(Received for publication, April 14, 1995)
From the
Saccharomyces cerevisiae contains at least five
acyl-CoA synthetases (fatty acid activation proteins, or Faaps). Four FAA genes have been recovered to date. Recent genetic studies
indicate that Faa1p and Faa4p are involved in the activation of
imported fatty acids, while Faa2p activates endogenous pools of fatty
acids. We have now purified Faa4p from S. cerevisiae and
compared its fatty acid substrate specificity in vitro with
the specificities of purified Faa1p, Faa2p, and Faa3p. Among C8-C18
saturated fatty acids, Faa4p and Faa1p both prefer C14:0. Surveys of
C14 fatty acids with single cis-double bonds at C2-C12
indicated that Faa4p and Faa1p prefer Z9-tetradecenoic acid,
although Faa4p's preference is much greater and also evident in
C16 and C18 fatty acids. Faa4p's selectivity for fatty acids with
a C9-C10 cis-double bond is a feature it shares with Faa3p and
is notable since in yeast Ole1p, a microsomal cis- desaturase, accounts for de novo production of monoenoic
acyl-CoAs from saturated acyl-CoA substrates. Faa4p has no detectable
acyl-CoA synthetase activity when incubated with tetradecenoic acids
having a trans-double bond at C2-3, C4-5,
C5-6, C6-7, C7-8, or C9-10. Faa3p can only use E9-tetradecenoic acid as a substrate, while E4-, E6- and E9-tetradecenoic acids can be used by Faa1p
and Faa2p. E2-tetradecenoic acid is an Faap inhibitor, with
Faa2p exhibiting the greatest sensitivity (IC
= 2.6
± 0.2 µM). Triacsin C
(1-hydroxy3-(E,E,E,2`,4`,7`-undecatrienylidine)-1,2,3-triazene)
has trans-double bonds at positions that correspond to those
in E2-, E5-, and E7-tetradecenoic acids.
This compound is a potent inhibitor of Faa2p (K
= 15 ± 1 nM; competitive with fatty
acid), less potent against Faa4p (K
= 2 µM), and not active against Faa1p or
Faa3p (IC
> 500 µM). Analysis of an n-tetradecanal plus a series of oximes (tridecanal oxime,
1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol) indicated that
the combination of an azenol moiety (R-CH&cjs0808;N-OH)
plus adjacent unsaturation are critical for triacsin C's
selective inhibition of Faa2p. Triacsin C and oxime derivatives appear
to be very useful for defining differences in molecular recognition
among S. cerevisiae acyl-CoA synthetases. The >25,000-fold
range in the inhibitory effects of triacsin C on these four Faaps
suggests that it may be possible to develop other selective inhibitors
of eukaryotic acyl-CoA synthetases.
Saccharomyces cerevisiae is an excellent, genetically
manipulatable model system to study the regulation of acyl-CoA
metabolism. De novo production of saturated acyl-CoAs from
acetyl-CoA and malonyl-CoA is catalyzed by the fatty acid synthetase
complex (Fas; (1) ). Palmitoyl-CoA and stearoyl-CoA are the
principal products of Fas. Ole1p, a microsomal cis- desaturase, accounts for all de novo production of
monoenoic acyl-CoAs from saturated acyl-CoA
substrates(2, 3, 4, 5) . Exogenously
derived and endogenous free fatty acids are activated to their acyl-CoA
derivatives by at least five long chain acyl-CoA synthetases encoded by
fatty acid activation (FAA) (
)genes(6, 7, 8) .
The
functions of four unlinked S. cerevisiae FAA genes have been
characterized by noting the phenotypes of isogenic strains of yeast
with all possible combinations of faa1, faa2, faa3, and/or faa4 (null) alleles(8) .
The results indicate that Faa1p and Faa4p are the principal acyl-CoA
synthetases responsible for activating imported fatty acids, while
Faa2p appears to be involved in activation of endogenous fatty acids to
their CoA derivatives. The physiologic role of Faa3p has not been
defined. The existence of FAA5 was inferred from the
observation that an faa1
faa2
faa3
faa4
strain is still able to survive on media containing myristate,
palmitate, or oleate as the sole carbon source, presumably because it
is able to import these fatty acids, activate them, and direct the
resulting acyl-CoAs to peroxisome-based
-oxidation
pathways(8) .
