Characterization of an acyl-CoA: carboxylate CoA-transferase from Aspergillus nidulans involved in propionyl-CoA detoxification


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Filamentous fungi metabolize toxic propionyl-CoA via the methylcitrate cycle. Disruption of the methylcitrate synthase gene leads to an accumulation of propionyl-CoA and attenuates virulence of Aspergillus fumigatus. However, addition of acetate, but not ethanol, to propionate-containing medium strongly reduces the accumulation of propionyl-CoA and restores growth of the methylcitrate synthase mutant. Therefore, the existence of a CoA-transferase was postulated, which transfers the CoASH moiety from propionyl-CoA to acetate and, thereby, detoxifying the cell. In this study, we purified the responsible protein from Aspergillus nidulans and characterized its biochemical properties. The enzyme used succinyl-, propionyl- and acetyl-CoA as CoASH donors and the corresponding acids as acceptor molecules. Although the protein displayed high sequence similarity to acetyl-CoA hydrolases this activity was hardly detectable. We additionally identified and deleted the coding DNA sequence of the CoA-transferase. The mutant displayed weak phenotypes in the presence of propionate and behaved like the wild type when no propionate was present. However, when a double-deletion mutant defective in both methylcitrate synthase and CoA-transferase was constructed, the resulting strain was unable to grow on media containing acetate and propionate as sole carbon sources, which confirmed the in vivo activity of the CoA-transferase.


Propionate is an abundant carbon source in soil (Conrad and Klose, 1999) and many aerobic growing microorganisms are able to use this short-chain carboxylic acid as a carbon and energy source. One of the major pathways for propionate metabolism is that via the methylmalonyl-CoA pathway (Brass and Ruff, 1989), which leads to the citric acid cycle intermediate succinyl-CoA. However, fungi do not possess the coenzyme B12-dependent methylmalonyl-CoA mutase (Ledley et al., 1991) and therefore cannot use the methylmalonyl-CoA pathway. Nevertheless, a huge number of fungi are able to grow on propionate as sole carbon and energy source and have therefore to use an alternative pathway for propionate catabolism (Brock et al., 2000).

We have previously shown that two members of the genus Aspergillus metabolize propionyl-CoA via the methylcitrate cycle (Brock et al., 2000; Maerker et al., 2005). Propionyl-CoA is generated by direct activation of propionate or by the breakdown of odd-chain fatty acids and of the amino acids valine, isoleucine and methionine (Zhang et al., 2004; Maerker et al., 2005; Ibrahim-Granet et al., 2008). Propionyl-CoA is condensed with oxaloacetate to yield methylcitrate. After isomerization to methylisocitrate and cleavage by a specific methylisocitrate lyase, succinate and pyruvate are formed. The release of pyruvate therefore resembles an α-oxidation of propionate (Brock et al., 2001; Brock, 2005).

In several studies the effect of propionyl-CoA accumulation on cellular metabolism has been investigated. Examples do not only derive from Aspergillus nidulans and Aspergillus fumigatus (Brock and Buckel, 2004; Maerker et al., 2005) but also from the bacterium Rhodopseudomonas sphaeroides (Maruyama and Kitamura, 1985) and from liver hepatocytes of humans (Brass, 1992). Although the latter two both metabolize propionyl-CoA via the methylmalonyl-CoA pathway rather than the methylcitrate cycle, the inability to remove the common toxic intermediate propionyl-CoA severely affects primary metabolism. Detailed biochemical characterizations have shown that especially the pyruvate dehydrogenase complex and the succinyl-CoA synthetase are competitively inhibited by propionyl-CoA (Brock and Buckel, 2004). Mutants of Aspergillus species, which carry a deleted methylcitrate synthase gene, accumulate significant amounts of propionyl-CoA in the presence of propionate and, in turn, growth is severely retarded (Brock et al., 2000; Maerker et al., 2005). In addition, the inability of A. fumigatus mutants to remove toxic propionyl-CoA, which most likely results from protein degradation during invasive growth, strongly reduces the capability to establish an invasive aspergillosis (Ibrahim-Granet et al., 2008).

Besides the negative effect on primary metabolism, secondary metabolism is also affected by accumulating propionyl-CoA. In A. nidulans polyketide synthesis of naphtopyrone, sterigmatocystin and ascoquinone A (Zhang and Keller, 2004; Zhang et al., 2004) and in A. fumigatus the synthesis of the polyketide DHN melanin (Maerker et al., 2005) are inhibited in the presence of high propionyl-CoA concentrations. Therefore, accumulation of propionyl-CoA in methylcitrate synthase mutant strains can easily be visualized by reduction of the colour of conidia harvested from different carbon sources (Brock et al., 2000; Maerker et al., 2005).

