Download On the mechanism of action of the antifungal agent propionate

Eur. J. Biochem. 271, 3227–3241 (2004) FEBS 2004
doi:10.1111/j.1432-1033.2004.04255.x
On the mechanism of action of the antifungal agent propionate
Propionyl-CoA inhibits glucose metabolism in Aspergillus nidulans
Matthias Brock1 and Wolfgang Buckel2
1
Laboratorium für Mikrobiologie, Universität Hannover; 2Laboratorium für Mikrobiologie, Fachbereich Biologie,
Philipps-Universität Marburg, Germany
Propionate is used to protect bread and animal feed from
moulds. The mode of action of this short-chain fatty acid
was studied using Aspergillus nidulans as a model organism.
The filamentous fungus is able to grow slowly on propionate, which is oxidized to acetyl-CoA via propionyl-CoA,
methylcitrate and pyruvate. Propionate inhibits growth of
A. nidulans on glucose but not on acetate; the latter was
shown to inhibit propionate oxidation. When grown on
glucose a methylcitrate synthase deletion mutant is much
more sensitive towards the presence of propionate in the
medium as compared to the wild-type and accumulates
10-fold higher levels of propionyl-CoA, which inhibits CoAdependent enzymes such as pyruvate dehydrogenase, succinyl-CoA synthetase and ATP citrate lyase. The most
important inhibition is that of pyruvate dehydrogenase, as
this affects glucose and propionate metabolism directly. In
contrast, the blocked succinyl-CoA synthetase can be circumvented by a succinyl-CoA:acetate/propionate CoAtransferase, whereas ATP citrate lyase is required only for
biosynthetic purposes. In addition, data are presented that
correlate inhibition of fungal polyketide synthesis by propionyl-CoA with the accumulation of this CoA-derivative.
A possible toxicity of propionyl-CoA for humans in diseases
such as propionic acidaemia and methylmalonic aciduria is
also discussed.
Sodium propionate is widely used as a preservative due to its
ability to inhibit fungal growth. Furthermore, this shortchain fatty acid (pion ¼ fat) prevents the biosynthesis of
polyketides such as ochratoxin A by Aspergillus sulphureus
and Penicillium viridicatum [1]. On the other hand, many
fungi are able to grow on propionate, although much more
slowly than on glucose or acetate. Recently we have shown
that in Aspergillus nidulans propionate is oxidized to
pyruvate via the methylcitrate cycle [2,3]. Propionyl-CoA
is formed from propionate, CoASH and ATP catalysed by
acetyl-CoA synthetase, FacA [4,5], and by an additional
acyl-CoA synthetase. The condensation of propionyl-CoA
with oxaloacetate inside the mitochondria yields (2S,3S)methylcitrate [2]. Isomerization of this tricarboxylic acid,
most likely via cis-2-methylaconitate [6], yields (2R,3S)-2methylisocitrate, which is cleaved to succinate and pyruvate
[3]. Studies with 13C-labelled propionate indicated that in
Escherichia coli the 2-oxo acid is further oxidized to
acetyl-CoA, which is either funnelled into the citrate cycle
or used for biosyntheses [7].
A clue to the mechanism of propionate toxicity was
the construction of an A. nidulans methylcitrate synthase
deletion strain (DmcsA), which was unable to grow on
propionate as sole carbon and energy source. Unexpectedly, growth of DmcsA on glucose was more
inhibited by propionate than that of a wild-type strain
[2]. This result indicated that (2S,3S)-methylcitrate or
(2R,3S)-2-methylisocitrate are unlikely to be responsible
for this inhibitory effect. At high levels of propionylCoA yeast citrate synthase catalyses the slow formation
of three of the four stereoisomers of methylcitrate [8],
but their concentrations (< 10 lM) are very low and it
remains controversial whether they may be able to act
as significant inhibitors. Therefore, whether the finding
that methylcitrate might be the causative agent of
propionate toxicity in Salmonella enterica [9] is also true
for eukaryotic cells, is questionable. Nevertheless, the
identification of these isomers by GLC/MS is used for
diagnosis of disorders in human propionate metabolism
such as propionic acidaemia and methylmalonic aciduria
[10,11].
The idea that propionyl-CoA itself could be the inhibitory
agent is supported by previous work on bacterial and
mammalian metabolism. The inhibition of growth of the
bacterium Rodopseudomonas sphaeroides by propionate was
most likely caused by propionyl-CoA, which acted as an
inhibitor of pyruvate dehydrogenase, competitive with
CoASH, Ki ¼ 0.84 mM. The addition of sodium bicarbonate increased the growth rate again, probably because it
stimulated the degradation of propionyl-CoA via methylmalonyl-CoA [12]. It was also shown that accumulation of
Correspondence to W. Buckel, Laboratorium für Mikrobiologie,
Fachbereich Biologie, Philipps-Universität Marburg, D-35032 Marburg, Germany. Fax: +49 6421 2828979, Tel.: +49 6421 2821527,
E-mail: Buckel@staff.Uni-Marburg.de
Abbreviations: ABTS, 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic
acid; ACS, acetyl-CoA synthetase; DTNB, 5,5¢-dithiobis(2-nitrobenzoic acid); GOD, glucose oxidase; LDH, lactate dehydrogenase;
MDH, malate dehydrogenase; POD, peroxidase.
(Received 22 April 2004, revised 4 June 2004, accepted 11 June 2004)
Keywords: acetate CoA-transferase; succinyl-CoA; polyketide synthesis; pyruvate dehydrogenase; pyruvate excretion.
FEBS 2004
3228 M. Brock and W. Buckel (Eur. J. Biochem. 271)
propionyl-CoA in rat liver hepatocytes led to a decrease in
the activity of pyruvate dehydrogenase [13].
In this investigation we examined carbon balances under
different growth conditions. We found that growth of
A. nidulans on glucose + propionate, especially of the
DmcsA strain, led to the excretion of pyruvate and to high
intracellular concentrations of propionyl-CoA, which inhibited pyruvate dehydrogenase, succinyl-CoA synthetase
(GDP forming) and ATP-citrate lyase. We conclude that
these observations can explain the toxicity of propionate
towards cells growing on glucose as sole carbon and energy
source. Furthermore, we were able to show a correlation
between inhibition of polyketide formation and intracellular
propionyl-CoA content.
Experimental procedures
Materials
Chemicals were from Sigma-Aldrich. Enzymes used for
determination of acetate, glucose and pyruvate were from
Roche. Columns and chromatographic media were, if not
otherwise indicated, from Amersham Pharmacia Biotech.
A. nidulans strains, growth conditions and carbon
balances
The A. nidulans strains used in this study are listed in
Table 1. Supplemented minimal and complete media
were prepared as described previously [14]. For the determination of specific enzyme activities on different carbon
sources, growth times were strain and medium specific.
Approximately 108 spores were used for inoculation of
100 mL medium and incubation was carried out in 250-mL
flasks at 37 C and 240 r.p.m. on a rotary shaker. On
media containing 50 mM glucose as sole carbon source
and 50 mM glucose + 100 mM acetate, all strains were
incubated for 20 h; on 50 mM glucose + 100 mM acetate + 100 mM propionate, all strains were incubated for
23 h; on 50 mM glucose + 100 mM propionate the strains
were incubated for 44 h, except strain SMB/acuA, which
showed much less inhibition in the presence of propionate
and was grown on this medium for 22 h. The presence of
residual glucose in the medium (> 20 mM) was determined
enzymatically. On 100 mM acetate and 100 mM acetate + 100 mM propionate all strains, with the exception
of strain SMB/acuA, were grown for 36 and 41 h,
respectively. To determine enzyme activities during growth
on 100 mM propionate, we added 10 mM glucose to the
medium to support initial growth. After total consumption
of glucose cells were grown further for at least 12 h.
Therefore, the wild-type strain was grown for 42 h, whereas
the methylcitrate synthase deletion strain and the facB
multi-copy strain were incubated for 94 h. Strain SMB/
acuA was always grown in the presence of glucose, because
the strain did not grow on acetate and growth on
acetate/propionate was very poor. Therefore, we used
the following composition of media and growth times:
10 mM glucose + 100 mM acetate harvest after 27 h;
10 mM glucose + 100 mM propionate harvest after 29 h;
10 mM glucose + 100 mM acetate + 100 mM propionate
harvest after 29 h. Determination of the residual glucose
concentration confirmed that the strains were incubated for
at least 12 h after total consumption of glucose. In addition,
we proved that acetate was still present under all conditions
where it was used as a carbon source. Growth at all
conditions and with all strains was replicated twice in order
to confirm the results.