The overall reaction catalyzed by an acyl-CoA synthetase is as follows:
Berg (9, 10) hypothesized that activation of acetate (and presumably other fatty acids) involved at least two steps:
assumes the formation of a mixed
phosphoryl-carboxyl anhydride
(R-CO-O-PO-O-ribose-adenine).
Bar-Tana and co-workers used partially purified rat liver microsomal
palmitoyl-CoA synthetase to present three lines of evidence that the
catalytic mechanism involves formation of an enzyme-bound
intermediate(11, 12, 13, 14, 15) .
First, the initial velocity pattern of the forward reaction suggested a
Bi Uni Uni Bi Ping Pong or a Bi Bi Uni Uni Ping Pong mechanism where
ATP and palmitate must bind to enzyme before any product is released (14) . Second,
O present in
[carboxy-
O]palmitate is incorporated
into AMP and palmitoyl-CoA but not into pyrophosphate(14) .
Third, an enzyme intermediate was recovered by gel filtration
chromatography after incubation of the acyl-CoA synthetase with
[
C]ATP and [
H]palmitate.
The molar ratio of fatty acid to nucleotide in the intermediate was
1:1(15) .
S. cerevisiae's four Faaps provide an opportunity to define common as well as divergent features in the structure/activity relationships of acyl-CoA synthetases. Faa1p, Faa2p, and Faa3p have been expressed in and purified from Escherichia coli strains that do not produce the single endogenous bacterial acyl-CoA synthetase encoded by the fadD gene(16, 17) . A survey (17) of their in vitro chain length selectivity using 4-24-carbon saturated fatty acids (C4:0-C24:0) indicated that Faa1p prefers C12:0 to C16:0 fatty acids, with C14:0 and C15:0 having the highest activities. C4:0-C9:0 and C20:0-C24:0 are inactive. Faa2p tolerates a broader range of fatty acyl chain lengths; C9:0-C13:0 are the most active substrates but C7:0-C8:0 and C14:0-C17:0 can be accommodated with less than a 2-fold reduction in acyl-CoA production. C22:0 and C24:0 are inactive. Faa3p has a 2-160-fold lower acyl-CoA synthetase activity with C9:0-C18:0 substrates compared with Faa1p and Faa2p. However, Faa3p is able to activate C22:0 and C24:0 (lignoceric acid). Surveys of C14, C16, and C18 fatty acids with and without cis-double bonds at C9-C10 indicate that Faa3p prefers palmitoleic and oleic acids as substrates. These are the two most abundant fatty acids in S. cerevisiae when it is grown on rich media (e.g. see (18) ). Faa4p has not been purified, nor has its acyl chain selectivity been defined. A recent genetic study indicated that a cDNA encoding rat liver acyl-CoA synthetase can complement at least some of the functions served by FAA4, namely activation of two exogenous fatty acids (myristate and palmitate), which are then utilized by intracellular phospholipid biosynthetic pathways(19) .
We have now isolated Faa4p from S. cerevisiae and compared its substrate selectivity with the selectivities of the other three known yeast Faaps using a series of saturated C8-C18 fatty acids, isomerically pure cis- and trans-tetradecenoic acids, a previously characterized triacsin inhibitor of mammalian acyl-CoA synthetases, and several aldoximes that are similar in some ways to the triacsin.
The functional effects of adding a 6xHis
tag to Faa4p were defined by comparing the growth of isogenic S.
cerevisiae strains under a variety of conditions. These strains,
YB332 (MATa NMT1 ura3 his3200 ade2 lys2-801 leu2 FAA1
FAA2 FAA3 FAA4) and YB525 (MATa NMT1 ura3 his3
200 ade2
lys2-801 leu2 faa1
::1.9::HIS3 FAA2 FAA3
faa4
0.3::LYS2), are described in (7) and (8) , respectively.