We have previously shown that the addition of acetate to propionate-containing growth medium has a beneficial effect on growth and polyketide synthesis of both Aspergillus species, accompanied with a decreased propionyl-CoA level (Brock and Buckel, 2004; Maerker et al., 2005). This was mainly attributed to the competition of acetate and propionate for the activation to the corresponding CoA-ester. For A. nidulans it was shown that the activation of acetate is strongly preferred over that of propionate (Brock and Buckel, 2004). Besides this preferred activation of acetate, a CoA-transferase activity was detected, which was able to transfer the CoASH moiety from succinyl-CoA and propionyl-CoA to propionate and acetate. Therefore, we supposed that a transfer of the CoASH moiety from succinyl-CoA to acetate could relieve the blockage of the citric acid cycle at the level of succinyl-CoA synthetase. In addition, the transfer of the CoASH moiety from propionyl-CoA to acetate was assumed to be involved in the decrease of propionyl-CoA accumulation in a methylcitrate synthase mutant (Brock and Buckel, 2004). Our preliminary data revealed that CoA-transferase activities were extremely low when cells were grown on glucose medium and strongly increased by the addition of acetate and/or propionate. However, due to the determination from crude extracts, it remained unclear, whether the observed transferase activities belonged to a single enzyme or to a group of transferases.

The aim of this study was to attribute CoA-transferase activities to the respective enzymes. Enzyme purification and biochemical characterization of the substrate specificity was used for the identification of the corresponding gene(s). Furthermore, we generated deletion mutants for phenotypic characterization and elucidation of the importance of the CoA-transferase activities in propionyl-CoA detoxification.


Initial purification of the CoA-transferase and gene identification

The methylcitrate synthase deletion strain RYQ11 was used for initial purification of the CoA-transferase. This strain was chosen, because previous investigations revealed that a methylcitrate synthase deletion background leads to increased CoA-transferase levels (Brock and Buckel, 2004). For maximum activity the strain was grown on media containing acetate and propionate as carbon sources. The protein lost its activity quite rapidly during the purification procedure, when stored in a buffer lacking ammonium sulphate or glycerol. Nevertheless, purification to approximately 60% homogeneity was achieved by fractionated ammonium sulphate precipitation, chromatography on Phenyl-Sepharose (high performance) and a 1 ml Resource Phenyl column. The major band at approximately 55 kDa, most likely responsible for the CoA-transferase activity, was cut from the gel and subjected to tryptic digestion. Eluted peptides were analysed by matrix-assisted laser desorption/ionization − time of flight (MALDI-TOF) and compared with the fungal database by the program mascot. Sixteen peptides exactly matched to the hypothetical protein AN1547.3 (Accession XP_659151; formerly denoted as a putative acetyl-CoA-hydrolase) with a Mascot score of 79 (peptide mass fingerprint; PMF) and a sequence coverage of 48% (PMF). In subsequent descriptions the corresponding gene sequence was denoted as coaT.

cDNA synthesis and in silico analysis of the CoA-transferase

In order to confirm the predicted coding sequence of the coaT gene, cDNA was amplified from glucose/propionate-grown mycelium of the methylcitrate synthase mutant RYQ11 with sequence-specific primers. The amplified cDNA was subcloned and independent clones were sequenced. Comparison of the cDNA sequence (Accession No. AM920694) with that of the genomic DNA confirmed the predicted open reading frame of AN1547.3. The gene contains two short intron sequences with 50 and 48 bp and analysis by MITOPROT ( identified a mitochondrial import sequence, comprising the first 14 amino acids with a probability for mitochondrial import of 0.9428 (maximum = 1.0). The mature protein possesses a theoretical isoelectric point (pI) of 5.95 and a molecular subunit mass of 56.388 kDa, which is in good agreement with the apparent subunit mass of the above partially purified protein. A blast search against the non-redundant fungal database ( revealed that the protein is highly conserved among both filamentous fungi and yeasts with over 90% identity to homologues in other Aspergillus species (94% identity to the Aspergillus oryzae Accession No. BAE55631, 93% identity to the Aspergillus terreus Accession No. XP_001216368, 92% identity to the A. fumigatus Accession No. XP_747359, 92% identity to the Aspergillus niger Accession No. CAL00484) and 75% identity to the Neurospora crassa Accession No. P15937 (Acu8), 67% identity to the Candida albicans Accession No. XP_714720 and 66% identity to the Saccharomyces cerevisiae Accession No. NP_009583 (Ach1p). In general these proteins were denoted as acetyl-CoA-hydrolases, because some weak hydrolase activity has been observed for the S. cerevisiae acetyl-CoA-hydrolase Ach1p and for the N. crassa enzyme Acu8. Mutants of N. crassa, which carry a defective acu8 gene, displayed an increased intracellular acetyl-CoA pool when grown in the presence of acetate (Connerton et al., 1992). Therefore, it appeared likely that such a hydrolase may be involved in controlling an acetyl-CoA overflow, which may derive from an imbalanced acetyl-CoA synthesis in association with an insufficient capacity to shunt this intermediate into the glyoxylate bypass and the citric acid cycle. However, CoA-transferase activity had not been tested. Therefore, the postulated function as an acetyl-CoA-hydrolase will be discussed in a later section.

