Eur. J. Biochem.

267, 2666±2671 (2000) q FEBS 2000

A novel function of yeast fatty acid synthase

Subunit a is capable of self-pantetheinylation

Fabienne Fichtlscherer1, Christian Wellein1, Maria Mittag2 and Eckhart Schweizer1

1
Lehrstuhl fuÈr Biochemie der UniversitaÈt Erlangen-NuÈrnberg, Erlangen, Germany; 2Botanisches Institut der UniversitaÈt MuÈnchen, Germany

The prosthetic group of yeast fatty acid synthase (FAS), 4 0 -phosphopantetheine, is covalently linked to Ser180 of
subunit a. It originates from coenzyme A and is transferred to the enzyme by a specific phosphopante-
theine:protein transferase (PPTase). The present study demonstrates that the FAS-activating PPTase of yeast
represents a distinct catalytic domain of the FAS complex and resides within the C-terminal portion of subunit a.
The autoactivation capacity of yeast FAS became evident from in vitro pantetheinylation studies using purified
apo-FAS preparations. These were readily converted to pantetheinylated holo-FAS simply upon addition of free
coenzyme A. Pantetheinylation-competent apo-FAS was prepared in vitro by constructing hybrid oligomers
containing a-subunits from two different pantetheine-less FAS-mutants. The respective mutants were selected
according to their ability to complement each other, in vivo. In vitro formation of hybrid apo-FAS complexes was
achieved by dimethylmaleic anhydride (DMMA) -induced reversible dissociation of mixtures of the two
constituent mutant enzymes. This treatment was both necessary and sufficient to produce pantetheinylation-
competent apo-FAS. Specific FAS activities were comparable independent of whether the apo-enzymes were
pantetheinylated in vivo or in vitro. Apart from the induction of overall FAS activity, incorporation of phos-
phopantetheine into apo-FAS was also demonstrated by the use of 3H-labelled coenzyme A, leading to the
formation of radioactively labelled FAS. It is concluded that pantetheinylation of yeast FAS is performed by an
intrinsic catalytic activity of the apo-enzyme proper. The endogenous PPTase acts in trans between different
subunits a in the a6b6 oligomer. The self-pantetheinylation of yeast FAS represents the first example of an apo-
enzyme being capable of post-translational autoactivitation.
Keywords: fatty acid synthase; self-pantetheinylation; interallelic complementation; reversible dissociation; a2
protomers.