YB332 (FAA1FAA4) was
transformed with the parental vector without FAA insert
(pBB358). Strain YB525 (faa1faa4
) was transformed
with pBB358 (GPD-vector), pBB364 (GPD-Faa4p without a
COOH-terminal 6xHis tag; (8) ), or pBB373 (GPD-Faa4p-6xHis). The strains were grown at 30 °C to an A
= 1 in supplemented minimal medium
without uracil (SMM-URA; 0.67% yeast nitrogen base with ammonium
sulfate (Bio101), 0.08% complete supplement minus uracil (Bio 101), 2%
dextrose). Aliquots (50 µl) were removed from the cultures and
added to 150 µl of sorbitol (1 M) and equivalent numbers
of cells were stamped with a multiprong applicator (Replaclone) onto
agar plates containing 1% yeast extract, 2% peptone, 2% dextrose (YPD)
with or without 25 µM cerulenin, 500 µM myristate, 500 µM palmitate, or 500 µM oleate. (Brij-58 (1% w/v) was added to media containing fatty
acids.) Plates were incubated at 24, 30, or 37 °C for 3-4
days. Experiments were performed in quadruplicate on two separate
occasions.
The rate of product formation was assessed using these assay conditions, myristate or oleate as substrates, each of the purified enzyme preparations, and incubation times of 1, 2, 4, 8, 16, 24, 32, and 40 min. The results established that formation of the triazolotetrazine dye was linear up to 32 min. Therefore, the activity of fatty acid analogs was surveyed using a 20-min incubation period. Each tetradecenoic acid was assayed in triplicate. All experiments were repeated at least three times.
Product
formation was assessed using these assay conditions, radiolabeled
oleate as a substrate, and incubation times of 2, 5, 10, 15, and 30
min. With each purified Faap-6xHis, acyl-CoA production was linear up
to 30 min. At 15 min, less that 20% of the fatty acid had been
converted to its acyl-CoA derivative. We selected this time point for
assaying other radiolabeled fatty acids. IC values were
determined using similar assay conditions except that the reactions
contained 5 µM [9,10-
H]oleate (10
Ci/mmol), 0-1.5 mM of the test compound, 0.2 µg of a
purified Faap-6xHis, and a 15-min incubation at 24 °C. (We
confirmed that < 20% of [
H]oleic acid is
converted to its CoA derivative under these conditions in the absence
of test compound.) All assays were done in triplicate on two or three
occasions.
The K of purified
Faa1p-6xHis and Faa2p-6xHis for oleate was defined by Eadie Hofstee
plots(27, 28) . These plots were also used to
calculate the K
for the inhibition of Faa2p-6xHis and
Faa4p-6xHis by triacsin C (Kameda Co.).
Figure 1:
A centromeric
plasmid encoding Faa4p, with or without a COOH-terminal histidine tag,
can rescue growth of an faa1faa4
strain of S.
cerevisiae when fatty acid synthetase is inhibited and the medium
is supplemented with fatty acids. An equal number of cells from YB332 (FAA1FAA4), transformed with the parental YCp-GPD vector (pBB358), or YB525 (faa1
faa4
),
transformed with either the parental vector, GPD-FAA4, or GPD-FAA4-6xHis, were plated onto YPD media
supplemented with 25 µM cerulenin (CER), with or
without 500 µM myristate (C14:0), palmitate (C16:0), or
oleate (C18:1
). Plates were incubated for 3-4
days at 24, 30, or 37 °C.
Ni-NTA-agarose affinity chromatography
of faa1
faa4
GPD-FAA4-6xHis cell lysates
resulted in an average 120-fold enrichment in oleoyl-CoA synthetase
activity (n = 6 experiments). In contrast, no
oleoyl-CoA synthetase activity was recovered from faa1
faa4
cells containing the GPD-vector
without insert. The affinity purification protocol is simple and rapid
(20 min from lysis of cells to assay). This rapidity is important;
Faa4p-6xHis is only stable for
60 min after purification is
completed.
Assay conditions were identified where
[H]oleoyl-CoA production by purified Faa4p-6xHis
was linear over a 30-min period (see ``Experimental
Procedures''). The conditions were then used to identify the
enzyme's pH and temperature optima. There is little change in
oleoyl-CoA synthetase activity at 24 °C from pH 6.5 to 7.9 (Fig. 2A). Moreover, enzyme activity varies <2-fold
between 24, 30 and 37 °C at pH 7.1 (Fig. 2B), in
general agreement with the in vivo studies shown in Fig. 1.
