Tài liệu Báo cáo khoa học: On the mechanism of action of the antifungal agent propionate Propionyl-CoA inhibits glucose metabolism in Aspergillus nidulans doc

On the mechanism of action of the antifungal agent propionate
Propionyl-CoA inhibits glucose metabolism in
Aspergillus nidulans
Matthias Brock
1
and Wolfgang Buckel
2
1
Laboratorium fu
¨
r Mikrobiologie, Universita
¨
t Hannover;
2
Laboratorium fu
¨
r Mikrobiologie, Fachbereich Biologie,
Philipps-Universita
¨
t Marburg, Germany
Propionate is used to protect b read and animal feed from
moulds. The mode of action of this short-chain fatty acid
was s tudied using Aspergillus nidulans as a model organism.
The filamentous fungus is able to grow slowly on propio-
nate, which is oxidized to acetyl-CoA via propionyl-CoA,
methylcitrate and pyruvate. Propionate inhibits growth of
A. nidulan s on glucose but not on acetate; the l atter 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 l evels o f p ropionyl-CoA, which inhibits CoA-
dependent enzymes such as p yruvate d ehydrogenase, s ucci-
nyl-CoA synthetase and ATP citrate lyase. The most
important inhibition is that of pyruvate dehydrogenase, as
this affects glu cose and propionate metabolism directly. In
contrast, the blocked succinyl-CoA synthetase can be cir-
cumvented by a succinyl-CoA:acetate/propionate CoA-
transferase, whereas A TP citrate lyase is r equired only for
biosynthetic purposes. In addition, data are presented that
correlate inhibition of fungal polyketide synthesis by pro-
pionyl-CoA with t he accumulation of this CoA-derivative.
A possible toxicity of propionyl-CoA for humans in diseases
such as propionic acidaemia and m ethylmalonic a ciduria i s
also discussed.
Keywords: a cetate CoA-transferase; succinyl-CoA; poly-
ketide synthesis; pyruvate dehydrogenase; pyruvate excre-
tion.
Sodium propionate is widely used as a preservative due to its
ability to inhibit fungal growth. Furthermore, this short-
chain fatty acid (pion ¼ fat) prevents the biosynthesis o f
polyketides such as ochratoxin A b y Aspergillus sulphureus
and Penicillium viridicatum [1]. On the other hand, many
fungi a re able to grow on propion ate, although much m ore
slowly than on glucose o r a cetate. 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 b y
acetyl-CoA synthetase, F acA [4,5], and by an additional
acyl-CoA synthetase. The condensation of p ropionyl-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)-2-
methylisocitrate, w hich is cleaved t o succinate and pyruvate
[3]. Studies with
13
C-labelled propionate indicated that in
Escherichia coli the 2-oxo acid is further oxidized to
acetyl-CoA, which is either funnelled i nto the citrate cycle
or used for biosyntheses [7].
A clue to the mechan ism 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. Unex-
pectedly, 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 b e responsible
for t his inhibitory effect. At h igh l evels of propionyl-
CoA yeast citrate synthase catalyses the slow formation
of three of the four stereoisomers of methylcitrate [8],
but their concentrations (< 10 l
M
) are very low and it
remains controversial whether they may be able to act
as significant inhibitors. Therefore, whether the finding
that methyl citrate might be the c ausative agent o f
propionate toxicity in Salmonella enterica [9] is also true
for eukaryotic cells, is questionable. Nevertheless, the
identification of these isomers b y GLC/MS i s used for
diagnosis of disorders in human propionate metabolism

such as propionic acidaemia and methylmalonic aciduria
[10,11].
The i dea t hat p ropionyl-CoA i tself c ould b e t he 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, K
i
¼ 0.84 m
M
. The addition of sodium bicarbon-
ate increased the growth rate again, probably because it
stimulated the degradation of propionyl-CoA via methyl-
malonyl-CoA [12]. It was also shown that a ccumulation of
Correspondence to W. Buc kel, Laboratorium fu
¨
r Mikrobiologie,
Fachbereich Biologie, Philipps-Universita
¨
t Marburg, D-35032 Mar-
burg, 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-nitro-
benzoic acid); GOD, glucose oxidase; LDH, lactate dehydrogenase;
MDH, malate deh ydrogenase; POD, peroxidase.
(Received 2 2 April 20 04, revised 4 June 2004, accepted 11 J u ne 2004)
Eur. J. Biochem. 271, 3227–3241 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04255.x

propionyl-CoA in rat liver hepatocytes led to a decrease in
the activity of pyruvate dehydrogenase [13].
In this investigation we e xamined c arbon balances under
different growth c onditions. We found that growth of
A. nidulan s on glucose + propionate, especially of the
DmcsA strain, led to the excretion of pyruvate a nd to high
intracellular c oncentrations of propionyl-CoA, which inhi-
bited pyruvate d ehydrogenase, succinyl-CoA s ynthetase
(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 u sed for
determination of acetate, gluco se and pyruvate were fr om
Roche. Columns a nd chromatographic m edia were, if not
otherwise indicated, from Amersham Pharmacia Biotech.
A. nidulans
strains, growth conditions and carbon
balances
The A. nidulans strainsusedinthisstudyarelistedin
Table 1 . Supplem ented minimal and complete media
were prepared as described previously [14]. For the deter-
mination of specific enzyme activities on different carbon
sources, growth t imes were strain and m edium specific.
Approximately 10
8

spores were used for inoculation of
100 m L medium a nd incubation was carried out i n 250-mL
flasks at 37 °C and 240 r.p.m. on a rotary shaker. O n
media containing 50 m
M
glucose as sole carbon source
and 50 m
M
glucose + 100 m
M
acetate, all strains were
incubated for 20 h; on 50 m
M
glucose + 100 m
M
acet-
ate + 100 m
M
propionate, all strains were incubated for
23 h; on 50 m
M
glucose + 100 m
M
propionate the strains
were incubated for 44 h, except strain SMB/acuA, which
showed much less inhibition in the presence of p ropionate
and was grown on this medium for 22 h. The p resence o f
residual g lucose in the medium (> 20 m
M
)wasdetermined

enzymatically. On 100 m
M
acetate and 100 m
M
acet-
ate + 100 m
M
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 m
M
propionate, we added 10 m
M
glucose to the
medium to support initial growth. After total c onsumption
of glucose cells were grown further for at least 12 h.
Therefore, the w ild-type strain was grown f or 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 t he 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 g rowth t imes:
10 m
M
glucose + 100 m
M
acetate harvest after 27 h ;
10 m

M
glucose + 100 m
M
propionate harvest after 29 h;
10 m
M
glucose + 100 m
M
acetate + 100 m
M
propionate
harvest after 29 h. Determination o f the residual glucose
concentration confirmed t hat the strains were i ncubated for
at least 12 h after total consumption of glucose. In addition,
we proved that acetate was still pres ent under all conditions
where it was used as a carbon source. Gr owth at all
conditions and with a ll strains was replicated twice i n order
to confirm the results.
For the determination of CO
2
production, A. nidulans was
grown a t 37 °C in a 1-L gas wash bottle c on taining 600 mL
medium (Schott, Mainz, Germany). The medium was stirred
at 350 r.p.m and bubbled with CO
2
-free air. The CO
2
was
removed by washing the a ir with 2
M