For the determination of CO2 production, A. nidulans was
grown at 37 C in a 1-L gas wash bottle containing 600 mL
medium (Schott, Mainz, Germany). The medium was stirred
at 350 r.p.m and bubbled with CO2-free air. The CO2 was
removed by washing the air with 2 M NaOH followed by
sterile water to avoid the transfer of NaOH to the growth
medium. The CO2 produced was trapped in a fourth wash
bottle containing 400 mL 0.2 M Ba(OH)2. The insoluble
BaCO3 that formed was dried at 60 C for 20 h and weighed.
Residual glucose and acetate contents in the growth medium
were determined by enzymatic methods (see below). The
mycelium was pressed to remove any liquid, frozen with
liquid nitrogen, lyophilized, weighed, and ground to a fine
powder. The CHN content of the mycelium was determined
by elemental analysis (Zentrale Routineanalytik, PhilippsUniversität Marburg, Lahnberge, Germany). Results from
Table 1. A. nidulans strains used in this study. Strain RYQ11 was used throughout all experiments. Strain SDmcsA1 was used in a previous work
was taken as a control to confirm the results of spore colour formation, enzyme activities and carbon consumption.
Strain
Genotype
Source
SMB/acuA
MH2671
Fab4-J3
A637
A634
A627
A26
SMI45
SRF200
RYQ11a
SDmcsA1a
SMB/B1
facA303, yA2; veA1
pabaA1; prn-309, cnxJ1
MH2671 cotransformed with pFAB4 and pAN222 (approx. 4–8 copies facB)
yA2, pabaA1, pdhA1
yA2, pabaA1; pdhB4
yA2, pabaA1; pdhC1
biA1; veA1
yA2, pabaA1; wA3; veA1
pyrG89; DargB::trpCDB; pyroA4; veA1
DmcsA::argB, biA1; veA1
DmcsA::argB, pyrG89; DargB::trpCDB; pyroA4; veA1
pyrG89; DargB::trpCDB; pyroA4; veA1 (alcA::mcsA, argB)
[2]
[46]
[46]
FGSC, Kansas City, KS, USA
FGSC, Kansas City, KS, USA
FGSC, Kansas City, KS, USA
FGSC, Kansas City, KS, USA
M. Krüger, Marburg, Germany
[47]
N. Keller, UW-Madison, USA
[2]
[2]
a
Two different methylcitrate synthase mutants (DmcsA). FGSC, Fungal genetics stock center (http://www.fgsc.net).
FEBS 2004
Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3229
three independent samples were (%): N, 6.4 ± 0.1;
C, 47.2 ± 0.3; H, 8.2 ± 0.1. Thus 1 g dried mycelium
consists of 472 mg carbon equivalent to 39.3 mmol.
Sample preparation of intracellular acyl-CoA from
lyophilized mycelium
The dried mycelium was ground to a fine powder in a
mortar and suspended in 10 mL 2% HClO4 and 1 mL
0.1% trifluoroacetic acid. The suspension was sonicated
three times for 4 min each at 70% full power and 60%
pulses (Branson 250 sonifier; Branson, Dietzenbach, Germany) and neutralized to pH 4–5 by drop-wise addition of
2 M K2CO3. After incubation on ice for 15 min most of the
perchloric acid was precipitated as insoluble KClO4. The
solution was centrifuged at 120 000 g for 25 min and
the supernatant was collected. For concentration and
partial purification of the CoA-thioesters, the supernatant
was applied on a C18-cartridge (Chromafix C18 ec,
510 mg; Macherey-Nagel, Düren, Germany), previously
rinsed with methanol and washed with 0.1% trifluoroacetic
acid. The supernatant was slowly applied to the column and
washed with 10 mL 0.1% trifluoroacetic acid. Elution was
carried out with 1.5 mL 50% acetonitrile/0.1% trifluoroacetic acid and samples were collected in 2-mL micro
centrifuge cups. The acetonitrile was evaporated in a Speed
Vac Concentrator (Bachofer GmbH, Reutlingen, Germany)
without heating and the residual volume of 200–500 lL was
measured with an accuracy of ± 2 lL using a micropipette.
An aliquot of the samples was used for the enzymatic
determination of acetyl-CoA and propionyl-CoA concentrations.
Determination of the intracellular volume
Wet weight was determined after pressing the mycelium
between several sheets of absorbent paper until no further
liquid could be removed. Mycelium was dried for at least
20 h at 60 C and weighed again; thereby 3.51 g wet cells
yielded 1.0 g dry cells, the mean value of 20 independent
samples.
Partial purification of ATP-citrate lyase and succinyl-CoA
synthetase from A. nidulans
A. nidulans strain SMB/acuA [2] was grown for 20 h on
glucose minimal medium. Mycelium was harvested over a
Miracloth filter membrane (Calbiochem). The mycelium
was dry-pressed for removal of residual medium and
suspended in 50 mM Tris/HCl pH 8.0 containing 2 mM
dithiothreitol (buffer A). The mycelium was homogenized
by an Ultra Turrax (T25 basic, IKA Labortechnik, Staufen,
Germany). Cells were broken by ultrasonication three times
for 4 min at 80% full power and 60% pulses (Branson 250
sonifier). The extract was centrifuged at 96 000 g and the
supernatant was applied to a Q-Sepharose column (Pharmacia Biotech, bed volume 25 mL), previously equilibrated
with buffer A. The enzyme was eluted in buffer A with a
0–1 M NaCl gradient. Enzyme-containing fractions were
checked for activity, collected and concentrated in an
Amicon chamber over a PM 30 membrane (Millipore,
Eschborn, Germany). Purity was sufficient for inhibition
studies. Succinyl-CoA synthetase was partially purified as
described above, except that buffer A did not contain
dithiothreitol. No further column purification was necessary
for the described activity measurements.
Enzymatic determination of glucose, acetate and
pyruvate in the growth medium
Glucose concentrations were determined by the combined
action of glucose oxidase (GOD, from A. niger), peroxidase
(POD, from horseradish) and 2,2¢-azinobis(3-ethylbenzo-6thiazolinesulfonic acid). The test was a modification of a
described procedure [15]. The composition of the test
reagent was: 130 mM sodium phosphate, pH 7.0; 400 U
POD (2 mg; 200 UÆmg)1), 800 U GOD (4 mg; 200 UÆmg)1)
and 25 mg 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic
acid), final volume 50 mL. Each assay, which contained
900 lL reagent and 100 lL sample, was incubated for
15 min at 37 C and measured at 436 nm in a spectrophotometer. The assay was linear in a range of 0–30 lM glucose.
A standard was run for every freshly prepared reagent.
Pyruvate concentrations were determined by the use of
lactate dehydrogenase (LDH) from rabbit muscle. The
oxidation of NADH was followed at 340 nm until no
further change in absorbance was visible; e340 ¼
6.3 mM)1Æcm)1 [16]. The assay contained, in a final volume
of 1 mL, 50 mM potassium phosphate pH 7.0, 0.2 mM
NADH, 0.5 U LDH and 50–100 lL different dilutions of
the medium.
Acetate concentrations were determined with citrate
synthase and malate dehydrogenase [17]. Acetate was
activated by an acetyl-CoA synthetase (ACS) from Saccharomyces cerevisiae (Roche) and the resulting acetyl-CoA
was condensed with oxaloacetate by the use of citrate
synthase from pig heart. Oxaloacetate was continuously
provided from malate by use of NAD+ and malate
dehydrogenase (MDH) from pig heart. A typical assay in
a final volume of 1 mL contained (mM) 50 potassium
phosphate, pH 7.0; 10 L-malate, 0.2 CoASH, 2 NAD+,
2 ATP, 4 MgCl2, 0.5 dithiothreitol, 0.5 U MDH, 0.5 U
citrate synthase, 0.1 U ACS and 50–100 lL diluted
medium. All components were added with the exception
of MDH and citrate synthase and the resulting absorbance
at 340 nm was measured (A1). MDH was added and the
absorbance after reaching the equilibrium was taken as A2.
Citrate synthase was added and the reaction was monitored
until no further change in absorbance was visible (A3).
Concentrations were calculated by the formula below
[e, absorbance (extinction) coefficient; d, length of light
path of the cuvette], which considers the decrease of the
concentration of oxaloacetate in equilibrium with L-malate
during the formation of NADH (the concentrations of
malate and NAD+ remain almost constant):
A3 A2
A2 A1
[Acetate] ¼
1þ
ed
A3 A1
Determination of intracellular propionyl-CoA and
acetyl-CoA
Concentrations of acyl-CoA were determined by the use of
citrate synthase from pig heart and purified methylcitrate
3230 M. Brock and W. Buckel (Eur. J. Biochem. 271)
synthase from the overproducing A. nidulans strain SMB/
B1 [2] by two independent methods. One method was
performed as described above for the determination of the
concentration of acetate from the growth medium. A 1-mL
assay contained 50 mM potassium phosphate, pH 7.0;
10 mM L-malate, 2 mM NAD, 0.5 U MDH, 0.5 U citrate
synthase, 0.5 U methylcitrate synthase and 50–100 lL
sample. The concentration of acetyl-CoA was determined
first by the use of citrate synthase. The reaction was
followed at 340 nm until no further change in absorbance
was detected. Methylcitrate synthase was added and the
second change in absorbance was monitored.