Overproduction and purification of the A. nidulans CoA-transferase

In order to characterize the biochemical properties of the CoA-transferase in more detail, we aimed in the overproduction of the enzyme in the original host strain A. nidulans. For that purpose the gene was fused with the isocitrate lyase promoter from A. nidulans and transferred into the recipient wild-type strain RMS011. The argB gene was used as a selection marker in transformation and resulting transformants were able to grow without the addition of arginine. Conidia of transformants were used to inoculate media containing glucose (20 mM) and propionate (100 mM) as carbon sources as the isocitrate lyase promoter is strongly induced in the presence of propionate (Brock and Buckel, 2004). Crude extracts from different transformants were checked for CoA-transferase activity using propionyl-CoA and acetate as substrates. Strain CoaT 1/5, which displayed a 20 times increased CoA-transferase activity compared with the wild type, was analysed by Southern blot analysis, revealing integration of at least four copies of the overexpression construct into the genome. Additionally, transcript levels of CoA-transferase and methylcitrate synthase were compared by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and confirmed that strain CoaT 1/5 displayed significantly higher levels of the coaT transcript than two different wild-type strains (details are provided in the Figs S1 and S2). Therefore, strain CoaT 1/5 was used for purification and biochemical characterization of the CoA-transferase. An optimized purification strategy was developed, which enabled the purification of the CoA-transferase by ammonium sulphate precipitation, hydrophobic interaction chromatography on Phenyl-Sepharose and dye affinity chromatography on Cibacron-Blue 3GA (Sigma-Aldrich). The dye binds enzymes, which possess a high affinity to NAD or other adenosine-containing cofactors such as coenzyme A and turned out to be more suitable than Reactive Red 120 agarose (data not shown), which displays similar properties in protein binding. The addition of glycerol to a final concentration of 20% strongly stabilized the enzyme during purification. A typical purification table and an SDS-polyacrylamide gel analysis are shown in Table 1 and Fig. 1A. The native subunit composition of the purified protein was analysed by blue native electrophoresis. A single band corresponding to a molecular mass of approximately 115 kDa was detected (Fig. 1B), which is in good agreement with a homodimeric structure of two identical subunits (calculated molecular mass: 112.8 kDa).

Table 1.  Purification record of the CoA-transferase from strain CoaT 1/5.
Purification stepActivity (U)Protein (mg)Specific activity (U × mg−1)Yield (%)Purification factor
  1. Specific activity was determined with propionyl-CoA and acetate as substrates. One unit is the amount of enzyme producing 1 μmol of acetyl-CoA per minute.

Cell-free extract90.63150.291001
(NH4)2SO4 precipitation65.72360.28731
Cibacron-Blue 3GA30.910.62.93410
Figure 1.

Purification and molecular mass determination of CoA-transferase from Aspergillus nidulans strain CoaT 1/5.
A. SDS-PAGE analysis of fractions from the CoaT purification from glucose/propionate-grown cells. The purified protein shows an apparent molecular subunit mass of 55 kDa. Lane M: molecular mass marker; lane 1: cell-free extract (15 μg); lane 2: (NH4)2SO4 fractionation (10 μg); lane 3: Phenyl-Sepharose (4.8 μg); lane 4: Cibacron-Blue 3GA (3.8 μg).
B. Blue native gel analysis for molecular mass determination of the native protein. Lane M: native molecular mass marker; lane 1: purified CoA-transferase. The protein shows a native apparent molecular mass of 115 kDa indicating a homodimeric protein structure.

Substrate specificity of purified CoA-transferase

We tested the purified enzyme for its activity with different donor and acceptor molecules as well as the hydrolase activity, in which the acceptor was omitted from the assay mixture and the release of CoASH from the donor molecule was monitored. As shown in Table 2, only acetyl-CoA, propionyl-CoA, succinyl-CoA and their corresponding free acids served as donor and acceptor molecules respectively. The highest specific activity was observed with the substrate couple succinyl-CoA and acetate, followed by the couple succinyl-CoA and propionate and by propionyl-CoA and acetate. Hydrolase activity was only detectable with CoA-esters, which were also active in the transferase reaction. In addition, hydrolase activity coincided strongly with stability of the CoA-esters at alkaline pH. Acetyl-CoA and propionyl-CoA are rather stable at pH 8.0 and only a slight increase in CoASH release compared with the natural decay was observed by addition of the protein. In contrast, succinyl-CoA spontaneously decomposed with a quite significant rate at alkaline pH and the increase in CoASH release was clearly detectable by the addition of enzyme. However, as the ratio between CoA-transferase and hydrolase was always in favour of that of the CoA-transferase reaction, the observed hydrolase activity seems to be a side-product, which occurs by binding of the donor in the absence of a suitable carboxylic acid acceptor molecule. The transfer of the CoA moiety from acetyl-CoA and propionyl-CoA to succinate was only measurable indirectly by the formation of the instable product succinyl-CoA and was monitored as an increase in CoASH release in comparison with that observed with the substrate-CoA in the absence of succinate.