NaOH followed by
sterile water to avoid the t ransfer o f NaOH t o t he growth
medium. The CO
2
produced was trapped in a fourth wash
bottle containing 400 mL 0.2
M
Ba(OH)
2
. The insoluble
BaCO
3
that formed was dried at 60 °C for 20 h a nd weighed.
Residual glucose and acetate contents in the g rowth medium
were determined by enzymatic methods (see below). The
mycelium was pressed to remove any liquid, frozen with
liquid n itrogen, lyophilized, w eighed, and ground to a fine
powder. The CHN content o f the mycelium was determined
by elemental analysis (Zentrale Routineanalytik, Philipps-
Universita
¨
t Marburg, Lahnberge, Germany). Results from
Table 1. A. nidulans strains used in this s tudy. Strain RYQ11 was used throughout all e xperiments. Strain SDmcsA1 was used in a previous work
was t aken as a control t o confirm the re sults of s pore co lour formation, enzyme activities and carbon consumption.
Strain Genotype Source
SMB/acuA facA303, yA2; veA1 [2]
MH2671 pabaA1; prn-309, cnxJ1 [46]
Fab4-J3 MH2671 cotransformed with pFAB4 and pAN222 (approx. 4–8 copies facB) [46]
A637 yA2, pabaA1, pdhA1 FGSC, Kansas City, KS, USA
A634 yA2, pabaA1; pdhB4 FGSC, Kansas City, KS, USA

A627 yA2, pabaA1; pdhC1 FGSC, Kansas City, KS, USA
A26 biA1; veA1 FGSC, Kansas City, KS, USA
SMI45 yA2, pabaA1; wA3; veA1 M. Kru
¨
ger, Marburg, Germany
SRF200 pyrG89; DargB::trpCDB; pyroA4; veA1 [47]
RYQ11
a
DmcsA::argB, biA1; veA1 N. Keller, UW-Madison, USA
SDmcsA1
a
DmcsA::argB, pyrG89; DargB::trpCDB; pyroA4; veA1 [2]
SMB/B1 pyrG89; DargB::trpCDB; pyroA4; veA1 (alcA::mcsA, argB) [2]
a
Two different methylcitrate synthase mutants (DmcsA). FGSC, Fungal genetics stock center ().
3228 M. Brock and W. Buckel (Eur. J. Biochem. 271) Ó FEBS 2004
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 car bon 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 m L 2% HClO
4
and 1 mL
0.1% trifluoroacetic acid. The suspension was sonicated
three times for 4 min each a t 70% full power and 60%
pulses (Branson 250 sonifier; Branson, Dietzenbach, Ger-
many) and neutralized to p H 4–5 by drop-wise addition of
2

M
K
2
CO
3
. A fter incubation on ice for 15 min m ost of t he
perchloric acid was precipitated as insoluble KClO
4
.The
solution w as c entrifuge d at 120 00 0 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
Ò
,
510 m g; Macherey-Nagel, Du
¨
ren, Germany), previously
rinsed with methanol and washed w ith 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% trifluoro-
acetic acid and samples were collected in 2-mL micro
centrifuge c ups. T he acetonitrile was evaporated in a Speed
Vac Concentrator (Bachofer GmbH, Reutlingen, Germany)
without heating and the residual volume o f 200–500 lLwas
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 concen-
trations.

Determination of the intracellular volume
Wet weight was determined after pressing the mycelium
between several sheets o f a bsorbent paper until no further
liquid could be removed. Mycelium was dried f or at least
20 h at 60 °C a nd 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. nidulan s strain SMB/acuA [2] w as grown for 20 h on
glucose minimal medium. Mycelium was har vested over a
Miracloth filter membrane (Calbiochem). The mycelium
was dry-pressed for removal of residual medium and
suspended in 50 m
M
Tris/HCl pH 8.0 containing 2 m
M
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 (Phar-
macia B iote ch, b ed 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 fo r a ctivity, c ollected and concentrated in an

Amicon chamber o ve r 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 f urther 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 o xidase (GOD, from A. niger), peroxidase
(POD, from horseradish) and 2,2¢-azinobis(3-ethylbenzo-6-
thiazolinesulfonic acid). The test was a modification of a
described procedure [ 15]. The composition of the test
reagent was: 130 m
M
sodium phosphate, pH 7.0; 400 U
POD (2 mg; 200 UÆmg
)1
), 80 0 U GOD (4 m g; 200 U Æmg
)1
)
and 2 5 mg 2,2¢-azinobis(3-ethylbenzo-6-thiazolinesulfonic
acid), final volume 50 mL. Each assay, which contained
900 lL rea gent and 100 lL sample, was incubated for
15 min at 3 7 °C and measured at 436 nm in a spectropho-
tometer. The a ssay was linear in a range of 0–30 l
M
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 a t 340 nm until no
further change in absorbance was visible; e
340
¼
6.3 m
M
)1
Æcm
)1
[16]. The assay c ontained, in a final volume
of 1 mL, 50 m
M
potassium phosphate pH 7.0, 0.2 m
M
NADH, 0.5 U LDH a nd 50–100 lL d ifferent 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 Sac-
charomyces 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 h eart. A typical a ssay in
a final volume of 1 mL contained (m
M
) 50 potassium

phosphate, pH 7.0; 10
L
-malate, 0.2 CoASH, 2 NAD
+
,
2ATP, 4MgCl
2
, 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 t he exception
of MDH a nd citrate synthase and the r esulting absorbance
at 340 nm was measured (A
1
). MDH was added and the
absorbance after r eaching t he equilibrium was taken as A
2
.
Citrate synthase was added and the reaction w as monitored
until no further c hange in absorbance w as visible (A
3
).
Concentrations were calculated by the formula below
[e, absorbance (extinction) coefficient; d, length of light
path of the cuvette], which con siders the decrease of the
concentration of oxaloacetate in equilibrium with
L
-malate
during t he formation o f NADH (the concentrations of
malate and NAD
+

remain almost constant):
[Acetate] ¼
A
3
À A
2
e  d
1 þ
A
2
À A
1
A
3
À A
1

Determination of intracellular propionyl-CoA and
acetyl-CoA
Concentrations of acyl-CoA were determined by the u se of
citrate synthase from pig heart and purified methylcitrate
Ó FEBS 2004 Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3229
synthase from the overproducing A. nidu lans strain SMB/
B1 [2] b y two independent methods. One me thod was
performed as described above for the determination of the
concentration o f acetate from t he growth medium. A 1-mL
assay contained 50 m
M
potassium phosphate, pH 7.0;
10 m