The second method was based on the formation of a
nitrothiophenolate (2-mercapto-5-nitrobenzoate dianion)
during the reaction of 5,5¢-dithiobis-(2-nitrobenzoate)
(DTNB) with CoASH, which was released during the
condensation of oxaloacetate with acetyl-CoA or propionyl-CoA. The assay contained, in a final volume of 1 mL,
50 mM Tris/HCl, pH 8.0; 1 mM oxaloacetate, 1 mM DTNB,
0.5 U citrate synthase, 0.5 U methylcitrate synthase and
20–100 lL sample. Change in absorbance was monitored at
412 nm; e ¼ 14.2 mM)1Æcm)1 [18,19]. Acetyl-CoA concentrations were determined first. When no further change in
absorbance was visible, methylcitrate synthase was added.
Enzyme assays
ATP citrate lyase. The assay [20] contained (mM)
50 Tris/HCl, pH 8.0; 0.2 NADH, 5 ATP, 0.34 CoASH,
20 citrate, 2 dithiothreitol, 2 MgCl2, 0.5 U MDH from
pig heart, enzyme sample and water to a final volume of
1 mL. The reaction was started by addition of enzyme
sample and decrease in absorbance at 340 nm was
monitored. One unit of enzyme activity was defined as
the amount of enzyme reducing 1 lmol NADÆmin)1
under the assay conditions.
Succinyl-CoA synthetase was measured by a modified
method for the determination of citrate synthase activity
[21]. A typical assay contained 50 mM Tris/HCl, pH 7.5;
0.14 mM succinyl-CoA, 1 mM DTNB, 0.5 mM GDP, 2 mM
MgCl2, 5 mM potassium phosphate, enzyme sample and
water to a final volume of 1 mL. One unit of enzyme activity
was defined as the amount of enzyme producing 1 lmol
CoASHÆmin)1 under the assay conditions.
Isocitrate lyase. The assay [22] contained (mM) 50 potassium phosphate, pH 7.0; 1 threo-isocitrate, 10 phenylhydrazine HCl, 2 dithiothreitol, 2 MgCl2, 10–100 lL enzyme
sample and water to a final volume of 1 mL. The formation
of glyoxylate phenylhydrazone was followed at 324 nm;
e ¼ 16.8 mM)1Æcm)1. One unit of enzyme activity was
defined as the amount of enzyme producing 1 lmol
glyoxylate phenylhydrazoneÆmin)1 under the assay conditions.
2-Methylisocitrate lyase. The assay was based on the
reduction of pyruvate with NADH catalysed by LDH,
whereby the decrease in absorbance at 340 nm was recorded
[3]. The composition of the reaction was (mM) 0.20 threo2-methylisocitrate, 2 MgCl2, 2 dithiothreitol, 0.2 NADH,
1.5 U LDH, 50 potassium phosphate, pH 7.0; enzyme
FEBS 2004
sample and water to a final volume of 1 mL. One unit of
enzyme activity was defined as the amount of enzyme
producing 1 lmol NADHÆmin)1 under the assay conditions.
Citrate synthase and methylcitrate synthase. Citrate
synthase and methylcitrate synthase activity was determined
as described previously [2]. The reaction mixture contained
(in mM), in a final volume of 1 mL, 50 Tris/HCl, pH 8.0;
1.0 5,5¢-dithiobis-(2-nitrobenzoic acid), cell-free extract and
0.2 acetyl-CoA or propionyl-CoA, respectively. The assay
was started by the addition on 1 mM oxaloacetate (final
concentration) and monitored at 412 nm. One unit of
enzyme activity was defined as the amount of enzyme
producing 1 lmol CoASHÆmin)1 under the assay conditions.
Pyruvate dehydrogenase. Pyruvate dehydrogenase (PDH)
activity was measured according to a procedure described
previously [23] with some modifications. The assay contained (in mM), in a final volume of 1 mL, 50 Tris/HCl,
pH 8.0; 2 pyruvate, 0.8 thiamine pyrophosphate, 2.5 cysteine/HCl, 2 NAD, 2 MgCl2, cell-free extract and water to a
final volume of 990 lL. The reaction was started by the
addition of 0.02–0.17 mM CoASH and reduction of NAD+
to NADH was followed at 340 nm. One unit of enzyme
activity was defined as the amount of enzyme producing
1 lmol NADHÆmin)1 under the assay conditions. The
activity of 2-oxoglutarate dehydrogenase was determined by
the analogous procedure, in which pyruvate was replaced by
2-oxoglutarate [24].
Acetyl-CoA synthetase. Acetyl-CoA synthetase activity
was determined in a coupled assay by the use of MDH
and citrate synthase. In this method the acetyl-CoA
produced reacts via citrate synthase with oxaloacetate,
which is provided by MDH from malate. The assay
contained (in mM), in a final volume of 1 mL, 50 potassium
phosphate buffer, pH 7.0; 10 sodium acetate, 2 NAD,
20 D,L-malate, 0.4 CoASH, 2 dithiothreitol, 4 MgCl2, 6 U
MDH (pig heart, Roche), 2 U citrate synthase (pig heart,
Roche), cell-free extract and water to a final volume of
980 lL. The reaction was started by the addition of 20 lL
of a 100 mM ATP solution (final concentration 2 mM) and
the reduction of NAD was monitored at 340 nm. The
extincition coefficient was set as 0.5 · 6.3 mM)1Æcm)1,
which compensates for the initial decrease of the oxaloacetate concentration in the equilibrium due to the
accumulation of NADH [25]. Lineweaver–Burk diagrams
were obtained by use of the worksheet of the program EXEL
98 (Microsoft Inc.).
Propionyl-CoA synthetase. Propionyl-CoA synthetase
activity was determined by the same method as described
for the determination of acetyl-CoA synthetase activity,
except sodium acetate was replaced by sodium propionate
and citrate synthase by methylcitrate synthase (0.8 U) from
A. nidulans [2].
CoA-Transferase. CoA-Transferase activity was determined by using succinyl-CoA or propionyl-CoA as the
CoA-donor and acetate or propionate as the acceptor.
FEBS 2004
Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3231
When acetate was the acceptor the assay was monitored by
the use of citrate synthase, which released CoASH upon the
condensation of newly generated acetyl-CoA with oxaloacetate as described for the determination of citrate synthase
activity. When propionate was used as the acceptor, purified
methylcitrate synthase was used to measure the CoASH
release upon the condensation of propionyl-CoA with
oxaloacetate as described for the determination of methylcitrate synthase activity. A typical assay contained (in mM),
in a final volume of 1 mL, 50 Mops, pH 7.5; 0.4 CoAdonor (succinyl-CoA or propionyl-CoA, respectively), 2 U
citrate synthase or 0.8 U methylcitrate synthase, respectively, 1 oxaloacetate and 10 CoA-acceptor (acetate or
propionate, respectively) and cell-free extract.
Oxidative branch of the pentose phosphate pathway. This
was determined by the use of glucose-6-phosphate as the
substrate and NADP as the hydrogen acceptor. Due to the
use of cell-free extracts, not only the activity of glucose6-phosphate dehydrogenase but also the activity of the
6-phosphogluconate dehydrogenase was measured. The
described method was slightly modified [26]. A typical assay
in a final volume of 1 mL contained (in mM) 50 Mops,
pH 7.5; 1 glucose-6-phosphate, 1 NADP, 5 EDTA and
cell-free extract. The reaction was monitored at 340 nm and
specific activity was defined as the reduction of 2 lmol
NADPÆmin)1Æmg protein)1.
Determination of maintenance
In order to calculate the amount of glucose used for
maintenance, the wild-type strain A26 was used. Four
100-mL aliquots of glucose minimal media in 250-mL flasks
were inoculated with 4 · 108 spores and incubated for 13 h
at 37 C and 240 r.p.m. Two of the samples were harvested
and dried at 70 C to measure biomass formation as a
control. The other two samples were washed with sterile
0.6 M KCl and transferred to fresh glucose minimal medium
containing cycloheximide (200 lgÆmL)1), which inhibits
eucaryotic protein biosynthesis. The cultures were incubated
for further 9 h at 37 C and 240 r.p.m. The mycelium was
dried and the biomass was compared to that of control
samples. Glucose concentrations before and after the
incubation with cycloheximide were measured as described
above.