4 0 -Phosphopantetheine is the prosthetic group of several bio-
synthetic multienzymes such as fatty acid synthases [1,2], most
polyketide synthases [3] and several polypeptide or siderophore
synthetases [4,5]. In these enzymes, protein-bound phospho-
pantetheine extending about 20 A Ê in length binds the substrates
and reaction intermediates as thioesters to its terminal sulf-
hydryl. Subsequently it serves as a `swinging arm' allowing
their translocation between different catalytic sites of the
multienzyme. Apart from these complex multienzymes, phos-
phopantetheine is also suggested to act as a prosthetic group of
the monofunctional yeast enzyme, a-aminoadipate reductase
[5]. Depending on the particular enzyme system, phospho-
pantetheine is covalently linked either to a low molecular mass
acyl (ACP), peptidyl (PCP) or aryl (ArCP) carrier protein
(type II systems) or it is attached to the respective domain(s) of
a large multifunctional enzyme (type I systems). In all cases,
formation of the pantetheinylated holo-enzyme is mediated by
the action of a specific phosphopantetheine: protein transferase.
Correspondence to E. Schweizer, Lehrstuhl fuÈr Biochemie der
UniversitaÈt Erlangen-NuÈrnberg, Staudtstr. 5, 91058 Erlangen, Germany.
Fax: 1 49 9131 85 28254, Tel.: 1 49 9131 85 28255,
E-mail: eschweiz@biologie.uni-erlangen.de
Abbreviations: FAS, fatty acid synthase; PPTase,
phosphopantetheine:protein transferase; ACP, acyl carrier protein;
DMMA, dimethylmaleic anhydride.
Enzymes: fatty acid synthase (EC 2.3.1.85); phosphopantetheine:protein
transferase (EC 2.7.8.7).
(Received 11 January 2000, accepted 7 March 2000)
As was first demonstrated by Vagelos and coworkers [6],
PPTases transfer the phosphopanteine moiety of coenzyme A
to the hydroxyl group of a specific serine residue of the
apo-protein. Apparently, each phosphopantetheinylated protein
possesses its own, specific partner PPTase. Studies on the
limited number of known PPTases indicate that the enzymes
differentiate, in many cases, between polypeptide and poly-
ketide synthases and, even more strictly, between type I and
type II apo-proteins [5,7±10]. Thus, the ACP synthase of
Escherichia coli activates a variety of bacterial, and even
mitochondrial, type II acyl carrier proteins [5,9], but it is
inactive with the S. cerevisiae or Brevibacterium ammonia-
genes type I apo-FAS [11]. As an exception, the surfactin
synthetase-activating enzyme of Bacillus subtilis, sfp, exhibits a
remarkable cross-reactivity with a variety of different type I
and even some type II systems [5,7]. Due to these specificity
differences, several different PPTases may be present even
within the same cell.
In yeast, three different phosphopantetheinylated proteins are
known, i.e. the cytoplasmic FAS complex [2], a bacterial-type
mitochondrial ACP [9] and the lysine biosynthetic enzyme,
a-aminoadipate semialdehyde dehydrogenase [5,12]. Each of
these enzymes appears to be activated by its own, specific
PPTase as pantetheinylation-defective mutants are, in all cases,
specifically affected in only one of the three enzymes while the
other two remain functional [9,12,13]. For mitochondrial ACP,
pantetheine-less mutants have allowed the identification of its
partner PPTase and the respective gene, PPT2 [9]. In contrast,
mutations leading to pantetheine-free fatty acid synthase were
q FEBS 2000
not located at a distinct and FAS-independent PPTase gene
locus but mapped at the C-terminal end of the multifunctional
FAS2 gene which encodes FAS subunit a [14]. The pante-
theinylation deficiency of the respective mutants could be
explained by both their inability of accepting phospho-
pantetheine or by the defect of a FAS-specific PPTase in
Fas2p. Upon screening the S. cerevisiae genome for potential
PPTase sequences, a distinct similarity between the C-terminal
domain of Fas2p and several known PPTase sequences became
apparent [5,8]. These findings suggested that the FAS-
activating PPTase represents, in addition to the ACP, b-ketoacyl
reductase and b-ketoacyl synthase domains, a fourth catalytic
center within FAS subunit a. In the present study this hypo-
thesis was experimentally verified by preparing pantetheine-
free apo-FAS in vitro, and by demonstrating that this apo-FAS
is readily converted into holo-FAS upon incubation with
coenzyme A.
M AT E R I A L S A N D M E T H O D S
Many of the materials and methods employed in this study have
been described in previous publications, such as culture media
and microbial growth conditions [15], the transformation of
yeast cells [16], or the [14C]b-alanine labelling [13], purifica-
tion and enzymatic analysis of yeast FAS [17]. [1-14C]b-alanine
(spec. activity 55 mCi´mmol21 ) was purchased from Moravec
Biochemicals (Brea, CA, USA). 3H-labelled coenzyme A
(spec. activity 40 Ci´mmol21) was from Hartmann Analytic,
Braunschweig, Germany.
Yeast strains and plasmids
The S. cerevisiae wild-type strain X2180-1A [15] and the
FAS2-deletion mutant ABYS.Dfas2 [16] have been described
previously. The diploid strain SC1458 (MATa/a fas2-2808/
fas2-158), the haploid strain SC1474 (MATa fas2-158 his3