ML
-malate, 2 m
M
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 u se of citrate synthase. The reaction w as
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 CoAS H, which was released during the
condensation of oxaloacetate with acetyl-CoA or propio-
nyl-CoA. The assay contained, in a final volume of 1 mL,
50 m
M
Tris/HCl, pH 8.0; 1 m
M
oxaloacetate, 1 m
M
DTNB,
0.5 U citrate synthase, 0.5 U methylcitrate synthase and
20–100 lL sample. Change in absorbance was m onitored at
412 nm; e ¼ 14.2 m
M
)1
Æcm
)1

[18,19]. Acetyl-CoA concen-
trations were determined first. When n o further change in
absorbance was v isible, methylcitrate s ynthase was a dded.
Enzyme assays
ATP citrate lyase. The assay [20] contained (m
M
)
50 Tris/HCl, pH 8.0; 0.2 NADH, 5 A TP, 0.34 CoASH,
20 citrate, 2 dithiothreitol, 2 MgCl
2
,0.5UMDHfrom
pig heart, enzyme sample and water to a final volume of
1 m L. The reaction was started by a ddition of enzyme
sample a nd decrease in absorbance a t 340 nm was
monitored. One unit o f enzyme activity was defined as
the a mount of enzyme reducing 1 lmol NADÆmin
)1
under the assay conditions.
Succinyl-CoA synthetase was measured b y a modified
method for the determination of citrate synthase activity
[21]. A typical assay contained 50 m
M
Tris/HCl, pH 7 .5;
0.14 m
M
succinyl-CoA, 1 m
M
DTNB, 0 .5 m
M
GDP, 2 m

M
MgCl
2
,5m
M
potassium phosphate, enzyme sample and
water to a final volume of 1 mL. One unit of e nzyme a ctivity
was defined as th e amount of enzyme producing 1 lmol
CoASHÆmin
)1
under the assay conditions.
Isocitrate lyase. The assay [ 22] contained (m
M
) 50 potas-
sium phosphate, pH 7.0; 1 threo-isocitrate, 10 phenylhydr-
azine HCl, 2 dithiothreitol, 2 MgCl
2
, 10–100 lLenzyme
sample and water to a final v olume of 1 mL. T he formation
of glyoxylate phenylhydrazone was followed at 324 n m;
e ¼ 16.8 m
M
)1
Æcm
)1
. O ne unit of enzyme a ctivity w as
defined as the amount of enzyme producing 1 lmol
glyoxylate phenylhydrazoneÆmin
)1
under the assay condi-

tions.
2-Methylisocitrate lyase. The a ssay was based o n the
reduction of pyruvate with NADH catalysed by LDH,
whereby the decrease in abso rbance at 340 nm w as r ecorded
[3]. The composition o f the reaction was (m
M
)0.20threo-
2-methylisocitrate, 2 MgCl
2
, 2 dithiothreitol, 0.2 NADH ,
1.5 U LDH, 50 potassium phosphate, pH 7.0; enzyme
sample and w ater 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 condi-
tions.
Citrate synthase and methylcitrate synthase. Citrate
synthase and m ethylcitrate synt hase activity was d etermined
as described previously [2]. The r eaction m ixture contained
(in m
M
), in a final v olume of 1 mL, 50 T ris/HCl, pH 8.0;
1.0 5 ,5¢-dithiobis-(2-nitrobenzoic acid), cell-free extract and
0.2 a cetyl-CoA or propionyl-CoA, respectively. The assay
was started by the addition on 1 m
M
oxaloacetate ( final
concentration) and m onitored at 412 nm. One unit of
enzyme activity was defined as the amount of enzyme

producing 1 lmol CoASHÆmin
)1
under the assay condi-
tions.
Pyruvate dehydrogenase. Pyruvate dehydrogenase (PDH)
activity was measured according t o a pro cedure described
previously [23] with some modifications. The assay con-
tained (in m
M
), in a final volume of 1 mL, 50 T ris/HCl,
pH 8.0; 2 pyruvate, 0.8 thiamine pyrophosphate, 2.5 cys-
teine/HCl, 2 NAD, 2 MgCl
2
, cell-free extract and water t o a
final volume of 990 lL. The reaction was started by the
addition of 0.02–0.17 m
M
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 t he assay c onditions. 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 m
M
), in a final v olume 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 MgCl
2
,6U
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 t he addition of 20 lL
of a 100 m
M
ATP solution (final concentration 2 m
M
)and
the reduction of NAD was monitored at 340 nm. The
extincition coefficient was set as 0.5 · 6.3 m
M
)1
Æcm
)1
,
which compensates for the initial decrease of the oxalo-
acetate concentration in t he equilibrium due to the

accumulation of NADH [25]. Lineweaver–Burk diagrams
were obtained b y use of the worksheet of the program
EXEL
98 (Microsoft Inc.).
Propionyl-CoA synthetase. Propion yl-CoA synthetase
activity was determined by the same method as described
for the determination of a cetyl-CoA synthetase activity,
except s odium acetate was replaced by sodium propionate
and c itrate synthase by methylcitrate s ynthase ( 0.8 U) f rom
A. nidulan s [2].
CoA-Transferase. CoA-Transferase activity was deter-
mined by u sing succinyl-CoA or propionyl-CoA as the
CoA-donor and acetate or propionate as the acceptor.
3230 M. Brock and W. Buckel (Eur. J. Biochem. 271) Ó FEBS 2004
When acetate was the acceptor the assay was monito re d b y
the use of citrate s ynthase, which released CoASH upon the
condensation of n ewly generated acetyl-CoA with oxalo-
acetate as described for t he determination of citrate synthase
activity. When p ropionate w as used as the acceptor, purified
methylcitrate synthase was used to measure the CoASH
release upon the condensation of p ropionyl-CoA w ith
oxaloacetate as described for t he determination of methyl-
citrate synthase activity. A t ypical ass ay contained ( in m
M
),
in a final volume of 1 mL, 50 M ops, pH 7.5; 0.4 CoA-
donor (succinyl-CoA or propionyl-CoA, respectively), 2 U
citrate synthase or 0.8 U m ethylcitrate synthase, r espect-
ively, 1 oxaloacetate and 10 C oA-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 glucose-
6-phosphate dehydrogenase but also the a ctivity o f the
6-phosphogluconate dehydrogenase was m easured. The
described method was slightly modified [ 26]. A typical assay
in a final volume of 1 mL contained (in m
M
) 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
)1
Æmg 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 · 10
8
spores and incubated f or 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 c ultures w ere in cubated
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 s howed that growth on glucose + prop-
ionate resulted in significant excr etions of pyruvate i nto t he
medium (Table 2). I n o rder to exclude substantial excretions
of other c arbon compounds, w e measured t he total c arbon
balances of wild-type and methylcitrate synthase deletion
strain (DmcsA). Therefore, the consumption of substrates,
formation of CO
2
, as well a s excretion of pyruvate and t he
final pH were determined in media in which c ells had been
grown on different carbon sources. The measured carbon
balances add up to almost 100% (T able 3) indicating that
there was no substantial excretion of compounds other than
CO
2
and pyruvate or a significant consumption of prop-
ionate. The increase in the final pH (Table 2 ) correlated
with the consumption of t he carboxylates, by w hich protons