Results
Carbon balances on different growth media
Initial experiments showed that growth on glucose + propionate resulted in significant excretions of pyruvate into the
medium (Table 2). In order to exclude substantial excretions
of other carbon compounds, we measured the total carbon
balances of wild-type and methylcitrate synthase deletion
strain (DmcsA). Therefore, the consumption of substrates,
formation of CO2, as well as excretion of pyruvate and the
final pH were determined in media in which cells had been
grown on different carbon sources. The measured carbon
balances add up to almost 100% (Table 3) indicating that
there was no substantial excretion of compounds other than
CO2 and pyruvate or a significant consumption of propionate. The increase in the final pH (Table 2) correlated
with the consumption of the carboxylates, by which protons
are removed from the medium, whereas by oxidation of
glucose no change in pH was observed. When grown only
on glucose there was no significant difference between the
wild-type and the DmcsA strain. In the presence of only
acetate there was no difference between the strains; the
approximate growth rate was only 50% of that with glucose
and the increase in pH from 6.4 to 8.2 correlated with the
high consumption of acetate. Growth on propionate alone
was not included in this study, as the growth rate of the
wild-type was extremely low and the morphology of the
mycelium was quite different. Furthermore, on propionate
the DmcsA strain did not grow at all.
Table 2. Carbon consumption and pyruvate excretion of wild-type and DmcsA strain under different growth conditions. The wild-type strain was
SMI45 and the initial pH was 6.3–6.5. Consumption and excretion are data are given in mmol substrateÆg dried mycelium)1. In all experiments the
concentration of glucose was 50 mM and that of sodium propionate100 mM. The concentration of sodium acetate was 50 mM except when used in
combination with propionate in which case 100 mM was used. Mycelia were harvested in the linear growth phase. DmcsA, methylcitrate synthase
deletion mutant (RYQ11 and SDmcsA1). For experiments marked by an asterisk see also Table 3.
Strain
C-Source
(final pH at harvest
of mycelium)
Glucose consumption
(mmol/g)
Acetate consumption
(mmol/g)
Pyruvate excretion
(mmol/g)
Growth time
(h)
Wild-type *
DmcsA *
Wild-type
DmcsA
Wild-type *
DmcsA *
Wild-type
DmcsA
Wild-type
DmcsA
Wild-type
DmcsA
Glucose (6.6)
Glucose (6.7)
Glucose/acetate (7.3)
Glucose/acetate (7.9)
Glucose/propionate (6.8)
Glucose/propionate (6.3)
Glucose/propionate/acetate (7.5)
Glucose/propionate/acetate (7.4)
Acetate/propionate (8.0)
Acetate/propionate (8.5)
Acetate (8.2)
Acetate (8.2)
10.6
9.9
10.0
8.0
14.6
16.2
7.0
11.0
–
–
–
–
–
–
1.0
9.0
–
–
24
19
49
62
54
55
0.140
0.054
0.070
0.087
1.27
2.21
0.37
0.50
0.144
0.035
< 0.01
< 0.01
20
20
22
22
44
72
30
30
47
47
40
40
FEBS 2004
3232 M. Brock and W. Buckel (Eur. J. Biochem. 271)
Table 3. Carbon balances of wild-type and DmcsA strain. Balances are calculated for 1 g of dried mycelium. The concentrations of the substrates are
indicated in Table 2 (marked by asterisks). The wild-type strain was SMI45 and DmcsA strains were RYQ11 and SDmcsA1.
Strain/C-source
Glucose
consumed
(mmol C)
Pyruvate
(mmol C)
CO2
recovered
(mmol C)
Biomass
(mmol C)
Total amount
recovered
[mmol C (%)]
Wild-type/glucose
DmcsA/glucose
Wild-type/glucose + propionate
DmcsA/glucose + propionate
64
60
88
97
0
0
3
6
21
20
40
49
39
39
39
39
60
59
82
94
±
±
±
±
4
4
4
4
Addition of acetate to a medium containing glucose did
not change the growth rate significantly, but the lack of
methylcitrate synthase in the mutant strain induced acetate
consumption (Table 2). This observation is similar to strain
Fab4-J3, which carries multiple copies of the transcriptional
activator FacB of the acetate utilization genes. FacB is
induced by acetate and acetylcarnitine [27]. Growth experiments with strain Fab4-J3 revealed that in the presence of
both glucose and acetate, the latter substrate is mainly used.
Thus cells grown on 50 mM glucose + 100 mM acetate
consumed only 2.7 mmol glucose but 44.2 mmol acetateÆ
g dried cells)1. That means that the higher basal level of the
transcriptional activator FacB in a strain, which carries
multiple integrations of the facB-gene in the genome, leads
to preferred use of acetate as carbon source.
From our results we can conclude that propionate or an
intermediary metabolite, most likely propionyl-CoA, is able
to induce genes from propionate as well as from acetate
metabolism (Table 4, see Icl, Micl and McsA). Therefore, in
the DmcsA strain, accumulation of propionyl-CoA, derived
from amino acid degradation, can cause the higher
consumption of acetate as compared to the wild-type.
A dramatic effect on the growth rate was observed when
propionate was added to the glucose medium; the growth
time doubled with the wild-type and increased 3.6· with the
DmcsA mutant. In both strains propionate caused an
increase in glucose consumption and a huge enhancement of
pyruvate excretion. The carbon balance excluded a significant excretion of other substances such as alanine [28],
which may have escaped our analytical tools. Furthermore
we found that the observed additional amount of consumed
glucose was almost completely oxidized to CO2 (Table 3).
Probably the increase in CO2 production caused by
propionate (doubled with the wild-type and tripled with
the mutant) was due to energy production required for
maintenance (see below) during the extended growth times.
Upon addition of acetate to the media containing glucose
and propionate, the growth rate of both strains increased
and the effect of propionate became less apparent. Finally,
in media containing acetate and propionate but no glucose,
there was only a small delay (30%) in growth of the mutant
as compared to the wild-type [2]. The higher acetate
consumption of the mutant strain was probably due to
higher maintenance requirement (see below) or to the action
of a CoA-transferase, which is induced by propionate and
seems to transfer the CoA-moiety from succinyl-CoA
preferentially to acetate (see below and Table 5).
The observed excretion of pyruvate prompted us to check
strains, in each of which another of the three genes encoding
pyruvate dehydrogenase [29] was mutated (A637, pdhA1-
±
±
±
±
4
4
2
4
±
±
±
±
4
4
2
4
(94)
(98)
(93)
(97)
mutant ¼ lipoate acetyltransferase; A634, pdhB4 ¼ b-subunit of pyruvate decarboxylase; A627, pdhC1 ¼ a-subunit
of pyruvate decarboxylase). All three strains were unable to
grow on glucose or propionate, but grew well on acetate.
Growth of strain A627 on 50 mM acetate yielded 239 mg
dried mycelium after 23 h (59 mmol acetateÆg mycelium)1).
Interestingly, growth of this mutant was enhanced rather
than inhibited by the addition of 50 mM glucose, which led
to the production of 313 mg mycelium in 23 h, whereby
26 mmol acetate and 4 mmol glucose were consumed and
0.9 mmol pyruvate were excreted. This can be explained by
the fact that production of cell mass from glucose requires
less ATP than from acetate, because the energy consuming
gluconeogenesis via the glyoxylate cycle is not necessary. On
the other hand consumption of acetate together with
glucose was not expected, since CreA regulation should
prohibit such a cometabolism. In the presence of glucose the
wide-domain regulatory protein CreA forms a complex with
target DNA binding sites and leads to a reduced transcription of genes coding for degradation of alternative carbon
sources [30]. However, we cannot exclude the spontaneous
formation of creA mutants, which derive from our cultivation conditions. This event would lead to a relieved carbon
catabolite repression as also shown for other glyoxylate
cycle mutants [5].
Determination of maintenance
Maintenance is the energy that is used for survival of cells
without any biomass formation. Determination of maintenance was based on the inhibition of protein biosynthesis by the action of cycloheximide. Cycloheximide
binds to the 80S-subunit of eukaryotic ribosomes and
prevents the initiation and elongation reaction of protein
biosynthesis. The mycelium of pregrown cultures was
washed and transferred to fresh medium containing
cycloheximide (200 lgÆmL)1), which was sufficient to
prevent biomass formation. Cultures were incubated for
8 h and dry mass as well as glucose consumption was
determined. In this experiment significant glucose consumption was observed (8.75 ± 0.1 mmolÆh)1Æg dried
cells)1). We conclude that indeed the prolonged growth
time of both the wild-type and DmcsA strains on glucose/
propionate medium led to the increased consumption of
glucose as determined.