Table 2.  Substrate specificity of purified CoA-transferase.
CoASH donorCoASH acceptorCoA-hydrolase (U mg−1)CoA-transferase (U mg−1)Ratio of hydrolase : transferase
  • a. 

    Activity detected by increased CoASH release from succinyl-CoA (unstable at pH 8.0).

  • b. 

    None detectable activity is marked with n.d.

Propionyl-CoAAcetate< 0.0012.9≥ 1:2900
Propionyl-CoASuccinate< 0.001Presenta

We then determined the Km values for acetate, when propionyl-CoA or succinyl-CoA served as CoA donors as well as the Km values for propionyl-CoA and succinyl-CoA in the presence of acetate as the acceptor molecule (Table 3). Interestingly, the donor type seems to influence the acceptor binding site, because acetate is bound more than six times more efficiently in the presence of propionyl-CoA than observed with succinyl-CoA. Therefore, the molecular size of the CoA donor molecule seems to influence the ability to bind acceptor molecules, which is also supported by the fact that propionate is 11 times less efficiently bound compared with acetate, when succinyl-CoA is used as the CoASH donor.

Table 3. Km values for substrates of the CoA-transferase.
CoA donorCoA acceptorKm donor (μM)Km acceptor (mM)

Physical and biochemical parameters of the CoA-transferase

Purified CoA-transferase required glycerol or ammonium sulphate to prevent denaturation and was stable when stored at −20°C in the presence of 50% glycerol. However, the enzyme lost up to 10% of activity within 5 h when stored at room temperature. At 50°C the enzyme displayed a half life of only 7 min. However, the maximum activity was achieved in a temperature range between 47°C and 52°C. Analysis of the pH stability revealed a similar stability at a pH range between 6.5 and 10.0, whereas the enzyme rapidly denatured irreversibly at pH values below 5 and above 11. The highest activity was observed in a pH range between 8.0 and 10.0. All these physical parameters fit well to an enzyme of a fungal strain with a growth optimum at 37°C.

We furthermore investigated the effect of different divalent cations, chelators and nucleotide-containing cofactors on CoA-transferase activity. Metals tested were Mg2+ (> 5 mM), Cu2+ (80 μM), Zn2+ (350 μM) and Fe2+ (400 μM). None of the metal ions had a stimulatory effect but all showed some reduction of enzymatic activity in the following order: Cu2+ > Zn2+ > Fe2+ = Mg2+. EDTA, which can act as a chelator of divalent cations, showed no effect on CoA-transferase activity when applied at a concentration of 6 mM. The same was true for NAD, NADP and ATP (all applied in a concentration of 1.6 mM) as well as NADH and NADPH (both used at a concentration of 0.7 mM). Therefore, the CoA-transferase does not seem to be dependent on any metal ion or cofactor to display full activity. When 3-nitropropionate, a structural analogue to succinate, was added in a concentration of up to 7 mM, no inhibition was observed, indicating that the active site is strongly adapted to binding of specific substrates.

Classification of CoaT

Class I CoA-transferases are rapidly inactivated by either the addition of hydroxylamine or the addition of borohydride. To determine the effect of these inhibitors on enzyme activity of CoaT, the enzyme was pre-incubated for 30 min with a 1000-fold excess of propionyl-CoA to ensure the complete charging of the active site with the activated glutamyl-CoA-thioester. As both borohydride and hydroxylamine did not lead to inactivation of the enzyme (100% residual activity with borohydride after 30 min of subsequent incubation at 22°C and 95% residual activity with hydroxylamine after 70 min), we can exclude that CoaT belongs to class I of CoA-transferases.

Class II CoA-transferases generally consist of three different subunits, in which the α-subunit is defined as the CoA-transferase, the β-subunit as a lyase and the γ-subunit as an acyl-carrier protein (ACP). Members of this family are citrate and citramalate lyases. The substrate does not become covalently bound rather than reacts via a ternary intermediate (Heider, 2001). Although these CoA-transferases are not inhibited by hydroxylamine or borohydride, we can exclude this class of transferases for CoaT. Class II CoA-transferases do not act on free CoA-thioesters rather than containing and producing an internally activated CoA-thioester at the ACP. As CoaT was shown to consist of a homodimeric structure, which excludes the heterotrimeric structure described for class II CoA-transferases and additionally does not seem to contain an acyl-carrier domain, the class II definition does not fit to our enzyme.

Class III CoA-transferases have not been studied extensively. Only four enzymes have been described and all belong to prokaryotic organisms. It is presumed that the reaction mechanism proceeds similar to that of class II CoA-transferases, except that no acyl carrier protein is involved in the formation of a ternary complex, rather that the CoA-thioester itself is involved in this formation. All of the characterized CoA-transferases act on free diffusing CoA donor and acceptor substrates, which fits well with our observations of CoaT. Even more, CoaT displays 44% identity and 66% similarity to Cat1, a succinyl-CoA:coenzyme A transferase from Clostridium klyveri (Accession P38946), which is involved in the anaerobic succinate degradation (Sohling and Gottschalk, 1996). Therefore, we conclude that CoaT belongs to the class III of CoA-transferases providing the first evidence of a class III CoA-transferases from a eukaryotic organism.