are removed from the medium, whereas by oxidation of
glucose no change in pH w as observed. W hen grown only
on glucose t here was no significant d ifference b etween 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 t he increase in pH from 6.4 to 8.2 correlated with the
high consumption of acetate. Growth o n 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 co nditions. The wild-type strain was
SMI45 an d th e initial pH was 6.3–6.5. Consumption and excretion are data are given in m mo l su bstrateÆg dried mycelium
)1
. In all experiments t he
concentration of glucose was 50 m
M
and t hat o f sodium propionate100 m
M
. The co ncentration of s odium acetate was 50 m
M
except when used in
combinatio n with propio nate in which ca se 100 m
M
was used. Mycelia were harv ested in th e linear growth ph ase. DmcsA, methylcitrate synthase
deletion mutant (RYQ11 a nd SDmcsA1). For experi ments marked b y an a sterisk see a lso 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 * Glucose (6.6) 10.6 – 0.140 20
DmcsA * Glucose (6.7) 9.9 – 0.054 20
Wild-type Glucose/acetate (7.3) 10.0 1.0 0.070 22
DmcsA Glucose/acetate (7.9) 8.0 9.0 0.087 22
Wild-type * Glucose/propionate (6.8) 14.6 – 1.27 44
DmcsA * Glucose/propionate (6.3) 16.2 – 2.21 72
Wild-type Glucose/propionate/acetate (7.5) 7.0 24 0.37 30
DmcsA Glucose/propionate/acetate (7.4) 11.0 19 0.50 30
Wild-type Acetate/propionate (8.0) – 49 0.144 47
DmcsA Acetate/propionate (8.5) – 62 0.035 47
Wild-type Acetate (8.2) – 54 < 0.01 40
DmcsA Acetate (8.2) – 55 < 0.01 40
Ó FEBS 2004 Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3231
Addition of acetate to a medium contai ning glucose did
not change the growth rate significantly, but the lack of
methylcitrate synthase i n the mutant strain induced acetate
consumption (Table 2). This observation is similar t o strain
Fab4-J3, which carries multiple copies of the transcriptional
activator FacB of the acetate utilization genes. FacB is
induced by a cetate and acet ylcarnitine [27]. G rowth experi-
ments w ith s train Fab4-J3 revealed that in the p resence of

both glucose and acetate, the latter substrate is m ainly used.
Thus cells grown on 50 m
M
glucose + 100 m
M
acetate
consumed only 2.7 mmol glucose but 44.2 mmol acetateÆ
g d ried cells
)1
. T hat means that the h igher basal level of t he
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 w e can conclude that propionate or an
intermediary metabolite, most like ly propionyl-CoA, is able
to induce genes from propionate as well as from acetate
metabolism (Table 4, see Icl, Micl and McsA). Therefore, i n
the DmcsA strain, accumulation o f propionyl-CoA, derived
from amino acid degradation, can cause the higher
consumption of acetate as compared to the wild-type.
A d ramatic e ffect o n t he growth rate was observed w hen
propionate was a dded to t he glucose medium; the g rowth
time doubled with the w ild-type and increased 3 .6· with the
DmcsA mutant. In both s trains prop ionate caused an
increase in glucose consumption and a huge enhancement o f
pyruvate excretion. The carbon balance excluded a signifi-
cant excretion of other substances such as alanine [28],
which may have escaped our analytical tools. Furt hermore
we found that the observed additional amount of consumed
glucose was almost completely oxidized to CO

2
(Table 3).
Probably the increase in CO
2
production caused by
propionate (doubled with the w ild-type and tripled with
the mutant) was due to energy production required for
maintenance ( see b elow) d uring the extended g rowth 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 p yruvate prompted u s to c heck
strains, in each of which another of the three genes encoding
pyruvate dehydrogenase [29] was mutated (A637, pdhA1-
mutant ¼ lipoate acetyltransferase; A634, pdhB4 ¼ b-sub-
unit of p yruvate 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 m
M
acetate yielded 239 mg
dried m ycelium a fter 23 h (59 mmol acetateÆg myc elium

)1
).
Interestingly, growth of this mutant was enhanced rather
than inhibited b y t he addition of 50 m
M
glucose, which l ed
to the production of 313 mg mycelium in 23 h, whereby
26 mm ol acetate and 4 mmol glucose were consumed and
0.9 m mol p yruvate were excreted. This can be explained by
the fact that p roduction of cell mass f rom glucose req uires
less ATP than from a cetate, because th e energy c onsuming
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 a nd leads t o a reduced transcrip-
tion of genes c oding for degradation of alternative carbon
sources [30]. However, w e cannot exclude the spontaneou s
formation of creA m utants, which derive from our cultiva-
tion conditions. T his e vent would l ead to a relieved carbon
catabolite repression as also shown for other glyoxylate
cycle mutants [5].
Determination of maintenance
Maintenance is the energy that is u sed for survival of cells
without any biomass formation. Determination of main-
tenance was based on t he inhibition of protein biosyn-
thesis by the action of cycloheximide. Cycloheximide
binds to the 80S-subunit o f eukaryotic ribosomes and
prevents the initiation and elongation reaction of protein

biosynthesis. The mycelium of pregrown cultures was
washed and tran sferred to f resh 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. I n this e xperiment significant g lucose con-
sumption was observed ( 8.75 ± 0.1 mmolÆh
)1
Ægdried
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 w hether propionyl-CoA a ccumulates in t he
methylcitrate s ynthase deletion strain during growth on
Table 3. Carbon balances of wild-type and DmcsA strain. Balan ces are calculated f or 1 g of dried m yce lium. The concentrations of the sub strates are
indicated in Table 2 (marked by asterisks). The wil d-type strain was SMI45 and DmcsA strains were RYQ11 and SDmcsA1.
Strain/C-source
Glucose
consumed
(mmol C)
Pyruvate
(mmol C)
CO
2

recovered
(mmol C)
Biomass
(mmol C)
Total amount
recovered
[mmol C (%)]
Wild-type/glucose 64 ± 4 0 21 ± 4 39 60 ± 4 (94)
DmcsA/glucose 60 ± 4 0 20 ± 4 39 59 ± 4 (98)
Wild-type/glucose + propionate 88 ± 4 3 40 ± 2 39 82 ± 2 (93)
DmcsA/glucose + propionate 97 ± 4 6 49 ± 4 39 94 ± 4 (97)
3232 M. Brock and W. Buckel (Eur. J. Biochem. 271) Ó FEBS 2004
Table 4. Specific enzyme activities from c ell-free extracts of different strains and growth conditions. Data are g iven in m UÆmg protein
)1
.Acs,acetyl-
CoA synthetase; Pc s, propionyl-CoA s ynthetase; Icl , isocitrate lyase; Micl, 2-methylisocitrate l yase; McsA, m ethylcitrate synthase. C -sources :G,
glucose; A, acetate; P, propionate. Numbers denote the concentrations of C-sources (m
M
); G50/A100/P100 ¼ 50 m
M
glucose + 100 m
M
acetate + 100 m
M
propionate.
Enzyme C-Source in medium Wild-type (A26) Fab4-J3 DmcsA SMB/acuA
Acs G50 19 ± 3 19 ± 2 16 ± 3 0.5 ± 0.2
Acs G50/A100 47 ± 4 119 ± 10 26 ± 4 1 ± 0.2
Acs G50/P100 22 ± 2 54 ± 2 24 ± 2 2.3 ± 0.2
Acs G50/A100/P100 59 ± 1 137 ± 10 27 ± 3 2 ± 0.3