Figure 2:
The pH, temperature, and acyl chain
selectivity of purified Faa4p-6xHis. pH and temperature optima were
determined using [H]oleic acid as a substrate and
the HPLC assay described under ``Experimental Procedures.''
The effects of changing pH were assessed at 24 °C. The temperature
optimum was defined at pH 7.1. A panel of radiolabeled C8-C18 saturated
fatty acids plus palmitoleic (C16:1
) and oleic
(C18:1
) acids were used to generate the data shown in panelC (24 °C, pH 7.1). Mean values ± 1
S.D. are plotted (n = 2 experiments, each done in
duplicate). An asterisk indicates that a radiolabeled acyl-CoA
product was not detected.
Radiolabeled myristoleic acid (C14:1) was not
commercially available. However, we had previously synthesized a series
of myristic acid analogs with single cis-double bonds at all
possible positions in the alkyl chain (Z2-Z13-tetradecenoic acids; (25) ). This
family of compounds allowed us to explore the enzyme's cis-double bond selectivity. Because they were unlabeled, we
determined their relative activities as Faa4p-6xHis substrates in a
system that detects acyl-CoA products through a series of secondary
reactions: (i) Candida acyl-CoA oxidase is used to generate
hydrogen peroxide from acyl-CoA; (ii) the hydrogen peroxide is used,
together with catalase, to oxidize methanol to formaldehyde; (iii)
formaldehyde reacts with 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole
to form a purple triazolotetrazine dye in the presence of basic
periodate, and (iv) the concentration of the purple dye is determined
colorimetrically. Failure to detect product in this enzyme-coupled
assay may either reflect the fact that the tetradecenoic acid is not a
substrate for the purified acyl-CoA synthetase or that the resulting
analog-CoA may not be a substrate for the Candida acyl-CoA
oxidase. We could conclude that an analog was not a substrate for Faa4p
only if acyl-CoA production was detected with another Faap.
When the coupled assay system contained purified Faa4p-6xHis, Z9-tetradecenoic acid was the most active compound. The Z10 and Z11 isomers were 3-4-fold less active, while Z12 or the Z8-Z2-tetradecenoic acids did not yield detectable levels of product (Fig. 3A).
Figure 3: Comparison of the activities of Z-tetradecenoic acids as Faap substrates. Each purified Faap was incubated with members of the panel of unlabeled tetradecenoic acids with single cis-double bonds at various positions along the alkyl chain (e.g.Z2 = cis-double bond between C2 and C3; C1 = carboxyl). Acyl-CoA production was measured using the coupled enzyme assay described under ``Experimental Procedures.'' The mean value obtained with each analog has been plotted (± S.D.) from three independent experiments, each done in triplicate. Product, triazolotetrazine dye. An asterisk indicates that acyl-CoA production was below the limits of detection of the assay (<0.02 pmol/min/mg Faap). Note the difference in scale in the plot of product formation with Faa3p.
Figure 4: Comparison of the activities of E-tetradecenoic acids as Faap substrates. Unlabeled tetradecenoic acids with trans-double bonds at the positions indicated were each incubated with a purified Faap-6xHis, and acyl-CoA product formation was detected with the coupled enzyme assay. Asterisk, acyl-CoA production below the limits of detection of the assay. See the legend to Fig. 3for further details.
The ``inactive'' E2, E5, and E7
isomers were evaluated further with an HPLC assay that measures the
ability of these compounds to block conversion of
[H]oleic acid to its CoA derivative. Only E2-tetradecenoic acid showed detectable inhibitory activity
(at 10-100-fold molar excess compared with substrate). The degree
of inhibition by E2-tetradecenoic acid was enzyme-specific:
Faa2p was most sensitive (IC
= 2.6 ± 0.2
µM). Faa1p-6xHis was less affected (IC
= 148 ± 10 µM).
Faa4p's preference for a cis- but not a trans-double bond at C9-C10 highlights its stereoselectivity. The panel of E-tetradecenoic acids also revealed a stereoselectivity in Faa2p that was not apparent with the corresponding cis-isomers.