Regulation of gene expression on different carbon sources

Determination of the CoA-transferase activity from crude extracts was problematic because of high background activities observed on the CoA-substrates. Nevertheless, preliminary determinations of enzymatic activities suggested that induction of CoA-transferase requires the presence of carboxylic acids in the growth medium (Brock and Buckel, 2004). For a more detailed analysis of coaT induction the promoter sequence was cloned in frame with the β-galactosidase gene from Escherichia coli. A. nidulans transformants were screened by Southern blot analysis (data not shown) and one of the strains (SCF/lacZ9), containing a single-copy integration, was selected for the determination of promoter activity after growth on different carbon sources. Results are presented in Table 4. Mycelium grown on glucose medium only displayed a low β-galactosidase activity, indicating a basal level of expression. Mycelium grown on glycerol displayed an activity, which was increased by a factor of < 3, which leads to the suggestion that a possible carbon catabolite repression mediated by glucose is relieved. Ethanol led to an even stronger induction of promoter activity compared with glucose and glycerol. This indicates that the four putative binding sites of FacB, the transcriptional activator of acetate utilization genes (Todd et al., 1998), which were detected in the promoter region (not shown), may be involved in coaT regulation. However, the obtained values on ethanol were still much lower than those observed on acetate or propionate. Therefore, it seems quite likely that an additional, yet unidentified, transcriptional activator binds to the promoter region of the coaT gene. Interestingly, the presence of acetate or propionate together with glucose, glycerol or ethanol led to strong induction of promoter activity, which implies that sensing of one of the carboxylic acids is sufficient to upregulate promoter activity.

Table 4.  Induction of coaT promoter activity after growth on different carbon compositions.
Carbon sourcesGrowth time (h)Specific activity (mU mg−1)Fold induction (relative to glucose)
  • a. 

    Activity resembles that from propionate grown cells. Glucose was added to support initial growth and cultures were incubated for further 10 h after complete glucose consumption.

  • G, glucose; Gly, glycerol; EtOH, ethanol; P, propionate; Ac, Acetate. Numbers denote the concentration of the respective carbon sources in mM.

G 502079 ± 101
Gly 10021210 ± 82.7
EtOH 100211000 ± 4412.7
Ac 100223184 ± 9340.3
G 10/P 100a37.53155 ± 16239.9
Ac 100/P 100223770 ± 9947.7
G 50/P50211160 ± 35814.7
G 50/Ac 10037.52570 ± 8532.5
G 50/Ac 100/P 100202653 ± 22533.6
Gly 100/P 50412448 ± 5031.0
EtOH 100/P 50412763 ± 15235.0

Subcellular localization of CoaT

Sequence analysis revealed that the CoA-transferase contains a mitochondrial targeting sequence at the N-terminal part of the protein. To demonstrate the functionality of the mitochondrial import sequence, a 772 bp promoter fragment of coaT was used to control the expression of a fusion protein consisting of the 5′ coding region of the coaT gene fused in frame with the gene coding for the eGFP protein. As a selection marker for transformation the ornithine carbamoyl transferase gene argB from A. nidulans was used. Strain RMS011 was the recipient strain in transformation and positive clones were selected for fluorescence microscopy. Strains were grown on acetate medium, because this medium strongly induced coaT expression as shown above. For subcellular localization of the fusion protein MitoTracker red 580 was used as a comparative tool. As shown in Fig. 2, eGFP fluorescence displayed the same pattern of distribution as observed with the MitoTracker. From this experiment we conclude that the mitochondrial import sequence of the CoA-transferase is functional and that the protein indeed localizes to mitochondria.

Figure 2.

Subcellular localization of CoA-transferase using eGFP and MitoTracker. Left: Green fluorescence of eGFP from a strain carrying a fusion of eGFP downstream of the N-terminal region of CoaT, which spans the predicted signal peptide for mitochondrial import. Right: Same hyphae as shown on the left side but fluorescence emitted by the MitoTracker was recorded, which specifically stains mitochondria. Both pictures show a similar distribution of fluorescence signals, which indicates a mitochondrial localization of the fusion protein.

Deletion of the coaT gene and complementation of a mutant strain

In order to study the importance of the CoA-transferase during growth on different carbon sources a deletion mutant was generated. The deletion construct contained the argB gene from A. nidulans flanked by the upstream and downstream region of the coaT gene. Two independent constructs were used, which differed in the orientation of the argB gene between the flanking regions. After transformation of A. nidulans strain RMS011 several transformants were randomly selected and Southern blot analysis with a probe directed against the downstream flanking region of the coaT gene was performed. The expected fragment size in the wild type after EcoRV restriction was 10678 bp and a shift to either 3227 or 2770 bp was expected in deletion strains, depending on the orientation of the argB gene. In total, three deletion mutants were obtained (for details on the Southern analysis refer to Fig. S3). One of these deletion strains, denoted as ΔcoaT 1/12, was selected for subsequent phenotypic characterization.