ura3 leu2), the transformants SC1509 and 1509-A (ABYS.D-
fas2 transformed with YCp2MM and pCA51, respectively) and
SC1522 (SC1474 transformed with pCA46) were isolated in
this study. The S. cerevisiae fas2-mutants of complementation
group VII originated from our own collection [18]. Mutant
fas2MM was isolated by site-directed mutagenesis as described
earlier [19]. Similarly, plasmid YCp2MM was described pre-
viously [19]. Plasmids pCA51 and pCA46 containing, respec-
tively, the fas2MM and fas2-158 mutations in the multicopy
yeast plasmids YEp351 [20] and pRS423 [21] were constructed
in this work. In both plasmids, the FAS2 reading frame was
associated with 600 bp of its 5 0 -flanking and 300 bp of its
3 0 -flanking sequences. In addition, six histidine codons have
been added to the C-terminus of both mutant FAS2 genes. In
SC1509, 1509-A and 1522, the chromosomal background of the
transformants ensured that no wild-type FAS2 sequence
would originate by recombination between chromosomal and
extrachromosomal FAS2 DNA.
FAS enzyme isolation
The wild-type and mutant FAS proteins investigated in this
study were purified by either one of two different procedures.
One of them has been described previously [17] and was
applied to the majority of FAS enzymes isolated in this work.
Freshly harvested wet cells (300±500 g) were used as starting
material for this procedure. Alternatively, the FAS proteins
from mutants SC1509-A and SC1522 were purified, by affinity
chromatography on Ni21-agarose making use of the C-terminal
Autoactivation of yeast fatty acid synthase (Eur. J. Biochem. 267) 2667
6  His-tag modification of subunit a, in these complexes. For
this purification, 80±100 g wet cells were broken with glass
beads. After removal of cell debris by 5 min centrifugation at
5000 g the homogenate was cleared by filtration and the FAS
protein was subsequently adsorbed to 5 mL of Ni-nitriloacetic
acid Agarose (Qiagen) by the batch procedure described by the
manufacturer. The pH of the imidazole elution buffer was 7.5.
The eluate was dialyzed against 0.1 m potassium phosphate
pH 7.3 and the FAS protein was pelleted by 10 h centrifugation
at 100 000 g.
DMMA-induced reversible dissociation of yeast FAS and
activation of apo- to holo-FAS
A typical experiment was performed as follows: 5 mg of
purified FAS (or a mixture of two mutant FAS proteins
containing 2.5 mg of each enzyme) in 0.5 mL 0.3 m potassium
phosphate pH 7.5, 10 mm dithiotreitol were added with 5 mL of
freshly prepared 10% dimethyl maleic anhydride (DMMA;
Sigma) in anhydrous tetrahydrofurane. After 90 min incubation
at 0 8C, 0.3 mL of 1% bovine serum albumin were added. The
pH was then slowly adjusted to 5.8 by adding a 4 : 1 (v/v)
mixture of saturated ammonium sulfate and 1 m acetic acid.
The resulting precipitate was collected by 20 min centrifu-
gation at 30 000 g and 20 8C. The pellet was dissolved in
0.5 mL reactivation buffer (0.1 m potassium phosphate pH 7.5,
20 mm FMN, 0.5 mm EDTA) and the enzyme was allowed to
renature for 90 min at room temperature. Pantetheinylation of
apo-FAS was subsequently performed by adding coenzyme A
to a final concentration of 3 mm. After 10 min incubation at
20 8C, the FAS activity was determined with an aliquot.
R E S U LT S
Interallelic complementation between pantetheine-less FAS
mutants in vivo
4 0 -Phosphopantetheine is bound to serine-180 of FAS subunit
a. Consequently, the N-terminal part of Fas2p around this
position is considered as the acyl carrier domain of the
multienzyme. In wild-type yeast, [14C]b-alanine is readily
incorporated from the culture media into the enzyme-bound
phosphopantetheine (Fig. 1A). In contrast, replacement of
serine-180 by glycine in mutant fas2MM, leads to the loss of
b-alanine and, thus, pantetheine-incorporation into an other-
wise intact FAS complex (Fig. 1D). Similarly, mutations at the
C-terminal end of FAS2 such as fas2-158 are defective in
phosphopantetheine incorporation, too (Fig. 1C). The pante-
theine-less characteristics of mutants fas2MM and fas2-158
have already been reported in a different context [19]. Fas2MM
was constructed by site-directed mutagenesis and is contained
in the yeast plasmids, YCp2MM and pCA51. To preclude an
eventual alternative pantetheinylation of the adjacent threonine-
181 in the S180G mutant, both S180 and T181 were replaced
by glycine in fas2MM. In contrast to the site-directed mutations
S180G and T181G in fas2MM, most other pantetheine-less
FAS2 mutations available in this laboratory were isolated by
random mutagenesis and map close to the C-terminus of Fas2p
[14]. In previous studies [17,18], these mutants were assigned
to a unique complementation group (no. VII in Fig. 2).
Members of this group exhibit different complementation
characteristics compared to the N-terminal mutant, fas2MM
(Fig. 2). One of the respective alleles, fas2-158, has recently
been characterized by DNA sequencing and was identified as a
G/D-replacement at position 1770 of Fas2p [19]. In Fig. 3, the
2668 F. Fichtlscherer et al. (Eur. J. Biochem. 267) q FEBS 2000