Acs A100 153 ± 5 205 ± 10 124 ± 2 17 ± 1
Acs G10/P100 133 ± 4 128 ± 10 150 ± 10 22 ± 2
Acs A100/P100 135 ± 10 289 ± 15 167 ± 10 18 ± 2
Pcs G50 10 ± 1 9 ± 1 8 ± 1 1 ± 0.5
Pcs G50/A100 16 ± 2 50 ± 5 13 ± 2 2.3 ± 0.2
Pcs G50/P100 10 ± 1 21 ± 1 10 ± 1 6 ± 0.4
Pcs G50/A100/P100 26 ± 2 38 ± 4 13 ± 1 3.5 ± 0.5
Pcs A100 58 ± 1 67 ± 3 42 ± 2 29 ± 2
Pcs G10/P100 77 ± 2 63 ± 3 76 ± 6 31 ± 1
Pcs A100/P100 59 ± 1 90 ± 2 74 ± 1 30 ± 1
Icl G50 0.2 ± 0.1 0.1 ± 0.1 0.2 ± 0.1 0.6 ± 0.2
Icl G50/A100 23 ± 1 108 ± 4 14 ± 2 7 ± 1
Icl G50/P100 35 ± 2 62 ± 9 41 ± 1 24 ± 2
Icl G50/A100/P100 85 ± 3 170 ± 5 34 ± 3 26 ± 1
Icl A100 86 ± 5 225 ± 5 63 ± 3 71 ± 4
Icl G10/P100 130 ± 5 107 ± 7 294 ± 10 67 ± 5
Icl A100/P100 161 ± 1 287 ± 15 180 ± 10 82 ± 5
Micl G50 7 ± 1 6 ± 2 6 ± 2 4 ± 1
Micl G50/A100 10 ± 1 12 ± 1 9 ± 2 11 ± 1
Micl G50/P100 30 ± 2 31 ± 2 62 ± 4 44 ± 1
Micl G50/A100/P100 26 ± 1 27 ± 1 29 ± 2 33 ± 1
Micl A100 26 ± 1 20 ± 1 29 ± 2 24 ± 1
Micl G10/P100 74 ± 5 28 ± 2 132 ± 1 64 ± 1
Micl A100/P100 35 ± 1 36 ± 2 46 ± 1 63 ± 3
McsA G50 1 ± 0 2 ± 1 0 1 ± 0
McsA G50/A100 5 ± 1 14 ± 2 0 7 ± 1
McsA G50/P100 55 ± 2 52 ± 2 0 57 ± 4
McsA G50/A100/P100 37 ± 1 38 ± 1 0 41 ± 1
McsA A100 38 ± 2 20 ± 1 0 42 ± 3
McsA G10/P100 147 ± 6 72 ± 3 0 153 ± 5

McsA A100/P100 35 ± 1 83 ± 1 0 133 ± 6
Table 5. CoA-transferase ac tivity from wi ld-type and DmcsA grown o n different c arbon sources. Data are given in mUÆmg protein
)1
;1 Uisdefinedas
the relea se o f 1 lmol CoASHÆmin
)1
under t he assay conditions. The w ild-type strain was A26 and DmcsA was RYQ11. Suc cinyl-CoA > acetate,
succinyl-CoA:acetate CoA-transferase; Succinyl-CoA > p ropionate, succinyl-CoA:propionate CoA-transferase; Pr opionyl-CoA > ac etate,
propionyl-CoA:acetate CoA-transferase.
CoA-donor > acceptor
Medium used for growth
Glucose
Glucose/
acetate
Glucose/
propionate
Glucose/acetate/
propionate Acetate Propionate
Succinyl-CoA > acetate (WT) 5.8 38 78 49 86 65
Succinyl-CoA > acetate (DmcsA) 12.7 41 115 63 109 143
Succinyl-CoA > propionate (WT) 2.4 12.4 24 15 32 25
Succinyl-CoA > propionate (DmcsA) 5.1 14.7 46 16 28 54
Propionyl-CoA > acetate (WT) < 0.5 4.4 9.6 5.6 9.6 5.3
Propionyl-CoA> acetate (DmcsA) 0.9 5.8 11.7 7.1 8.7 13.3
Ó FEBS 2004 Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3233
different c arbon sources, mycelium w as 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 E xperimental p rocedure s.

The suitability of this method was checked by mixing
16.5 nmol acetyl-CoA and 1 6.1 nmol of propionyl-CoA
and performing the identical procedure as for the partial
purification of the acyl-CoA e ster from lyophilized
mycelium, including addition of perchloric acid , neutral-
ization, centrifugation, C
18
-cartridge and c oncentration.
The recovery was 15.1 nmol (91.5%) acetyl-CoA and
14.4 nmol (89.5%) propionyl-CoA w hich showed that the
method gave reliable results. Therefore we can conclude
that the r atio between a cetyl-CoA and p ropionyl-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 2 0 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 accumu-
lation of propionyl-CoA under these conditions, as the
thioester cannot be oxidized further. Addition of acetate
to the glucose/propionate medium reduced the propionyl-
CoA level of the cells, whereas an increase was observed
again a fter growth on acetate + propionate without
glucose. Despite this high level of propionyl-CoA, which
was most probably due to an unspecific action of acetyl-
CoA synthetase (described below), only a slight growth

inhibition was visible [2] a nd Table 2. Remarkably, under
the different gr owth conditions the i ntracellular acetyl-
CoA 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 e asiest 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 ml Æg dry cells
)1
,
which is in good agreement w ith that of Neurospora crassa
(2.54 mLÆg dried cells
)1
) [31]. Investigations on the intra-
cellular 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% o f the intrac ellular water i s
not availab le as a solvent for metabolites [32]. We t herefore

used this latter value for the calculation of the internal
propionyl-CoA c oncentration o f t he methylcitrate synthase
mutant and the wild-type after growth on 5 0 m
M
glu-
cose + 100 m
M
propionate. Thus the DmcsA strain accu-
mulated 0.21 m
M
propionyl-CoA, whereas i n t he wild-type
strain only 0.03 m
M
propionyl-CoA could be found.
Nevertheless, concentrations given here are just a simple
mathematical calculation. Due to t he very high concentra-
tion 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 i n t he cytoplasm. For transport to t he mitochon-
dria a conversion into a carnitine-ester and a back-
conversion to the CoA-ester inside the mitochondria has
to be involved, which is most likely performed by cytoplas-
mic and mitocho ndrial ac yl-carnitine transferases (AcuJ [33]
and FacC [27]). The transporter involved in t hat 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 m echanism
Fig. 1. Intracellular contents o f acetyl-CoA
and pro pionyl-CoA from A. nidulans wild-type