For complementation of the mutant ΔcoaT 1/12 two independent strategies were chosen. One strategy involved the backtransfer of the gene into the deletion strain by transformation and use of the pabaA gene encoding p-aminobenzoic acid synthase as an auxotrophic marker. A single transformant was obtained, which was prototroph for both arginine and p-aminobenzoic acid, indicating an ectopic integration of the complementation construct (Southern analysis provided in Fig. S4). In the second strategy a sexual crossing of the deletion mutant with the biotin and arginine auxotrophic strain SCF 3 was performed. Offsprings were selected for arginine and p-aminobenzoic acid prototrophy, which was indicative for sexual crossing of both strains and a replacement of the chromosome carrying the coaT deletion. Although the latter complemented strains SCF 7.1 and SCF 7.2 behaved identical to arginine auxotrophic wild-type strains, they were not included in subsequent descriptions, because addition of arginine to prototrophic strains influenced the conidial colour, which was a central point in phenotypic characterization.

Phenotypic characterization of the coaT deletion strain

To investigate the effect of the carbon composition on morphology and growth of the coaT deletion strain agar plates containing different carbon compositions were inoculated with an arginine prototrophic wild-type strain (SCF 1.2), the deletion strain ΔcoaT 1/12 and the complemented strain obtained by re-transformation of the mutant. No phenotype was observed with the mutant, when grown on glucose, acetate, glycerol or ethanol used as the sole carbon sources. However, the mutant was more sensitive to the addition of propionate, especially when glycerol was the second carbon source (Fig. 3). On these media the colour of conidia was lost in dependence of the amount of propionate added, indicating some accumulation of propionyl-CoA in the coaT deletion strain. On glucose/propionate medium no enhanced phenotype was observed, indicating a low activation of propionate and a sufficiently high methylcitrate synthase activity to remove propionyl-CoA. On acetate, propionate induced a phenotype but higher propionate concentrations were required compared with glycerol, which is consistent with a competition of acetate and propionate for activation to the respective CoA-esters by acyl-CoA synthetases. This hypothesis was furthermore confirmed by the observation that an excess of acetate restored the phenotype of the mutant on glycerol and propionate-containing medium. As the mutant was also able to grow on propionate as sole carbon and energy source (not shown), we can conclude that the enzyme is not essential for growth under any of the investigated conditions but it seems to be involved in release of propionyl-CoA toxicity in the presence of alternative carbon sources.

Figure 3.

Phenotypic characterization of wild type (WT), coaT deletion mutant 1/12 (ΔcoaT) and complemented mutant (Comp) grown on solid media containing different carbon sources. G, glucose; P, propionate; Ac, acetate; Gly, glycerol. Numbers denote the millimolar concentration of the respective carbon source. The wild-type strain and the complemented strain hardly reveal any differences, whereas the mutant strain shows some reduction in spore colour formation when propionate was used in combination with acetate or glycerol, indicating an accumulation of propionyl-CoA.

Phenotypic characterization of a coaT and mcsA double-deletion strain

The phenotypic characterization of the coaT deletion strain together with the substrate specificity of the purified protein led to the assumption that the CoA-transferase is involved in the reduction of the propionyl-CoA level under certain growth conditions. Interestingly, the phenotype of the ΔcoaT strain on acetate/propionate medium was rather weak, although β-galactosidase assays showed that the gene was strongly transcribed. We therefore speculated that acetate is preferably activated to the corresponding CoA-ester and the small amounts of activated propionate become removed by the synchronous action of the methylcitrate cycle. However, previous investigations on a methylcitrate synthase mutant showed that the level of propionyl-CoA accumulation remained low when the mutant was grown on acetate/propionate-containing medium. This observation was accounted to the action of a CoA-transferase, which efficiently transfers the CoASH moiety from propionyl-CoA to acetate and thereby reduces the toxification by propionyl-CoA (Brock and Buckel, 2004). To prove this assumption a double-deletion strain (SCF 11), defective in both coaT and mcsA, was generated by sexual crossing of the single mutants. After verification of the deletions by marker segregation, PCR and Southern blot analysis (Fig. S5), a wild-type strain (SCF 1.2), the single mutant ΔcoaT (SCF 8), the single mutant ΔmcsA (SCF 9) and a double mutant ΔcoaTmcsA (SCF 11) were compared for their growth behaviour on different media (Fig. 4). As expected, no phenotype of either one of the strains was observed, when glucose, acetate, ethanol or glycerol were used as sole carbon source. The addition of propionate to the single-carbon sources led to a complete loss of spore colour formation of the methylcitrate synthase mutant except on acetate/propionate-containing medium, on which it behaved similar to the wild type, confirming previous investigations. However, the phenotype of the ΔmcsA strain had not previously been tested on ethanol/propionate and it was surprising that the strain grew poorly on this carbon composition. This indicates that ethanol cannot compete for the activation of propionate to propionyl-CoA and, furthermore, no acceptor molecule is present in sufficient amounts to which the CoASH moiety could be transferred. However, a determination of the exact propionyl-CoA level was not possible due to the extremely low growth rate under this condition. The ΔcoaTmcsA double mutant mainly behaved like the ΔmcsA strain when grown on glucose/propionate and glycerol/propionate medium. However, the phenotype on ethanol/propionate was slightly enhanced. Most important, outgrowth of this double mutant on acetate/propionate medium was completely abolished. This was also confirmed by inoculation of liquid media. Even when high conidia concentrations (1 × 107 conidia ml−1) were applied to acetate/propionate-containing liquid media, no outgrowth was observed after 1 week of incubation (not shown). This clearly demonstrates an essential role of the CoA-transferase on removal of toxic propionyl-CoA especially in a methylcitrate synthase negative background and confirms the speculations on the role of such a CoA-transferase on acetate/propionate-containing medium (Brock and Buckel, 2004).