Intracellular acetyl-CoA and propionyl-CoA contents
To investigate whether propionyl-CoA accumulates in the
methylcitrate synthase deletion strain during growth on
FEBS 2004
Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3233
Table 4. Specific enzyme activities from cell-free extracts of different strains and growth conditions. Data are given in mUÆmg protein)1. Acs, acetylCoA synthetase; Pcs, propionyl-CoA synthetase; Icl, isocitrate lyase; Micl, 2-methylisocitrate lyase; McsA, methylcitrate synthase. C-sources: G,
glucose; A, acetate; P, propionate. Numbers denote the concentrations of C-sources (mM); G50/A100/P100 ¼ 50 mM glucose + 100 mM
acetate + 100 mM propionate.
Enzyme
C-Source in medium
Wild-type (A26)
Fab4-J3
DmcsA
Acs
Acs
Acs
Acs
Acs
Acs
Acs
Pcs
Pcs
Pcs
Pcs
Pcs
Pcs
Pcs
Icl
Icl
Icl
Icl
Icl
Icl
Icl
Micl
Micl
Micl
Micl
Micl
Micl
Micl
McsA
McsA
McsA
McsA
McsA
McsA
McsA
G50
G50/A100
G50/P100
G50/A100/P100
A100
G10/P100
A100/P100
G50
G50/A100
G50/P100
G50/A100/P100
A100
G10/P100
A100/P100
G50
G50/A100
G50/P100
G50/A100/P100
A100
G10/P100
A100/P100
G50
G50/A100
G50/P100
G50/A100/P100
A100
G10/P100
A100/P100
G50
G50/A100
G50/P100
G50/A100/P100
A100
G10/P100
A100/P100
19
47
22
59
153
133
135
10
16
10
26
58
77
59
0.2
23
35
85
86
130
161
7
10
30
26
26
74
35
1
5
55
37
38
147
35
19
119
54
137
205
128
289
9
50
21
38
67
63
90
0.1
108
62
170
225
107
287
6
12
31
27
20
28
36
2
14
52
38
20
72
83
16
26
24
27
124
150
167
8
13
10
13
42
76
74
0.2
14
41
34
63
294
180
6
9
62
29
29
132
46
0
0
0
0
0
0
0
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
3
4
2
1
5
4
10
1
2
1
2
1
2
1
0.1
1
2
3
5
5
1
1
1
2
1
1
5
1
0
1
2
1
2
6
1
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
2
10
2
10
10
10
15
1
5
1
4
3
3
2
0.1
4
9
5
5
7
15
2
1
2
1
1
2
2
1
2
2
1
1
3
1
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
SMB/acuA
3
4
2
3
2
10
10
1
2
1
1
2
6
1
0.1
2
1
3
3
10
10
2
2
4
2
2
1
1
0.5
1
2.3
2
17
22
18
1
2.3
6
3.5
29
31
30
0.6
7
24
26
71
67
82
4
11
44
33
24
64
63
1
7
57
41
42
153
133
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
±
0.2
0.2
0.2
0.3
1
2
2
0.5
0.2
0.4
0.5
2
1
1
0.2
1
2
1
4
5
5
1
1
1
1
1
1
3
0
1
4
1
3
5
6
Table 5. CoA-transferase activity from wild-type and DmcsA grown on different carbon sources. Data are given in mUÆmg protein)1; 1 U is defined as
the release of 1 lmol CoASHÆmin)1 under the assay conditions. The wild-type strain was A26 and DmcsA was RYQ11. Succinyl-CoA > acetate,
succinyl-CoA:acetate CoA-transferase; Succinyl-CoA > propionate, succinyl-CoA:propionate CoA-transferase; Propionyl-CoA > acetate,
propionyl-CoA:acetate CoA-transferase.
Medium used for growth
CoA-donor > acceptor
Glucose
Glucose/
acetate
Glucose/
propionate
Glucose/acetate/
propionate
Acetate
Propionate
Succinyl-CoA > acetate (WT)
Succinyl-CoA > acetate (DmcsA)
Succinyl-CoA > propionate (WT)
Succinyl-CoA > propionate (DmcsA)
Propionyl-CoA > acetate (WT)
Propionyl-CoA> acetate (DmcsA)
5.8
12.7
2.4
5.1
< 0.5
0.9
38
41
12.4
14.7
4.4
5.8
78
115
24
46
9.6
11.7
49
63
15
16
5.6
7.1
86
109
32
28
9.6
8.7
65
143
25
54
5.3
13.3
FEBS 2004
3234 M. Brock and W. Buckel (Eur. J. Biochem. 271)
different carbon sources, mycelium was harvested, directly
frozen in liquid nitrogen and lyophilized. After opening
the cells by sonication in the presence of perchloric acid,
CoA-thioesters were partially purified and determined
enzymatically as described in Experimental procedures.
The suitability of this method was checked by mixing
16.5 nmol acetyl-CoA and 16.1 nmol of propionyl-CoA
and performing the identical procedure as for the partial
purification of the acyl-CoA ester from lyophilized
mycelium, including addition of perchloric acid, neutralization, centrifugation, C18-cartridge and concentration.
The recovery was 15.1 nmol (91.5%) acetyl-CoA and
14.4 nmol (89.5%) propionyl-CoA which showed that the
method gave reliable results. Therefore we can conclude
that the ratio between acetyl-CoA and propionyl-CoA
remained constant during the procedure and the total
yield was about 90% assuming that all cells were opened
by the procedure described above.
After 20 h of growth on glucose as the sole carbon
source, neither the wild-type nor the methylcitrate
synthase deletion strain showed significant accumulation
of propionyl-CoA (Fig. 1). Addition of propionate to the
glucose medium led to an increase of the propionyl-CoA
level in the wild-type strain. The methylcitrate synthase
deletion strain showed an up to tenfold higher accumulation of propionyl-CoA under these conditions, as the
thioester cannot be oxidized further. Addition of acetate
to the glucose/propionate medium reduced the propionylCoA level of the cells, whereas an increase was observed
again after growth on acetate + propionate without
glucose. Despite this high level of propionyl-CoA, which
was most probably due to an unspecific action of acetylCoA synthetase (described below), only a slight growth
inhibition was visible [2] and Table 2. Remarkably, under
the different growth conditions the intracellular acetylCoA concentrations were kept constant in a relatively
narrow range (20–60 nmolÆg)1 dried cells), even in the
mutant strain.
Determination of the intracellular volume
In order to obtain the intracellular concentration of
accumulated acyl-CoA esters, it was necessary to know
the internal volume in relation to the mass of dried
mycelium. The easiest way to calculate this volume was to
measure the water content from the difference between the
mass of wet and dry A. nidulans cells. Thus the internal
volume was determined to be 2.51 ± 0.13 mlÆg dry cells)1,
which is in good agreement with that of Neurospora crassa
(2.54 mLÆg dried cells)1) [31]. Investigations on the intracellular concentrations of different metabolites of A. niger
considered only the free intracellular water not bound to
proteins, rather than the total water content, which was also
similar to that of N. crassa. This content of free water was
determined as 1.20 mLÆg dried mycelium)1 by the use of
xylitol and showed that 50% of the intracellular water is
not available as a solvent for metabolites [32]. We therefore
used this latter value for the calculation of the internal
propionyl-CoA concentration of the methylcitrate synthase
mutant and the wild-type after growth on 50 mM glucose + 100 mM propionate. Thus the DmcsA strain accumulated 0.21 mM propionyl-CoA, whereas in the wild-type
strain only 0.03 mM propionyl-CoA could be found.
Nevertheless, concentrations given here are just a simple
mathematical calculation. Due to the very high concentration of macromolecules within the cell, accompanied by
high viscosity, local concentrations may differ from that
shown here. In addition, propionyl-CoA is supposed to be
generated in the cytoplasm. For transport to the mitochondria a conversion into a carnitine-ester and a backconversion to the CoA-ester inside the mitochondria has
to be involved, which is most likely performed by cytoplasmic and mitochondrial acyl-carnitine transferases (AcuJ [33]
and FacC [27]). The transporter involved in that process is
most likely AcuH [34]. Mutants of the corresponding genes
were unable to grow on propionate as sole carbon and
energy source (data not shown). This transport mechanism
Fig. 1. Intracellular contents of acetyl-CoA
and propionyl-CoA from A. nidulans wild-type
and DmcsA strain grown under different conditions. Carbon and energy sources were: 50 mM
glucose; 50 mM glucose and 100 mM sodium
propionate; 50 mM glucose, 100 mM sodium
acetate, and 100 mM sodium propionate;
100 mM sodium acetate and 100 mM sodium
propionate. The CoA-thioesters were released
from the cells and determined as described in
Experimental procedures.
FEBS 2004
Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3235
implies a higher concentration of propionyl-CoA within the
mitochondria. However, the fact that propionyl-CoA
cannot be converted in a methylcitrate synthase deletion
strain would lead to the formation of an equilibrium
between propionyl-CoA and propionyl-carnitine in mitochondria and cytoplasm. Since the equilibrium constant
between these two propionate esters is close to 1.0, we
assume for our calculations that the concentration of
propionyl-CoA is similar in all compartments.