Fig. 2. Complementation characteristics of pantetheine-less fas-2

mutants. Mutants of opposite mating types, a or a, were crossed and
growth of the resulting diploids was scored on fatty acid-free YPD media.
The PPTase-defective group VII-mutants are indicated. ±, no growth;
1, wild-type-like growth

apo-FAS. The restitution of overall FAS activity with the hybrid

enzyme synthesized by the fas2-158/fas2-2808 complementing
diploid is evident from the data shown in Table 1. It is further
seen from Fig. 2 that interallelic complementation being excep-
tional between the PPTase-mutants of group VII, occurs in all
crosses between those and the ACP-mutant, fas2MM. Thus,
among pantetheine-less fas2-mutants, a variety of allele combi-
nations was identified allowing the production of pantetheiny-
lated holo-FAS, in vivo. These findings provided the basis
for constructing potential pantetheinylation-competent apo-FAS
hybrids, in vitro.

Fig. 1. Sucrose density gradient profiles of purified FAS from wild-type

(A), fas2-158 (C) and SC1509 (D) cells after growth in [1-14C] In vitro constructed apo-FAS is pantetheinylated to holo-FAS
b-alanine-labeled YPD-FA media [13]. In (B) fas2-158/fas2MM hybrid by an intrinsic PPTase activity of the multienzyme
apo-FAS had been reassociated, after DMMA-treatment, from 4.4 mg In previous studies, hybrid fatty acid synthases had been
fas2-158 and 4.4 mg fas2MM FAS and was subsequently pantetheinylated constructed in this laboratory, in vitro, by dissociation and
to holo-FAS by the addition of 20 nmoles of [3H] coenzyme A
subsequent reassociation of mixtures of two differently mutated
(40 Ci´mmol21) in a volume of 0.5 mL. The activated enzyme was
FAS enzymes [23]. Reversible dissociation of yeast FAS may
sedimated for 10 h at 100 000 g and subsequently placed on the gradient.
be achieved by transient acylation of the enzyme, under strictly
Centrifugation occurred, in all cases, for 15 h at 100 000 g in a 10±30%
controlled conditions, with dimethylmaleic anhydride. The
sucrose density gradient. Radioactivity is represented by closed circles,
resulting hybrid enzymes exhibited restituted FAS activities
protein concentration (A280) by open circles.
according to the in vivo complementation characteristics of
the respective mutants. In the present study, the technique of
DMMA-induced reversible dissociation of purified FAS
relative positions of the two mutations, fas2MM and fas2-158,
proteins was applied to selected pantetheine-less fatty acid
within FAS2 are depicted. Although both mutations lead to
synthases in order to construct pantetheinylation-competent
the same phenotype, i.e. the biosynthesis of pantetheine-less
apo-FAS, they nevertheless map at opposite ends of FAS2.
Mutation of FAS subunit a usually affects specifically either
one of the three FAS2-encoded functions, i.e. b-ketoacyl syn-
thase, b-ketoacyl reductase or pantetheine-binding. In hetero-
allelic crosses, fas2-mutants with different functional lesions
complement each other while those being defective in the same
function do not [18,22]. As an exception, complementation
between isofunctionally defective fas2-mutants is observed
with a few specific allele combinations. Thus, among the
pantetheine-less mutants shown in Fig. 2, interallelic comple-
mentation occurs between mutants fas2-158, fas2-2808, fas2- Fig. 3. Amino-acid replacements of the fas2-158 and fas2MM muta-
750 and fas2MM. Due to this effect, the fas2-158/fas2-2808 tions within FAS2. Mutated nucleotide and amino-acid (in parenthesis)
and fas2-158/fas2-750 heterozygous diploids synthesize func- positions are numbered and marked by a dot. Arrows indicate approximate
tionally active and, hence, pantetheine-containing holo-FAS, extensions of the acyl carrier (ACP), b-ketoacyl reductase (RED),