Figure 4.

Phenotypic characterization of different A. nidulans strains grown on various carbon sources. Wild-type SCF 1.2 = WT; CoA-transferase deletion mutant SCF 8 = ΔcoaT; methylcitrate synthase deletion mutant SCF 9 = ΔmcsA; double-deletion mutant SCF 11 = ΔcoaTmcsA. Carbon sources utilized consisted of: G, glucose; P, propionate; Ac, acetate; EtOH, ethanol; Gly, glycerol. Numbers denote the millimolar concentration of the respective carbon source. Note that the double-deletion mutant is not able to germinate on acetate/propionate-containing medium, whereas all other strains are able to grow on this medium (highlighted by a box).


In this study we identified a CoA-transferase, which is produced in the presence of carboxylic acids, mainly acetate and propionate. The enzyme is classified as a class III CoA-transferases, because it displays a homodimeric structure, contains no ACP as in class II CoA-transferases and, unlike class I CoA-transferases, is not inhibited by hydroxylamine or borohydride. In class I CoA-transferases an active site glutaryl residue performs a nucleophilic attack on the donor CoA-thioester. This leads to the formation of an enzyme-bound acyl-glutamyl anhydride and the CoA moiety stays in the anionic form within the active site pocket. This anion performs a nucleophilic attack on the acyl-glutamyl anhydride, which leads to the formation of an enzyme bound glutamyl-CoA-thioester and the release of the free acid of the donor CoA-thioester. By entering of the acceptor carboxylic acid into the active site the reaction proceeds into the reverse reaction, which completes the transfer of the CoA moiety (Heider, 2001). As both hydroxylamine and borohydride attack the intermediate glutamyl-CoA-thioester in class I CoA-transferases, enzymatic activity becomes irreversibly inhibited. Hydroxylamine leads to the formation of a hydroxamate at the activated glutamyl-CoA, whereas borohydride reduces the glutamyl residue to a glutamyl-alcohol, whereby the CoA residue stays covalently bound to the enzyme (Selmer et al., 2002). In class III CoA-transferases the CoA-thioester forms a ternary complex without becoming covalently bound to the enzyme, which eliminates a possible inhibition by borohydride or hydroxylamine and is in agreement with our observations on CoaT. Therefore, this study provides the first report of a class III CoA-transferase from a eukaryotic source which, however, seems to be widespread among fungi, as highly homologous proteins are present in all fungal genomes that we analysed.

We had previously speculated the existence of a CoA-transferase, because a methylcitrate synthase mutant showed a strong accumulation of propionyl-CoA when grown on glucose/propionate medium but only a slight accumulation on acetate/propionate medium (Brock and Buckel, 2004). Additionally, CoA-transferase activity was detected acting on succinyl- and propionyl-CoA as donor and acetate and propionate as acceptor molecules. However, as enzyme activities were only determined from crude extracts it was not clear whether all activities belonged to a single or a group of enzymes and, furthermore, the impact of this activity on propionyl-CoA detoxification was unresolved.

Due to the purification and characterization of CoaT we can now clearly state that all activities derived from a single enzyme. Acetyl-, propionyl- and succinyl-CoA were the sole CoASH donors and only their corresponding acids acted as acceptors. Although the highest specific activity was observed with succinyl-CoA and acetate, the transfer reaction with this substrate couple seems unlikely to occur under in vivo conditions because the Km value for the substrate acetate in combination with succinyl-CoA was 2.5 mM. Furthermore, the transfer between succinyl-CoA and propionate seems unlikely to occur under in vivo conditions as 28 mM propionate was needed to reach the Km value for propionate. The reaction most likely proceeding under in vivo conditions is that of the substrate couple propionyl-CoA and acetate. Although this reaction displayed the lowest specific activity, the Km values for both substrates were in a realistic range for in vivo conditions (10 μM for propionyl-CoA and 0.38 mM for acetate). This assumption is furthermore supported by the growth behaviour of the methylcitrate synthase/CoA-transferase double-deletion mutant on acetate/propionate medium. Although both single mutants grew on acetate/propionate medium, growth of the double mutant was completely abolished. We conclude that in the wild-type situation propionyl-CoA is detoxified on this medium by transfer of the CoASH moiety to acetate without the urgent need of the methylcitrate synthase, which is in agreement with the minor phenotype of the methylcitrate synthase deletion strain. In contrast, when only the CoA-transferase is deleted, methylcitrate synthase is sufficiently active to remove the propionyl-CoA. The double deletion of both genes in A. nidulans therefore completely blocks the removal of the toxic compound, leading to a high accumulation of propionyl-CoA and an inability to grow. Unfortunately, we were not able to determine the propionyl-CoA level of the double-deletion strain on acetate/propionate medium as conidia did not germinate.

