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 (http://www.fgsc.net). 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 (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 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 [...]... 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 Ó 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... 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-CoA g dry weight)1 (ratio 1 : 2.2); when the medium contained 100 mM... [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... 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... 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... 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... 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. .. 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... 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 ÔuncouplingÕ 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. .. 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 . On the mechanism of action of the antifungal agent propionate Propionyl-CoA inhibits glucose metabolism in Aspergillus nidulans Matthias. 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