Formation of acetyl-CoA and propionyl-CoA
For the determination of the substrate specificity of acetylCoA synthetase and a putative propionyl-CoA synthetase
we used the acetate-grown strain Fab4-J3 and glucose/
propionate grown SMB/acuA cells (10 mM glucose/100 mM
propionate; 29 h). The high expression of the acetate
utilization genes in the Fab4-J3 strain seemed to be suitable
to measure mainly the acetate and propionate activating
activity of acetyl-CoA synthetase. In comparison SMB/
acuA carries a defective acetyl-CoA synthetase gene, which
means that the activating activity must derive from alternative acyl-CoA synthetases, most likely a propionyl-CoA
synthetase.
The kinetic constants were determined with an extract
from acetate grown Fab4-J3 cells with acetate as substrate:
Vmax ¼ 205 mUÆmg)1 protein and Km ¼ 44 lM (Vmax/
Km ¼ 4700 UÆg)1ÆmM)1); with propionate as substrate the
values were: Vmax ¼ 67 mUÆmg)1 and Km ¼ 640 lM
(Vmax/Km ¼ 100 UÆg)1ÆmM)1); hence the enzyme is 47 times
more specific for acetate than for propionate. In comparison, an extract from propionate grown SMB/acuA cells
gave following values with acetate as substrate: Vmax ¼
22 mUÆmg protein)1 and Km ¼ 880 lM (Vmax/Km ¼ 25
UÆg)1ÆmM)1) and with propionate as substrate: Vmax ¼
31 mU mg protein)1 and Km ¼ 90 lM (Vmax/Km ¼ 344
UÆg)1ÆmM)1); specificity ratio of acetate: propionate ¼
0.073. These data indicate that A. nidulans possesses both a
highly active specific acetyl-CoA synthetase, and at least one
additional synthetase which prefers propionate 14 times
over acetate as substrate. The existence of two functional
acetyl-CoA synthetases, ACS1 and ACS2, displaying
different kinetics towards propionate, has also been shown
in Sc. cerevisiae [35]. Furthermore, some bacteria such as
E. coli and Salmonella typhimurium carry a specific propionyl-CoA synthetase, which is distinct from the acetyl-CoA
synthetase [36]. A candidate for such a propionyl-CoA
synthetase from A. nidulans is the hypothetical protein
AN5833.2 (Accession No. EAA58342) from the conceptual
translation of the A. nidulans genome (http://www.broad.
mit.edu/annotation/fungi/aspergillus/geneindex.html). The
protein possesses a conserved AMP-binding domain, which
is also present in acetyl-CoA synthetases and shows 63%
similarity (43% identity) to propionyl-CoA synthetases
from bacterial sources such as Brucella melitensis (Accession
No. AAL51488) or Vibrio parahaemolyticus (Accession No.
BAC59907).
To determine the extent of acetate activation in comparison to propionate activation in the presence of both
substrates we used the wild-type strain A26 grown on a
medium containing 100 mM acetate + 100 mM propionate
(Table 6). The cell-free extract was used to determine the
inhibition of acetyl-CoA synthetase activity by propionate.
The acetyl-CoA formed was measured in a coupled assay
with citrate synthase, which displays no significant activity
with propionyl-CoA. Therefore we exclusively monitored
the activity for activation of acetate. In the presence of
0.5 mM acetate and 10 mM propionate (ratio 1 : 20) we
observed still 50% acetyl-CoA synthetase activity. Therefore we conclude that in a wild-type background the
activation of acetate is much favoured over the activation of
propionate or, vice versa, acetate inhibits the formation of
propionyl-CoA. This observation readily explains the
decreased propionyl-CoA levels found in cells grown on
glucose/acetate/propionate as compared to glucose/propionate.
Inhibition of CoASH-dependent enzymes of glucose
metabolism
The high levels of propionyl-CoA in the mutant strain
raised the question of whether the thioester might inhibit
CoA-dependent enzymes in glucose metabolism. Initial
experiments showed that pyruvate dehydrogenase, ATP
citrate lyase and succinyl-CoA synthetase were inhibited by
propionyl-CoA, but that 2-oxoglutarate dehydrogenase and
also the acetyl-CoA dependent citrate synthase exhibited no
effect with propionyl-CoA.
Pyruvate dehydrogenase. In order to investigate the
inhibitory effect of propionyl-CoA on the in vitro activity
of the pyruvate dehydrogenase complex, cell-free extracts of
glucose-grown wild-type cells (strain A26) were used.
Activity was monitored by the reduction of NAD+ in the
presence of pyruvate and CoASH. At low concentrations of
CoASH (0.021 mM) and relatively high propionyl-CoA
concentrations (0.32 mM) the formation of NADH from
the complex was inhibited by 88%. At equimolar concentrations of both (0.17 mM CoASH and 0.16 mM propionylCoA), the inhibitory effect of propionyl-CoA was still
around 50%. The Km for CoASH (7.2 lM) increased in the
presence of 0.1 mM propionyl-CoA 3.6-fold (25 lM),
whereas Vmax was reduced only by 30%, which demonstrated a mainly competitive inhibition with an apparent Ki of
50 lM. Addition of high concentrations of propionate
Table 6. Acetyl-CoA synthetase activity from wild-type strain A26
grown on 100 mM acetate + 100 mM propionate. 100% acetyl-CoA
synthetase activity refers to (135 ± 10) mUÆmg protein)1.
Substrates
Acetate
(mM)
Propionate
(mM)
Ratio
Acetate : propionate
Activity
(%)
10
60
10
10
5
1
0.5
0.1
0
5
20
40
40
10
10
10
–
12 : 1
1:2
1:4
1:8
1 : 10
1 : 20
1 : 100
100
91
86
75
61
66
50
25
3236 M. Brock and W. Buckel (Eur. J. Biochem. 271)
(20 mM) did not produce any significant inhibition. Therefore we can conclude that the excretion of pyruvate during
growth on glucose/propionate medium is caused by a direct
inhibition of the pyruvate dehydrogenase complex by
propionyl-CoA. Furthermore, the elevated pyruvate
excretion of the methylcitrate synthase mutant is in
agreement with the higher intracellular propionyl-CoA
concentrations.
ATP citrate lyase and succinyl-CoA synthetase. In order
to measure the activities of ATP citrate lyase and succinylCoA synthetase more precisely, we partially purified both
enzymes by chromatography over a Q-Sepharose column.
Inhibition of ATP citrate lyase by acetyl-CoA, propionylCoA and butyryl-CoA was measured by addition of
different concentrations of single acyl-CoA to the in vitro
assay in the presence of 0.34 mM CoASH. Activity without
addition of acyl-CoA (10 mUÆmL)1) was set to 100%
(Fig. 2A). Propionyl-CoA showed the strongest inhibitory
effect, followed by acetyl-CoA and butyryl-CoA.
Succinyl-CoA
synthetase. Succinyl-CoA
synthetase
(10 mUÆmL)1) was assayed with succinyl-CoA, inorganic
phosphate and GDP by trapping the liberated CoASH with
5,5¢-dithiobis-2-nitrobenzoate (Fig. 2B). At concentrations
of 0.4 mM acetyl-CoA or 0.4 mM propionyl-CoA the
succinyl-CoA synthetase was inhibited by 70%. A combination of 0.2 mM acetyl-CoA and 0.4 mM propionyl-CoA,
however, caused a 95% inhibition, whereas in the presence
of 0.6 mM acetyl-CoA the inhibition was only 80%.
Therefore, accumulation of propionyl-CoA in the mutant
strain ( 0.2 mM) might lead to a partial block of the citric
acid cycle at the level of succinyl-CoA synthetase.