although the respective haploids contain pantetheine-less b-ketoacyl synthase (CON) and PPTase (PPT) domains.
q FEBS 2000
Table 1. In vivo and in vitro pantetheinylation of hybrid apo-FAS.
In vivo synthesized hybrid holo-FAS was isolated from strains SC1522
(fas2-158/fas2MM) and SC1458 (fas2-158/fas2-2808). In vitro, apo-FAS
hybrids were constructed by reassociating DMMA-treated mixtures of
2.9 mg fas2-158 and 2.3 mg fas2-2803 FAS subunits in the one case, and of
0.56 mg fas2-158 and 1.9 mg fas2MM FAS subunits, in the other. The latter
two enzymes had been purified by Ni21-agarose affinity chromatography.
Experimental details of the dissociation and reassociation treatment were as
described in Materials and methods. FAS activity was determinded after
90 min reassociation both before and 10 min after the addition of 3 mm
coenzyme A.
Specific FAS
activity
Enzyme [mU´(mg protein)21]
Wild-type FAS 2500
In vivo formed hybrid FAS:
fas2-158/fas2MM 1100
fas2-158/fas2-2808 1600
In vitro formed hybrid FAS:
fas2-158/fas2MM without CoASH addition 0
fas2-158/fas2MM after CoASH addition 560
fas2-158/fas2-2808 without CoASH addition 0
fas2-158/fas2-2808 after CoASH addition 850
Mutant FAS mixture:
fas2-158/fas 2MM without CoASH addition 0 3
fas2-158/fas 2MM after CoASH addition 0 the enzyme. As is evident from Fig. 1B, radioactive phos-
hybrid apo-FAS complexes. According to the results depicted in
Fig. 2, hybrids from mutants fas2-158 and fas2-2808 or from
fas2-158 and fas2MM were expected to give pantetheinylation-
competent apo-FAS. In a first series of experiments, conditions
were optimized using wild-type FAS to give reproducibly high
reactivation rates ranging between 50 and 80% of the original
overall FAS activity (data not shown). These conditions were
subsequently applied to equimolar mixtures of the pantetheine-
less mutant apo-FAS proteins listed in Table 1. The resulting
apo-FAS hybrids were still inactive with respect to overall
FAS activity (Table 1). Upon additon of 3 mm coenzyme A,
however, activation of apo- to holo-FAS was initiated very
Fig. 4. Hypothetical dimeric structure of FAS subunit a allowing
interallelic complementation in the fas2-158/fas2MM hybrid. Mutation-
ally defective domains are indicated as open circles. The zig-zag line
represents the phosphopantetheine prosthetic group. Abbreviations are as
explained in Fig. 3.
Autoactivation of yeast fatty acid synthase (Eur. J. Biochem. 267) 2669
efficiently. No such activation was observed with an untreated
mixture of the two enzymes, i.e. if dissociation/reassociation
was omitted prior to the addition of coenzyme A (Table 1).
Table 1 lists representative data from one out of three inde-
pendent experiments performed in this context. Thus, formation
of hybrid FAS oligomers is obviously necessary for the
activation of apo- to holo-FAS. Furthermore, it is evident
from Table 1 that apart from apo-FAS no external PPTase is
required for the pantetheinylation of subunit a.
The specific FAS activities obtained by the in vitro
pantetheinylation reaction were 850 mU´mg21 with the fas2-
158/fas2-2808 hybrid and 560 mU´mg21 with the fas2-158/
fas2MM hybrid (Table 1). These activities correspond, in both
cases, to about 50% of the specific activities exhibited by the
respective enzymes if they were synthesized by complementing
diploids, in vivo (Table 1). As reactivation rates of DMMA-
treated FAS may be as low as 50%, the activities of the in vitro
activated enzymes compare well to those of the respective
in vivo synthesized complexes. The specific FAS activity of
the fas2-158/fas2MM hybrid complex is about half of that
exhibited by wild-type FAS (Table 1). This reduction is exactly
what should be expected, theoretically, as in the hybrid