and Dmc sA strain gro wn under d ifferent condi-
tions. Carbo n and energy s ourc es were: 5 0 m
M
glucose; 50 m
M
glucose and 100 m
M
sodium
propionate; 50 m
M
glucose, 100 m
M
sodium
acetate, and 100 m
M
sodium propionate;
100 m
M
sodium acetate and 100 m
M
sodium
propionate. T he CoA-thioesters were re leased
from the cells and determined as d escribed in
Experimental procedures.
3234 M. Brock and W. Buckel (Eur. J. Biochem. 271) Ó FEBS 2004
implies a higher concentration of propionyl-CoA within t he
mitochondria. However, the fact that propionyl-CoA
cannot be converted in a methylcitrate synthase deletion
strain wo uld l ead to the formation of an equilibrium
between propionyl-CoA and propionyl-carnitine in mito-

chondria 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 acetyl-
CoA synthetase and a putative propionyl-CoA s ynthetase
we u sed the a cetate-grown strain Fab4-J3 and g lucose/
propionate grown S MB/acuA cells (10 m
M
glucose/100 m
M
propionate; 29 h ). The high expression of the acetate
utilization g enes in th e Fab4-J3 strain seemed to be suitable
to measure mainly the acetate and propionate activating
activity of acetyl-CoA synthetase. I n comparison SMB/
acuA carries a defective acetyl-Co A synthetase gene, which
means that the activating activity must derive from alter-
native acyl-CoA synthetases, most likely a propionyl-CoA
synthetase.
The kinetic constants were determined with an extract
from acetate grow n Fab4-J3 cells with acetate as substrate:
V
max
¼ 20 5 mUÆmg
)1
protein and K
m
¼ 44 l
M

(V
max
/
K
m
¼ 4700 UÆg
)1
Æm
M
)1
); with propionate as substrate the
values were: V
max
¼ 67 mUÆmg
)1
and K
m
¼ 64 0 l
M
(V
max
/K
m
¼ 100 UÆg
)1
Æm
M
)1
); hence the enzyme is 47 times
more specific for acetate than for propionate. In compar-

ison, an extract from propionate grown SMB/acuA cells
gave following values with acetate as substrate: V
max
¼
22 mUÆmg protein
)1
and K
m
¼ 880 l
M
(V
max
/K
m
¼ 25
UÆg
)1
Æm
M
)1
) and with propionate as substrate: V
max
¼
31 mU mg protein
)1
and K
m
¼ 90 l
M
(V

max
/K
m
¼ 34 4
UÆg
)1
Æm
M
)1
); specifi city r atio of acetate: propionate ¼
0.073. These data indicate that A. nidulans possesses both a
highly active specific acetyl-CoA synthetase, a nd at least one
additional synthetase which prefers propionate 14 times
over acetate as substrate. The existence of two functional
acetyl-CoA synthetases, ACS1 and A CS2, displaying
different kinetics towards propionate, has also been shown
in Sc. c erevisiae [35]. Furthermore, some bacteria such as
E. coli and Salmonella typhimurium carry a specific propio-
nyl-CoA synthetase, which is distinct f rom the acetyl-CoA
synthetase [36]. A candidate for such a propionyl-CoA
synthetase from A. nidulans is the h ypothetical protein
AN5833.2 ( Accession No. E AA58342) f rom the concept ual
translation of the A. nidulans genome (ad.
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 s ynthetases
from bacterial sources such as Brucella melitensis (Accession
No. AAL51488) or Vibr io parahaemolyticus (Acce ssion No.
BAC59907).

To determine the extent of acetate activation in compar-
ison to propionate activation in the p resence o f both
substrates we used the wild-type strain A26 grown o n a
medium containing 100 m
M
acetate + 100 m
M
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 m
M
acetate and 10 m
M
propionate (ratio 1 : 20) we
observed still 50% acetyl-CoA syntheta se activ ity. There-
fore we conclude that in a wild-type b ackground 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/pro-
pionate.
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 s howed that pyruvate dehydrogenase, ATP
citrate l yase a nd succinyl-CoA synthetase were inhibited by
propionyl-CoA, but th at 2-oxoglutarate dehydrogenase and
also the acetyl-CoA dependent citrate s ynthase exhibited no
effect with propionyl-CoA.
Pyruvate dehydrogenase. In order to i nvestigate 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 b y the reduction of NAD
+
in the
presence of pyruvate and CoASH. At low concentrations of
CoASH (0.021 m
M
) and rel atively high propionyl-CoA
concentrations (0.32 m
M
)theformationofNADHfrom
the complex was inhibited by 8 8%. A t equimolar concen-
trations of both ( 0.17 m
M
CoASH and 0.16 m
M
propionyl-
CoA), the inhibitory effect of propionyl-CoA w as still
around 50%. The K
m

for C oASH (7.2 l
M
)increasedinthe
presence of 0.1 m
M
propionyl-CoA 3 .6-fold (25 l
M
),
whereas V
max
was r educed only b y 3 0%, which demonstra-
ted a mainly competitive inhibition with an apparent K
i
of
% 50 l
M
. Addition of high concentrations of propionate
Table 6. Acetyl-CoA synthetase activity from wild-type strain A26
grown on 100 m
M
acetate + 100 m
M
propionate. 100% acetyl-CoA
synthetase activity refers to (135 ± 10) mUÆmg protein
)1
.
Substrates
Activity
(%)
Acetate

(m
M
)
Propionate
(m
M
)
Ratio
Acetate : propionate
10 0 – 100
60 5 12 : 1 91
10 20 1 : 2 86
10 40 1 : 4 75
540 1:8 61
110 1:10 66
0.5 10 1 : 20 50
0.1 10 1 : 100 25
Ó FEBS 2004 Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3235
(20 m
M
) did not produce any significant inhibition. There-
fore we can conclude that the e xcretion of pyruvate during
growth on glucos e/propionate medium is caused by a d irect
inhibition of the pyruvate dehydrogenase complex by
propionyl-CoA. Furthermore, the elevated pyruvate
excretion o f 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 succinyl-