CoA-transferase activity
As mentioned above, succinyl-CoA synthetase is almost
completely blocked by the combined action of propionyl-
FEBS 2004
CoA and acetyl-CoA. In the presence of both thioesters
one might expect an accumulation of succinyl-CoA in the
cell and a deadlock of further reactions of the citric acid
cycle. The carbon balances revealed, however, that
glucose is almost completely decomposed to CO2 and,
furthermore, the oxidation of acetate is not inhibited by
propionate. Therefore, we searched for an alternative
reaction converting succinyl-CoA into succinate. For this
purpose we determined the ability of cell-free extracts to
transfer the CoA-moiety from succinyl-CoA to acetate or
propionate as well as the ability to decompose propionylCoA by the transfer of the CoA-moiety to acetate by the
action of a CoA-transferase. The wild-type and the
methylcitrate synthase deletion strain were grown on
different carbon sources and the presence of such a CoAtransferase was tested using succinyl-CoA + acetate,
succinyl-CoA + propionate and propionyl-CoA + acetate
as substrates (Table 4). In both strains highest CoAtransferase activity was determined by use of succinylCoA as the CoA-donor and acetate as the acceptor,
followed by the transfer from succinyl-CoA to propionate
( 35% of the former activity) and the transfer from
propionyl-CoA to acetate ( 11%). The enzyme was
most active in strains grown in the presence of propionate
and always higher in the DmcsA strain as compared to
the wild-type. These CoA-transferase levels resemble the
expression pattern of the gene encoding 2-methylisocitrate
lyase, a specific enzyme of the methylcitric acid cycle
(compare Table 4 to Table 3). Therefore, we conclude
that an efficient transfer of the CoA-moiety from
succinyl-CoA to acetate in the presence of both acetate
and propionate is possible. In addition this might explain
the low accumulation of propionyl-CoA during growth
on glucose/acetate/propionate medium especially of the
DmcsA strain, which is consistent with the higher growth
rate and the elevated acetate consumption of both strains
(Table 1). In the absence of acetate (glucose/propionate
medium) the CoA-moiety, however, can only be trans-
Fig. 2. Inhibition of ATP citrate lyase (A) and
succinyl-CoA synthetase (B) from A. nidulans
by different CoA-thioesters. Both enzymes
were partially purified by chromatography
over Q-Sepharose. Activity without addition
of CoA-thioesters ( 10 mUÆmL)1) was set as
100%.
FEBS 2004
Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3237
ferred to propionate, which would on the one hand
enable a completion of the citric acid cycle, but on the
other hand produce even more propionyl-CoA, which
accumulates especially in the DmcsA strain. CoA-transferases are already known from pro- and eukaryotic
sources. However, the transfer of the CoA-moiety from
succinyl-CoA to acetate or propionate has not been
shown before in any organism at a reasonable rate.
Further investigations on a purified enzyme will need to
prove the substrate specificity and intracellular localization of the enzyme to manifest these observations.
The oxidative branch of the pentose phosphate pathway
We mentioned above that cells grown on glucose/propionate medium released approximately twice as much CO2 for
the formation of 1 g dried mycelium; we attributed this
mainly to the reduced growth rate and the consequent high
consumption via maintenance (8 mmolÆg)1Æh)1). Another
explanation of this apparent uncoupling of glucose oxidation from growth could be the pentose phosphate cycle, in
which no ATP is conserved. This pathway is involved in the
metabolism of glucose and is essential for the generation of
NADPH and ribose, which are necessary for biosynthetic
processes such as fatty acid and nucleotide synthesis. If only
NADPH is required, glucose can be completely oxidized via
this pathway to CO2 without ATP formation. It was
demonstrated that glucose-6-phosphate dehydrogenase, the
first enzyme of this pathway, is essential for the viability of
fungal cells, most likely due to its important biosynthetic
role [37,38]. As shown in Table 7, A. nidulans contains
relatively high amounts of glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase, which
were measured together in the same assay. The data indicate
that the presence of propionate in the medium reduces the
activity by 50% in the wild-type as well as in the DmcsA
strain. Therefore it appears unlikely that an enhanced
oxidation of glucose via the pentose phosphate cycle is
responsible for the observed uncoupling of glucose oxidation and growth inhibition caused in the presence of
propionate.
Correlation of spore colour formation to propionyl-CoA
levels and enzymatic activities
The spore colour of conidia from A. nidulans derives from
the polyketide naphtopyrone [39]. We have assumed a
Table 7. Determination of the oxidative steps of the pentose phosphate
pathway. Wild-type and DmcsA were grown on different carbon
sources and the combined activity of glucose-6-phosphate dehydrogenase and gluconate-6-phosphate dehydrogenase was determined.
One unit (U) is defined as the reduction of 1 lmol of NADP+ per min.
The wild-type strain was A26 and DmcsA was RYQ11.
Growth
condition
Wild-type
(UÆmg protein)1)
DmcsA
(UÆmg protein)1)
Glucose
Glucose/acetate
Glucose/propionate
Glucose/acetate/propionate
1.35
1.12
0.87
0.85
1.36
1.05
0.73
1.18
negative effect of propionyl-CoA on spore colour formation
in an earlier study, without the knowledge about the
accumulation of propionyl-CoA [2]. Recently, by screening
for A. nidulans mutants with a defect in the synthesis of the
polyketide sterigmatocystin (ST) a methylcitrate synthase
deletion strain was identified. Further analysis of this
mutant showed that it was not only disturbed in ST
production but also in the formation of ascoquinone A,
a polyketide, which is responsible for the red pigment of
sexual spores (ascospores). Both polyketides are formed
under conditions when carbon sources become limited
(‡ 70 h of growth). Therefore, an accumulation of propionyl-CoA was predicted, which derives from the degradation
of amino acids such as isoleucin, valine and methionine
during starvation [40]. In this study we tried to correlate the
inhibition of spore colour formation directly to the level of
propionyl-CoA under different growth conditions.
In A. nidulans spore colour formation is prevented
especially in a methylcitrate synthase deletion strain by the
addition of propionate (Fig. 3, lines III, IV, V and VI). This
effect is not observed upon the addition of acetate to the
growth medium (Fig. 3, line II) and implies that the
presence of propionyl-CoA or methylmalonyl-CoA inhibits
polyketide synthases, for which fungi apparently only use
acetyl-CoA and malonyl-CoA as substrates. As shown in
lines III–VI of Fig. 3, the addition of increasing amounts of
propionate also affects the wild-type and the facB multicopy strain but not strain SMB/acuA, which carries a
defective acetyl-CoA synthetase (n.b. acuA ¼ facA). The
order of the inhibitory effect on spore colour formation was:
methylcitrate synthase deletion strain, followed by the facB
multi-copy strain and the wild-type. This observation is in
agreement with the activities for propionate activation in
comparison to methylcitrate synthase activity (Table 3:
compare Pcs and McsA on media G50/P100 and G10/
P100). Strain SMB/acuA shows lowest propionyl-CoA
synthetase activity but significant methylcitrate synthase
activity. The facB multi-copy strain shows elevated propionyl-CoA synthetase activity without increasing methylcitrate synthase activity and therefore reacts more sensitively
than the wild-type. However, it is noteworthy that strain
Fab4-J3 in comparison to the wild-type shows similar
activities of Acs, Pcs and Icl on propionate medium (G10/
P100 of Table 3) but reduced levels of propionate specific
enzyme activities such as methylcitrate synthase and methylisocitrate lyase (a canditate gene is AN8755.2 from the
conceptual translation of the A. nidulans genome, which
shows 46% identity to the methylisocitrate lyase from
Sc. cerevisiae). This implies that the activating effect on
glyoxylate cycle enzymes mediated by propionate is FacB
independent and furthermore, higher basal levels of FacB
seem to have a negative effect on methylcitrate cycle
enzymes.
The inability of the methylcitrate synthase mutant to
remove propionyl-CoA via the methylcitrate pathway leads
to loss of spore colour formation even at low propionate
concentrations. As shown in lines VII and IX of Fig. 3, the
addition of acetate to glucose/propionate medium releases
suppression of spore colour formation especially in the
methylcitrate synthase mutant and the wild-type. The facB
multi-copy strain Fab4-J3, however, is inhibited even more.
This strain shows strongly increased acetyl-CoA and
3238 M. Brock and W. Buckel (Eur. J. Biochem. 271)
FEBS 2004
Fig. 3. Spore colour formation of different A. nidulans strains. Growth conditions are given on the right (G, glucose; P, propionate, A, acetate; e.g.
G50/P10 ¼ the medium contained 50 mM glucose + 10 mM propionate). Strains are A26, wild-type; Fab4-J3, facB multi copy strain; DmcsA,
methylcitrate synthase deletion strain; SMB/acuA, facA303 mutation in the acetyl-CoA synthetase.
propionyl-CoA synthetase activity on media containing
both acetate and propionate (Table 3), which leads to the
accumulation of propionyl-CoA. On a medium containing
50 mM acetate and 10 mM propionate, the levels were
18 nmol acetyl-CoA and 40 nmol propionyl-CoAÆg dry
weight)1 (ratio 1 : 2.2); when the medium contained
100 mM acetate and 100 mM propionate, the levels rose to
20 nmol acetyl-CoA and 66 nmol propionyl-CoA Æg dry
weight)1 (ratio 1 : 3.3). Furthermore lines VII and VIII
show that this strain behaved very similarly on media
without glucose, which is in agreement with the observation
that the strain hardly uses glucose and acetate in parallel (see
section entitled Carbon balances on different growth
media). Nevertheless, the spore colour of strain Fab4-J3 in
lanes IX and X is hard to visualize, because the number of
spores at these growth conditions is greatly reduced. This is
also true for the DmcsA-strain on G50/P100, which implies
that at high propionyl-CoA concentrations not only spore
colour formation but also conidiation is affected.
