolor actinorhodin PKS, displaying the canonical ACP fold. The 3-oxobutyl group, which does not enter the ACP pocket, is shown in stick
representation.
remains likely that these systems will continue to be an important
target for the development of new antibacterial compounds.
The two antibacterial compounds that are currently in use are
triclosan and isoniazid, which both inhibit the enoyl reductase
FabI [8]. Triclosan is widely used in household products such as
hand soap, whereas isoniazid is an anti-tuberculosis drug. It has
also been shown that the pantothenamide class of antibiotics is
metabolized to form non-productive ACP, which contributes to the
antibiotics’ activity [144]. Pantothenate is a precursor for CoA,
and the pantothenamides are inactive analogues thereof [145].
Through the same biosynthetic pathway that is used to make
CoA, an inactive derivatized pantothenamide is made instead and
added on to ACP in place of a phosphopantetheine group [144]. FA
synthesis is consequently shut down because this form of ACP is
not capable of binding the fatty acyl groups. There is also a broad
range of natural products that inhibit various steps in the FA
synthesis process. For example, cerulenin inhibits FabB and FabF
by binding at their active sites [146,147]. Considerable interest has
been placed on platensimycin and numerous analogues have been
developed [148]. As researchers continue to improve the understanding of how to specifically target bacteria [112], it only seems
a matter of time until these will become available commercially.
More recently, the connection of FA synthesis to two pervasive
diseases in our society have further sparked interest in FA
metabolism. Administration of C75, an analogue of the natural
FAS inhibitor cerulenin, led to drastic weight loss in mice [149].
Since other areas of FA metabolism are influenced by C75 as well,
it is not clear how much the inhibition of FA synthesis contributes
to this effect and as such, additional investigation is needed in
this area [12,13]. Furthermore, the commercially available drug
Orlistat has been shown to inhibit the thioesterase domain in
human FAS and thereby it halts tumour progression in mice [150].
This illuminates a very intriguing possibility of using human FAS
inhibitors for targeted cancer treatment, as FA synthesis is only
c The Authors Journal compilation c 2010 Biochemical Society
16
D. I. Chan and H. J. Vogel
active in adipose and liver tissues and is largely not needed in
normal tissue function [12]. Additionally, FAS inhibitors have
not displayed any toxicity in chemoprevention studies, making
them attractive targets for both prevention and tumour treatment
options.
CONCLUSIONS
ACP and FAS have long been prominent proteins for study in biochemistry [1]. Their central role and importance in all cells, as
well as the abundance and small size of ACP have undoubtedly
contributed to their appeal for investigation. Nevertheless, there
are many areas in this field that need further detailed study.
Only now are we beginning to understand how type II ACP
accommodates acyl chains of various lengths and excitingly, the
structures of the type I mega-synthases have shed some light on
the organization and mechanism of FAS function. Furthermore,
with the continued improvements made to computational docking
protocols, this approach has become a feasible alternative to
X-ray crystallography for the determination of ACP–protein
complexes [151,152]. Input data to drive the docking can stem
from a wide range of techniques, such as NMR chemicalshift perturbations and cross-saturation experiments [153],
mutagenesis and hydrogen/deuterium exchange data or even
bioinformatic information can be included. This further enhances
the applicability and accessibility of this technique. In light
of the potential role FAS inhibitors may play as various therapeutic
agents, it is crucial to continue to further our understanding
of FASs. FA synthesis is also being investigated for use in
biofuel production. Some researchers have recently engineered
photosynthetic cyanobacteria to efficiently over produce FAs and
secrete them. This enables ready harvesting of FAs for renewable
biofuel production [154]. Improving our understanding of FASs
will also contribute to our ability to utilize PKS and NRPS systems
for our benefit. To date, E. coli has already been used as the
host organism for the synthesis of various active natural, nonendogenous products such as macrolides [155], cyclic peptides
[156] and aromatic polyketides [157], which are all made using
either PKS or NRPS systems. Variations of the native protein
clusters found in these systems have been made to synthesize
libraries of unnatural, natural products [158]. However, many
aspects are still poorly understood, such as the importance of
linker regions between the subunits and the basis for subunit
preference, as yields are often low in these modified systems
[159]. With a better understanding of ACP and, importantly, the
factors that govern effective ACP–protein interactions, we will be
able to engineer more proficient molecular assembly lines [160–
162]. Clearly, there are many avenues still to explore in the field
of FA synthesis and with the significant developments in recent
years, the field will be able to continue to move forward swiftly.
FUNDING
Research in this laboratory on ACPs was supported by a grant from the Canadian Institutes
for Health Research. H.J.V. holds a Scientist award from the Alberta Heritage Foundation
for Medical Research.
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Received 31 March 2010/19 May 2010; accepted 24 May 2010
Published on the Internet 28 July 2010, doi:10.1042/BJ20100462
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