CoA synthetase more precisely, we partially purified both
enzymes by c hromatography over a Q-Sepharose column.
Inhibition of ATP citrate lyase by a cetyl-CoA, propionyl-
CoA and butyryl-CoA was measured by addition of
different concentrations of single acyl-CoA to the in vitro
assay i n t he presence of 0.34 m
M
CoASH. Activity without
addition of acyl-CoA (10 mUÆmL
)1
)wassetto100%
(Fig. 2 A). Propionyl-CoA showed the strongest inhibitory
effect, followed by acetyl-CoA and butyryl-CoA.
Succinyl-CoA synthetase. Succinyl-CoA s ynthetase
(10 mUÆmL
)1
) was assayed with succinyl-CoA, inorganic
phosphate and GDP by trapping the liberated CoASH w ith
5,5¢-dithiobis-2-nitrobenzoate (Fig. 2 B). At concentrations
of 0.4 m
M
acetyl-CoA or 0.4 m
M
propionyl-CoA the
succinyl-CoA synthetase was inhibited by 70%. A combi-
nation of 0.2 m
M
acetyl-CoA and 0.4 m
M
propionyl-CoA,

however, caused a 95% inhibition, whereas in t he presence
of 0.6 m
M
acetyl-CoA the inhibition was only 80%.
Therefore, accumulation of propionyl-CoA in the mutant
strain ( % 0.2 m
M
) might lead to a partial block o f t he 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-
CoA a nd acetyl-CoA. In the presence of both thioesters
one might expect an accumulation of succinyl-CoA in t he
cell and a deadlock of further reactions of the citric acid
cycle. The carbon balances revealed, however, that
glucose is almost completely decomposed to CO
2
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 propionyl-
CoA 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 CoA-
transferase was tested using s uccinyl-CoA + acetate,

succinyl-CoA + propionate and propionyl-CoA + acetate
as substrates (Table 4). In both strains highest CoA-
transferase activity was determined by use of succinyl-
CoA as the CoA-donor and a cetate 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 o f th e gene encoding 2-methylisocitrate
lyase, a specific enzyme of the m ethylcitric a cid cycle
(compare Tab le 4 to Table 3). Ther efore, we conclude
that an efficient transfer of the CoA-moiety f rom
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
Dmc sA strain, which is consistent with the higher growth
rate and the elevated acetate consumption of both strains
(Table 1). In the absence o f acetate (glucose/propionate
medium) t he CoA-moiety, however, can only be trans-
Fig. 2. Inhibition of ATP c itrate lyase (A) and
succinyl-CoA synthe tase (B) f rom A. nidulans
by differ ent CoA-thioesters. Both enzymes
were partially purified by chroma tography
over Q- Sepharose. Ac tivity without ad dition
of CoA-thioesters ( % 10 m UÆmL
)1
)wassetas

100%.
3236 M. Brock and W. Buckel (Eur. J. Biochem. 271) Ó FEBS 2004
ferred to propionate, which would on the one hand
enable a completion of the citric acid cycle, but on the
other h and p roduce e ven m ore p ropionyl-CoA, which
accumulates especially in the DmcsA strain . CoA-trans-
ferases 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 a nd intracellular l ocaliza-
tion of the enzyme to m anifest these observations.
The oxidative branch of the pentose phosphate pathway
We mentioned above that cells gr own on glucose/propion-
ate medium r eleased a pproximately t wice as much CO
2
for
the formation of 1 g dried mycelium; we attributed this
mainly to the reduced growth r ate and the consequent high
consumption via maintenance (8 mmolÆg
)1
Æh
)1
). Another
explanation of this apparent ÔuncouplingÕ of glucose o xida-
tion from growth could be the pentose phosphate cycle, in
which no ATP is conserved. This pathway is involved i n the
metabolism o f g lucose and is essential for the g eneration of
NADPH and ribose, which are necessary for biosynthetic

processes such as fatty acid and nucleotide s ynthesis. If only
NADPH is required, glucose can be completely oxidized via
this pa thwa y to CO
2
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 dehydro-
genase and gluconate-6-phosphate dehydrogenase, which
weremeasuredtogetherinthesameassay.Thedataindicate
that the p resence of propionate in the medium reduces the
activity by % 50% in the w ild-type as well a s in the DmcsA
strain. T herefore it appears unlikely that a n enhanced
oxidation of glucose via the pentose phosphate cycle is
responsible for the observed uncoupling of glucose oxida-
tion and g rowth inhibition caused in the presence of
propionate.
Correlation of spore colour formation to propionyl-CoA
levels and enzymatic activities
The spore c olour of conidia from A. nidulans derives f rom
the polyketide naphtopyrone [39]. We have assumed a
negative effect of propionyl-CoA on spore colour formation
in an earlier study, w ithout the knowledge about the
accumulation of propionyl-CoA [2]. Recently, by screen ing
for A. nidulans mut ants with a defect in the synthesis of the
polyketide sterigmatocystin (ST) a methylcitrate s ynthase
deletion strain was i dentified. 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 c onditions when carbon sources become limited
(‡ 70 h of g rowth). Therefore, an accumulation o f propio-
nyl-CoA was predicted, which derives fr om the degradation
of amino acids such as isoleucin, valine a nd methionine
during starvation [ 40]. In this study we tried t o correlate the
inhibition of spore colour formation d irectly to the l evel of
propionyl-CoA under different growth conditions.
In A. nidu lans 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 m ethylmalonyl-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, th e addition of increasing amounts of
propionate also affects the wild-type and the facB multi-
copy strain but not strain SMB/acuA, w hich carries a
defective acetyl-CoA synthetase (n.b. acuA ¼ facA). The
order of t he inhibitory effect on spore colour formation was:
methylcitrate synthase d eletion s train, followed b y t he fa cB
multi-copy strain and the wild-type. This observation is in
agreement with the activities for propionate activation in
comparison t o met hylcitrate s ynthase a ctivity (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 fa cB multi-co py strain shows elevated propio-
nyl-CoA synthetase a ctivity w ithout increasing methyl-
citrate synthase activity and th erefore reacts more sensitive ly
than the wild-type. However, it is noteworthy that strain
Fab4-J3 in c omparison to the wild-type s hows similar
activities of Acs, Pcs and Icl on propionate med ium (G10/
P100 of Table 3) but reduced levels of propionate specific
enzyme activities such as methylcitrate synthase and meth-
ylisocitrate lyase ( a canditate gene is AN8755.2 from the
conceptual translation of the A. nid ulans genome, which
shows 46% ide ntity to the m e thylisocitrate lyase f rom
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 ha ve a n egative effect on methylcitrate cycle
enzymes.
The inability of the methylcitrate synthase mutant to
remove propionyl-CoA via the m ethylcitrate pathway l eads
to loss of spore colour formation even at low propionate
concentrations. A s s hown i n lines VII a nd IX of Fig. 3, the
addition of acetate to glucose/propionate medium releases
suppression of spore c olour formation especially in the
methylcitrate synthase mutant and the w ild-type. The facB
multi-copy strain Fab4-J3, however, i s inhibited e ven more.
This strain shows strongly increased acetyl-CoA and
Table 7. Determination of the o xidative steps o f the pentose phosphate
pathway. Wild-type and DmcsAweregrownondifferentcarbon
sources an d t he combined activity o f glucose-6-phosphate dehyd ro-
genase and g luco nate-6-pho sphate dehydrogenase was determined.
One u nit (U) is defined as the reduction of 1 lmol of NADP

+
per min.
The wild-type strain was A26 and DmcsA wa s RYQ11.
Growth
condition
Wild-type
(UÆmg protein
)1
)
DmcsA
(UÆmg protein
)1
)
Glucose 1.35 1.36
Glucose/acetate 1.12 1.05
Glucose/propionate 0.87 0.73
Glucose/acetate/propionate 0.85 1.18
Ó FEBS 2004 Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3237
propionyl-CoA synthetase activity on media containing
both acetate a nd propionate (Table 3), which leads t o the
accumulation of propionyl-CoA. On a medium containing
50 m
M
acetate and 10 m
M
propionate, the l evels were
18 nmol acetyl-CoA and 40 nmol propionyl-CoAÆgdry
weight
)1
(ratio 1 : 2.2); when the medium contained