Utilization of acetate by the acetyl-CoA synthetase
mutant is strictly dependent on the activity of the
predicted propionyl-CoA synthetase. Strain SMB/acuA
shows better growth on media containing only 10 mM
propionate and 50 mM acetate instead of equimolar
FEBS 2004
Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3239
concentrations of these. This can be explained by the
necessity of the presence of 10 mM propionate to induce
propionyl-CoA synthetase activity (Table 3), which is
then able to activate acetate. At equimolar concentrations
of acetate and propionate, activation of propionate by the
propionyl-CoA synthetase is much more likely than that
of acetate (see section entitled Formation of acetyl-CoA
and propionyl-CoA; Vmax/Km (acetate) ¼ 25 UÆg)1ÆmM)1;
Vmax/Km (propionate) ¼ 344 UÆg)1ÆmM)1). From these results we conclude that the acetyl-CoA/propionyl-CoA
ratio and also the ability to activate propionate to
propionyl-CoA has to be well balanced with the methylcitrate synthase activity for successful spore colour
formation and growth.
The data in Table 3 further imply that propionyl-CoA
might be a direct inducer of methylcitrate cycle genes. On
media G50/P100 and G10/P100, which lead to a strong
accumulation of propionyl-CoA in the DmcsA-strain, the
activity of methylisocitrate lyase is twice as high than that of
the wild-type. The addition of acetate to these media not
only lowered the propionyl-CoA level, but also that of
methylisocitrate lyase activity. Therefore, a putative transcriptional activator of the methylcitrate cycle genes seems
to be activated by propionyl-CoA (or propionyl-carnitine)
rather than by methylcitrate as suggested for the procaryotic
regulator of the propionate utilization genes from
S. typhimurium [41].
Discussion
Growth of A. nidulans on glucose medium is inhibited by
propionate in a concentration-dependent manner. In a
strain carrying a defective methylcitrate synthase gene, this
effect is even much more pronounced. When acetate was the
main carbon source, addition of propionate had no growth
inhibitory effect on the wild-type and little effect on the
methylcitrate synthase deletion strain. One might assume
that the inhibition observed on glucose is caused by a
reduced glucose uptake, due to the presence of carboxylic
acids. We were able to show that acetate and propionate did
not inhibit uptake of glucose by measuring the total carbon
consumption and carbon balances from different carbon
sources. Measurements clearly indicated that in the presence
of glucose and propionate, despite the reduced growth rate,
an elevated level of glucose was required for the formation
of 1 g dried mycelium. Furthermore, on glucose/acetate/
propionate, which should inhibit glucose uptake even more,
the growth rate was increased and was actually higher than
that observed with acetate as sole carbon source [2].
On the other hand, we were able to correlate the growth
inhibitory effect of propionate on glucose medium with the
intracellular concentration of propionyl-CoA. Since this
CoA-derivative also accumulated on acetate/propionate
medium without showing significant growth retardation, we
concluded that propionyl-CoA inhibits enzymes mainly
involved in glucose rather than in acetate metabolism. We
found that activities of CoA-dependent enzymes such as
ATP citrate lyase, succinyl-CoA synthetase and the pyruvate dehydrogenase complex were strongly inhibited in the
presence of propionyl-CoA.
ATP citrate lyase from A. nidulans provides cytosolic
acetyl-CoA required for the biosynthesis of fatty acids and
polyketides. The enzyme level was shown to be regulated by
the carbon source present in the media: high levels on
glucose and low levels on acetate. Unfortunately, the effect
of propionate on enzyme levels was not investigated and
remains unclear [42]. Therefore, further studies will also
have to focus on the activity pattern of this enzyme on
propionate containing media. We cannot evaluate the direct
effect of a partial inhibition of ATP citrate lyase by
propionyl-CoA on the metabolism, because this enzyme is
not involved in glucose degradation. It is also not clear
whether inhibition of ATP citrate lyase indirectly diminishes
polyketide synthesis or whether a direct interaction of
propionyl-CoA with polyketide synthetase is responsible for
this effect.
Succinyl-CoA synthetase is directly involved in the
degradation of glucose, acetate and propionate via the
Krebs cycle. Therefore an inhibition of this enzyme would
block the oxidation of all three substrates, which was not
observed with acetate. An elegant way to bypass the
inhibition of this synthetase is the transfer of the CoAmoiety from succinyl-CoA to either acetate or propionate.
We were able to show the existence of such a CoAtransferase, which indeed seems to be induced by propionate
but prefers acetate to propionate as CoA acceptor (Table 4).
Hence, the CoA-transferase explains the higher growth rate,
which was always observed when acetate was added to a
medium containing propionate. In the absence of acetate,
however, the transferase enhances the formation of propionyl-CoA, which traps the system into a loop.
A very important inhibition is attributed to the pyruvate
dehydrogenase complex. The low Ki of 50 lM propionylCoA (compare to 840 lM for pyruvate dehydrogenase of
R. sphaeroides) not only clarified the growth inhibition of
both organisms but also the observed excretion of pyruvate,
which was dependent on the intracellular propionyl-CoA
content. The excretion of pyruvate clearly demonstrates that
the target of propionyl-CoA is pyruvate dehydrogenase
rather than Krebs cycle enzymes. Since pyruvate dehydrogenase catalyses an irreversible reaction, the inhibition of
any enzyme of the cycle cannot lead to an accumulation of
pyruvate. The inhibition of pyruvate dehydrogenase also
explains the low growth rate on propionate. We showed
that in addition to a functional methylcitrate cycle pyruvate
dehydrogenase is required for the pathway of propionate
oxidation. Therefore activation of propionate and the
subsequent oxidation of propionyl-CoA to acetyl-CoA
has to be well balanced and does not allow high turnovers.
Despite the different metabolism of propionyl-CoA in
fungi and humans (methylcitrate cycle vs. methylmalonylCoA pathway) we conclude from our results that accumulation of propionyl-CoA might show severe effects not only
on fungal but also on human cells, which carry defective
genes of the methylmalonyl-CoA pathway. Mutated genes
encoding propionyl-CoA carboxylase and methylmalonylCoA mutase cause the diseases propionic acidemia and
methylmalonic aciduria, respectively. Both are generally
diagnosed by the determination of methylcitrate in the urine
generated from accumulated propionyl-CoA, especially in
liver hepatocytes [43,44]. Hence, phenotypes of the diseases
(dehydration, lethargy, nausea and vomiting as well as a
risk for neurologic sequelae) might be caused not only by
metabolites derived from propionyl-CoA as are propionate,
3240 M. Brock and W. Buckel (Eur. J. Biochem. 271)
b-hydroxypropionate, b-hydroxybutyrate, methylmalonylCoA and methylcitrate, but also directly by propionyl-CoA
inhibiting pyruvate dehydrogenase as described in this
study.
Besides the impairment caused by propionyl-CoA we
cannot exclude a depletion of free CoASH, which would
also lead to a strong disturbance of the metabolism and a
reduction of pyruvate oxidation. However, the fact that the
DmcsA strain also accumulates significant amounts of
propionyl-CoA on acetate/propionate medium without
showing a significant reduction in biomass formation
compared to acetate as sole carbon source [2] seems to
exclude this effect.
In order to get further insights into the mechanism of
growth inhibition mediated by propionate, future work will
focus on the phenotypic characterization of other mutants
carrying defective genes of the methylcitrate cycle. Analysis
of the fatty acid composition from the DmcsA strain grown
on different carbon sources might also give an insight into
substrate specificity of acetyl-CoA carboxylase and fatty
acid synthases, depending on the existence of branched and
odd chain fatty acids. Furthermore, we are trying to identify
and purify the transcriptional activator of the propionate
utilization genes and analyse its DNA recognition sequence.
Knowledge of this sequence will facilitate the screening of
other promoters for putative regulation by propionate,
which might be helpful in the understanding of metabolic
networks.
In summary the data presented here demonstrate how
metabolites are shuttled between different pathways in
fungal cells. However, exact flow rates cannot be
determined by these methods. Flux measurements by
13
C-NMR-spectroscopy could be helpful but are certainly
difficult to interpret due the simultaneous use of mixtures
of two or three substrates. Analysis of different mutants
will give supporting evidence, but definite conclusions
cannot be drawn, because every change of enzyme
activity in a metabolic network is able to disturb the
metabolism [45].
Acknowledgements
This work was supported by grants of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. We thank Jennifer
Beier (Universität Hannover, Germany) for her technical assistance
during activity determination, Richard B. Todd (The University of
Melbourne, Australia) for providing strain Fab4-J3 and Professor
Nancy Keller (University of Wisconsin-Madison, USA) for providing
strain RYQ11.
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