enzyme harboring the S180G replacement only three out of
six a-subunits are susceptible to pantetheinylation (cf. Fig. 4).
In addition to the enzymatic activation of apo-FAS upon
incubation with unlabelled coenzyme A, self-pantetheinylation
of apo-FAS was also demonstrated by the incorporation of
H-labelled phosphopantetheine from tritiated coenzyme A into
phopantetheine became very effectively incorporated into the
FAS protein upon incubation of the purified fas1-158/fas2MM
apo-FAS hybrid with 3H-labelled coenzyme A. Concomitantly
with the incorporation of the radioactive label, induction of
overall FAS activity was also observed in this experiment (not
shown).
DISCUSSION
The present study describes the preparation of pantetheinyl-
ation-competent apo-FAS and its use in characterizing the
FAS-activating PPTase of yeast. Using a defined in vitro
pantetheinylation system, this PPTase was demonstrated to be a
constituent activity of the FAS multienzyme proper. The
respective active site was located next to the C-terminus of
FAS subunit a within a region where, in previous studies, the
bulk of pantetheine-less FAS mutations had been mapped [14].
Its ability of self-pantetheinylation, even in heterologous
expression systems, had so far prevented the isolation of
yeast apo-FAS. Although the intrinsic PPTase activity of yeast
FAS had already been suggested previously by a variety of
biochemical and genetic data [8,13], experimental evidence
remained indirect as long as a defined in vitro activation system
was not available. In an earlier report from this laboratory,
apo-FAS had been prepared by a similar approach as described
here and was activated, to a limited extent, by the addition of
coenzyme A and yeast cell extract [23]. It is now clear that, in
these experiment, addition of the cell homogenate was not
relevant and that the low efficiency of activation was due to
inappropriate renaturation conditions of the hybrid enzyme.
Thus, a major achievement and, at the same time, a prerequisite
of the work reported in the present study was the optimization
of experimental conditions for the reversible disssociation of
yeast FAS allowing the recovery of up to 100% native FAS.
Using this technique, two different types of apo-FAS hybrid
enzymes were prepared. The one hybrid, fas2-158/fas2MM,
2670 F. Fichtlscherer et al. (Eur. J. Biochem. 267)
Fig. 5. Genetic organization of the type I FAS multigenes in yeast and
B. ammoniagenes. Bold arrows indicate contiguous and independently
expressed reading frames. Abbreviations: ACE, acetyl transferase; ENR,
enoyl reductase; DEH, dehydratase; MAL/PAL, malonyl/palmityl trans-
ferase; ACP, acyl carrier protein; RED, b-ketoacyl reductase; CON,
b-ketoacyl synthase; PPT, PPTase.
contained PPTase-defective together with ACP-defective
a-subunits. The hypothetical arrangement of the two types of
subunits within a heterodimeric a2 protomer is depicted in
Fig. 4. In this model, the a2 dimer contains one active and one
inactive ACP/PPTase domain-combination. The restoration of
about 50% wild-type FAS activity in the fas2158/fas2MM
hybrid is in good agreement with this model (cf. Table 1). At
the same time, the model supports, once more, an a2 dimer as
the minimal functional entity within the a6b6 FAS complex of
yeast. In earlier studies, dimeric protomers had been proposed
for both the FAS a- and b-subunits [24]. This idea was
originally based on the experimentally observed frequency and
predictability of interallelic complementation between hetero-
functionally defective fas-mutants which is best explained if
catalytic FAS domains interact, quite generally, between
different rather than within the same multidomain monomer.
The second apo-FAS hybrid constructed in this work, fas2-158/
fas2-2808, comprizes two fas2-mutations affecting both the