100 m
M
acetate and 100 m
M
propionate, the levels rose to
20 nmol acetyl-CoA and 66 nmol propionyl-CoA Ægdry
weight
)1
(ratio 1 : 3.3). Furthermore lines VII and VIII
show that this strain behaved very similarly on media
without glucose, which i s in agreement w ith 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 t o visualize, because the number o f
spores at these g rowth conditions is greatly reduced. This is
also true for the DmcsA-strain on G50/P100, which i mplies
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 s trictly dependent on the activity of t he
predicted propionyl-CoA synthetase. Strain SMB/acuA
shows better growth on media containing only 10 m
M
propionate and 50 m
M
acetate instead of equimolar
Fig. 3. Spore colour formation of different A. nidulans str ain s. Growth conditio ns a re g iven on the right (G, g lu cose; P , p ropiona te, A , a cetate; e .g.
G50/P10 ¼ the medium contained 50 m
M

glucose + 10 m
M
propionate). Strains a re A26, wild-type; Fab4-J3, facB multi copy strain; DmcsA,
methylcitrate s ynthase deletion s train; SMB/acuA, facA303 mutation in the ac etyl-CoA syn thetase.
3238 M. Brock and W. Buckel (Eur. J. Biochem. 271) Ó FEBS 2004
concentrations of these. This can be e xplained by the
necessity of the presence of 10 m
M
propionate to induce
propionyl-CoA synthetase activity (Table 3), which is
then able to act ivate acetate. A t equimolar concentrations
of acetate and propionate, a ctivation of p ropionate by the
propionyl-CoA synthetase is much more likely than that
of acetate (see section entitled Formation of acetyl-CoA
and propionyl-CoA; V
max
/K
m (acet ate)
¼ 25 UÆg
)1
Æm
M
)1
;
V
max
/K
m ( propionate)
¼ 344 UÆg
)1

Æm
M
)1
). From these re-
sults 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 methyl-
citrate 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 l evel, but also that of
methylisocitrate lyase activity. Therefore, a putative tran-
scriptional a ctivator of the methylcitrate cycle genes see ms
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 g ene, this
effect is even much more pronounced. When a cetate was the
main carbon source, addition of propionate had no growth
inhibitory effect on the w ild-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 g lucose by measuring the total carbon
consumption a nd carbon balances from different carbon
sources. Measurements clearly indicated that in the presence
of glucose and propionate, despite the r educed 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 g lucose uptake even more,
the g rowth r ate was increased a nd was actually higher than
that observed with acetate as sole carbon source [2].
On the o ther hand, we were able to correlate the g rowth
inhibitory effect of propionate o n glucose m edium with the
intracellular concentratio n o f propionyl-CoA. Since t his
CoA-derivative a lso a ccumulated on acetate/propionate
medium without showing sign ificant growth r etardation, 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 pyru-
vate dehydrogenase complex were strongly inhibited in t he
presence of propionyl-CoA.
ATP citrate lyase f rom A. nidulans provides cytosolic
acetyl-CoA required for the biosynthesis of fatty acids a nd
polyketides. The enzyme level was shown to be r egu lated by
the carbon source present in the media: high levels on
glucose and low l evels on a cetate. Unfortunately, t he effe ct
of propionate on enzyme levels was not investigated and
remains unclear [42]. Therefore, further studies will also

have to focus on the ac tivity p attern of this enzyme on
propionate containing media. We cannot evaluate the d irect
effect of a p artial inhibition of ATP citrate lyase b y
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 s ynthetase i s responsible for
this effect.
Succinyl-CoA synthetase is directly involved in the
degradation of g lucose, acetate and p ropionate via the
Krebs c ycle. Therefore an inhibition of this enzyme would
block the oxidation of all three substrates, which was not
observed with a cetate. An elegant way to bypass the
inhibition of this synthetase is the transfer of the CoA-
moiety from succ inyl-CoA to either acetate or p ropionate.
We were able to show the existence of such a CoA-
transferase, which indeed seems to be i nduced 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, t he transferase enhances t he formation of p ropio-
nyl-CoA, which traps the system into a loop.
A very i mportant inhibition is attributed to the p yruvate
dehydrogenase c omplex. The low K
i
of % 50 l
M
propionyl-

CoA (compare to 840 l
M
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 o f propionyl-CoA i s pyruvate d ehydrogenase
rather than Krebs cycle enzymes. Since p yruvate dehydro-
genase catalyses an irreversible reaction, the inhibition of
any enzyme o f 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 ad dition to a f unctional methylcitrate cycle pyruvate
dehydrogenase is required for the pathway of propionate
oxidation. Therefore activation of propionate and the
subsequent oxidation o f p ropionyl-CoA to acetyl-CoA
has to b e well b alanced and does not allow high t urnovers.
Despite the different metabolism of propionyl-CoA in
fungi and humans (methylcitrate cycle vs. m ethylmalonyl-
CoA pathway) we conclude from our results that accumu-
lation of propionyl-CoA might show severe effects not only
on fungal but also on human cells, which carry defective
genes of the methylmalonyl-CoA p athway. M utated genes
encoding propionyl-CoA carboxylase and methylmalonyl-
CoA mutase cause the diseases propionic acidemia and
methylmalonic a ciduria, r espectively. Both are g enerally
diagnosed by the d etermination of methylcitrate in the urine
generated from accumulated propionyl-CoA, especially in
liver hepatocytes [ 43,44]. H ence, 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 a s are propionate,
Ó FEBS 2004 Propionyl-CoA inhibits glucose metabolism (Eur. J. Biochem. 271) 3239
b-hydroxypropionate, b-hydroxybutyrate, methylmalonyl-
CoA 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 o f the metabolism a nd a
reduction of pyruvate oxidation. However, the fact t hat 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 i nsights into the mechanism of
growth inhibition mediated by propionate, future work will
focus o n the phenotypic characterization of other mutants
carrying defe ctive g enes of the methylcitrate cycle. Analysis
of the fatty acid composition f rom the DmcsA strain grown
on different c arbon sources m ight 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 t his sequence w ill 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, e xact flow r ates 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 o f different mutants
will give supporting evidence, but definite conclusions
cannot be drawn, because Ôevery change of enzyme
activity in a metabolic network i s able to d isturb the
metabolismÕ [45].
Acknowledgements
This work was s upported by g rants of t he Deutsche Forschungsge-
meinschaft and the Fonds der Chemischen Industrie. We thank Jennifer
Beier (Universita
¨
t Hannover, Germany) for her technical assistance
during activity determination, RichardB.Todd(TheUniversityof
Melbourne, Australia) for p roviding strain Fab4-J3 and Professor
Nancy K elle r (University of Wisconsin-Madison, USA) fo r providing
strain RYQ11.
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