Figure 5: Comparison of the structures of triacsins A-D with tetradecanoic acid, n-tetradecanol, tridecanal oxime, 1-azadeca-1,3,5-trienol, and 1-azaundeca-1,3,5-trienol. The triazenol group in the triacsins has been boxed and aligned with comparable regions of the other compounds.
Triacsin C has trans-double bonds at positions that
correspond to those in E2-, E5-, and E7-tetradecenoic acids (Fig. 5). As noted above, one of
these isomers, E2-tetradecenoic acid, is a selective inhibitor
of Faa2p. This led us to ask whether triacsin C is also a selective
Faap inhibitor. Since oleic acid is a substrate for each Faap, we
measured the effects of triacsin C on their oleoyl-CoA synthetase
activities. The triacsin is not active against purified Faa1p-6xHis or
Faa3p-6xHis (IC > 500 µM). It is a potent
inhibitor of Faa2p, with an IC
of 80 ± 10 nM and a K
of 15 ± 1 nM (the enzyme's K
for
C18:1
is 25 ± 1 µM; Fig. 6). Eadie-Hofstee plots indicated that triacsin C is
competitive with respect to oleate (Fig. 6). We were not able to
determine whether triacsin C is noncompetitive with ATP or CoA, as is
the case with the bacterial acyl-CoA synthetase, because Faa2p-6xHis
has to be stored with these compounds (concentration of 2 mM)
to remain stable. Triacsin C also inhibits purified Faa4p-6xHis but is
less potent (IC
= 4.5 ± 0.5 µM; K
= 2 µM) than is the
case with Faa2p.
Figure 6:
Triacsin C is a potent competitive
inhibitor of Faa2p. A, Eadie-Hofstee plots of the inhibition
of Faa2p with varying concentrations of triacsin C using tritiated
oleic acid as the substrate. Note that with increasing concentrations
of triacsin C, the K for oleate (slope = -K
)
becomes greater while the V
(the y intercept) remains unaffected. This finding indicates that
triacsin C is competitive with respect to fatty acid. B, plot
of the concentration of triacsin C versus the K
values determined from panelA. The K
of triacsin C is
15 ± 1 nM. n = 3 independent
experiments, each done in duplicate.
The oxime derivatives of 2,4-nonadienal and
2,4-decadienal were also synthesized, yielding 1-azadeca-1,3,5-trienol
and 1-azaundeca-1,3,5-trienol, respectively (Fig. 5). They were
obtained as E-isomers. These compounds may be considered to be
bis(desaza)triazenols (i.e. they contain a domain that
resembles the triazenol functional group including two conjugated trans-double bonds (triazenol:
R-CH&cjs0808;N-N&cjs0808;N-OH;
bis(desaza)triazenol:
R-CH&cjs0808;CH-CH&cjs0808;N-OH)). The
compounds were obtained as E-isomers. 1-Azadeca-1,3,5-trienol
and 1-azaundeca-1,3,5-trienol both inhibit Faa2p (IC = 6 ± 0.5 and 16 ± 1 µM,
respectively). As with triacsin C itself, they show selectivity between
the Faaps, i.e. IC
> 1500 µM for
Faa1p (Faa1p's C18:1
K
= 62 ± 3 µM).
Activation of imported fatty acids during growth
at 24-37 °C on rich media is principally the function of
Faa1p since deletion of FAA1 but not FAA4 impairs the
ability of yeast to grow on YPD/cerulenin/fatty acid media(8) .
The impairment is temperature- and fatty acid-dependent; growth
retardation worsens with higher temperatures and when C16:0 or
C18:1 is substituted for C14:0(8) . The
combination of faa1 and faa4 null alleles produces
inviable cells on YPD/cerulenin/fatty acid media when fatty acid
= 14:0, 16:0, or C18:1
. However, no other
combination of two faa null alleles results in this lethal
phenotype, and faa2
faa4
and faa3
faa4
cells demonstrate no diminution of growth on YPD/cerulenin/fatty
acid media (8) .