PPTase activity of subunit a. Thus, interallelic complementa-
tion leads in this enzyme, in contrast to the fas2-158/fas2MM
hybrid, to the mutual correction of the mutationally induced
misfolding of the two constituent a-subunits. Overall FAS
activity being restituted by this mechanism depends on the
particular allele combination and may well exceed 50%
wild-type FAS activity. The experimentally observed higher
specific activity of the fas2-158/fas2-2808 hybrid compared to
fas2-158/fas2MM, agrees with this prediction.
The fas2-158 mutation representing a G1770D replacement
in the C-terminal part of Fas2p affects a GxV signature motif
which is highly conserved in all known PPTases [5]. The
resulting PPTase deficiency conforms with the functional
importance of this position which had also been observed
with another PPTase, sfp from B. subtilis. According to Quadri
et al. [7] nonconservative substitutions of the respective glycine
are not tolerated in this system, either.
In contrast to the dimeric animal FAS multienzymes, the
hexameric type I FAS complexes of yeast and B. ammonia-
genes exhibit the same linear arrangement of their constituent
catalytic domains (Fig. 5). Whether this conservation reflects
functional constraints or simply an evolutionary relationship
between the two microbial FAS multienzymes remains open, to
date. In both systems, the hypothetical fusion of all FAS
component enzymes into a contiguous multienzyme is inter-
rupted at one specific position. This position, though, is
different in the bacterial and yeast FAS multigenes (cf. Fig. 5).
As a result, yeast FAS is encoded by two unlinked genes, FAS1
and FAS2, being located on two different chromosomes. In
B. ammoniagones, all of the FAS component enzymes are
linked but the PPTase is encoded by an autonomous gene.
Nevertheless, this PPTAse gene is closely linked to one of the
two FAS multigenes, fasB. A similar linkage of apo-protein
genes with their partner PPTase genes has been observed with
q FEBS 2000
the surfactin synthetase of B. subtilis and the gramicidin
synthetase of B. brevis [7]. In B. ammoniagenes, the PPT1-
encoded PPTase obviously activates not only the product of the
linked FAS gene, fasB, but also the structurally homologous
FAS complex encoded by the unlinked locus, fasA. This
suggests that chromosomal linkage of apo-protein and
PPTase genes may not be of functional importance. According
to the model depicted in Fig. 4, the PPTase in FAS subunit a
pantetheinylates not its own ACP site but acts, in trans, on the
ACP domain of the second a-subunit in the dimer. This
intermolecular activation mechanism implies the post-transla-
tional pantetheinylation of subunit a whith both monomers in
the a2 dimer being folded and associated prior to their
modification. Our findings that activation of apo-FAS was
most efficient if coenzyme A was added after rather than during
the reassociation process (data not shown) supports this
conclusion.
In conclusion, the reported presence of an additional, PPTase
domain within the array of catalytic sites of the yeast FAS
multienzyme represents a novel biosynthetic principle not only
in the biosynthesis of pantetheine-containing multienzymes but,
quite generally, in the formation enzymes with covalently
bound prosthetic groups. This mechanism ensuring exhaustive
modification of all ACP domains in the complex by non-
Mendelian kinetics is certainly most efficient whenever the
respective PPTase is not required for additional cellular
pantetheinylation reactions.
ACKNOWLEDGEMENTS
This work was supported by the Deutsche Forschungsgemeinschaft and by
the Fonds der Chemischen Industrie. We thank Dr JoÈrg Hofmann for his
assistance in designing the figures.
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