These observations beg the obvious
question, ``Why is there an Faa4p?'' Faa4p does not have to
form a heterodimer with Faa1p for either enzyme to function since GPD-FAA1 or GPD-FAA4 episomes can rescue growth of faa1faa4
cells on YPD/cerulenin/fatty acid media ( Fig. 1and (8) ). The remarkable instability of purified
Faa4p raises the possibility that it may normally reside in a cellular
membrane. However, the enzyme does not appear to have a distinctive
role in fatty acid transport across the yeast's plasma membrane.
Measurement of the rate and extent of import of C14:0 or C16:0 into
isogenic FAA1,FAA4 and faa1
faa4
strains
harvested during exponential growth indicated that (i) S.
cerevisiae contains a saturable fatty acid transport system (K
for the two fatty acids
50
µM, where K
represents the
substrate concentration at which the transport rate is half its maximal
value) and (ii) fatty acid uptake is not coupled to activation by
either Faa1p or Faa4p (i.e. there is no difference in the
ability of FAA1FAA4 and faa1
faa4
strains to
take up fatty acids, nor does overexpression of Faa1p or Faa4p produce
a detectable increase in C14:0 or C16:0 import(19) ). There is
no dramatic difference in the fatty acid selectivities of the two
purified enzymes in vitro; both prefer C14:0 among C8-C18
saturated fatty acids, and both favor a cis-double bond at
C9-C10. It is likely that the physiologic function of Faa4p has not
been determined because the effects of genetic manipulations have only
been assessed under conditions that allow exponential growth (e.g. analysis of isogenic strains containing single faa null
alleles indicates that during exponential growth when Fas is active, none of the FAA genes is essential). However, there
is at least one circumstance when both FAA1 and FAA4 are needed, even when Fas is active. Wild type strains of S.
cerevisiae can grow using glycerol as the sole carbon source if
glycerol can be efficiently converted to glyceraldehyde-3-phosphate for
entry into the glycolytic pathway. This requires adequate mitochondrial
respiration so that the NADH produced by glycolysis can be removed.
Isogenic strains with single faa
alleles grow on
YP/glycerol at rates comparable to wild type cells(8) .
Analysis of cells with all possible combinations of two faa
alleles revealed that only faa1
faa4
strains
exhibit diminished growth. This diminution is temperature-dependent; at
30 °C there is mild growth retardation, while at 37 °C no
growth is observed(8) . These findings raise the possibility
that FAA1 and FAA4 influence mitochondrial function,
either directly or indirectly.
Additional studies of isogenic
strains with various combinations of faa null alleles are
clearly required, including an analysis of their viability/longevity
during stationary phase when marked changes in lipid metabolism are
known to occur(34) . Given the predilection of Faa1p and Faa4p
for C14:0, an additional parameter to monitor in stationary phase cells
with faa1 and/or faa4
is the effect of
introducing mutations in the essential myristoyl-CoA:protein N-myristoyltransferase gene (NMT1), specifically
mutations that reduce Nmt1p's affinity for its acyl-CoA substrate
and hence produce a requirement for augmentation of cellular
myristoyl-CoA pools to maintain survival (35, 36) .
The azatrienols lack two of the five double bonds present in triacsin C. Thus, the fourth conjugated double bond found in triacsins A-D (Fig. 5) is not absolutely required for its inhibitory effect on Faa2p.
It is remarkable that the azatrienols ``preserve'' the Faap selectivity found with triacsin C. It is difficult to imagine that the chemical mechanism of catalysis is different between the Faaps. Our data with the unsaturated tetradecenoic acid analogs reveal distinct differences in acyl chain recognition (e.g. Faa2p is the most sensitive to the presence of a trans-double bond at C2-C3 while being least selective for double bonds at the other positions tested (C4-C12)). Differences in the positioning of the conjugated oxime within Faa2p (and Faa4p) compared with the other Faaps may determine its ability to prevent proper positioning of a fatty acid substrate for reaction with ATP and thus prevent formation the mixed anhydride (R-CO-O-AMP). Alternatively, the conjugated oxime may itself react with ATP. In this sense, triacsin and oxime derivatives may help define differences in molecular recognition among members of this family of four yeast acyl-CoA synthetases. The >25,000-fold range in the inhibitory effects of triacsin C on the four yeast Faaps suggests that it may be possible to develop other selective inhibitors of eukaryotic acyl-CoA synthetases. These may be useful therapeutically (38) .