We also investigated the subcellular localization of the enzyme. The protein contains a mitochondrial import sequence and the fusion of the N-terminal part of the protein with the green fluorescent protein localized to mitochondria, confirming the functionality of the leader peptide. The mitochondrial localization of the CoA-transferase implies that the transfer reaction also occurs within this compartment. Uptake of acetate and propionate by filamentous fungi has not been studied in detail. However, both acetate and propionate may enter the cytoplasm by a specific transport system. A blast search of the A. fumigatus and A. nidulans genome revealed the existence of GPR/FUN34 family proteins (AFUA_2G04080A and AN5226.2), which display 53% and 50% identity to the yeast acetate transporter Ady2p, which was shown to be essential for the active uptake of acetate (Paiva et al., 2004) and may also be able to transport propionate. Propionyl-CoA is formed within the cytoplasm and is shuttled over the mitochondrial membrane via the carnitine transport system (M. Brock, unpubl. data) providing the CoASH donor inside of this compartment. However, acetate is also activated within the cytoplasm and uses the same carnitine transport system to enter mitochondria and it remains unclear which amounts of free acetate might be present within the mitochondria to act as a CoASH acceptor.

Interestingly, the amino acid sequence of our CoA-transferase showed a high identity to the acetyl-CoA hydrolases Ach1p from S. cerevisiae and Acu8 from N. crassa (66% and 75% respectively). Although CoaT also displayed some hydrolase activity, this hydrolase activity was always much weaker than that of the CoA-transferase reaction. However, CoA-transferase activity was not tested for the N. crassa enzyme and a preliminary attempt on the yeast enzyme Ach1p to measure such an activity failed (Buu et al., 2003). Nevertheless, detailed analysis on the purified proteins from N. crassa and S. cerevisiae are required to exclude a transferase activity for these enzymes. The explanations for the requirement of such an energy wasting acetyl-CoA hydrolase, e.g. for the prohibition of autoacetylation of other enzymes in the presence of high acetyl-CoA levels (Lee et al., 1990) or for balancing the acetyl-CoA levels between different compartments (Connerton et al., 1992), are not satisfying and the existence of such an enzyme was generally denoted as a biochemical conundrum (Lee et al., 1996; Buu et al., 2003). However, both acetyl-CoA hydrolase mutants showed a phenotype, which was never observed in our coaT deletion mutant and which may point to different functions of the enzymes. Both the S. cerevisiae mutant and the N. crassa mutant were impaired in growth when acetate was used as the sole carbon and energy source, showing a delay of several days until exponential growth started. However, the overall biomass yield of the mutants and their respective wild-type strains was comparable (Lee et al., 1990; Marathe et al., 1990). Therefore, at least a regulatory role in trafficking of internal metabolites could be attributed to these hydrolases. However, this phenotype was restricted to acetate as ethanol used as a carbon source did not lead to this growth defect, which points to an importance of free acetate in the function of the hydrolase, which is similar to our observation on the CoA-transferase. Currently we are trying to re-investigate the substrate specificity and activity of the yeast Ach1p to obtain deeper insights on the function of this enzyme. Detailed analysis of Ach1p will show whether the enzyme also acts as a CoA-transferase. Additionally, we will try to complement the A. nidulansΔcoaTmcsA double mutant with the yeast gene to study the phenotype of the resultant strain.

In conclusion, the coaT gene from A. nidulans codes for a CoA-transferase with only a minor hydrolase activity. The enzyme is mainly required for the transfer of the CoASH moiety from propionyl-CoA to acetate, thereby reducing the amount of toxic propionyl-CoA. A CoA-transferase mutant in a wild-type background only displays minor phenotypes on media containing propionate and behaves like the wild type when no propionate is present. As the growth inhibition and reduction of spore colour formation of a methylcitrate synthase mutant is enhanced in a coaT mutant background, we attribute a supporting function in propionyl-CoA detoxification to the CoA-transferase.

Experimental procedures


Chemicals were obtained from Sigma-Aldrich (Taufkirchen, Germany), and columns and chromatographic media were, if not indicated otherwise, from GE Healthcare (Freiburg, Germany). Suppliers of other materials and enzymes are indicated in the text.

Growth and maintenance of Aspergillus strains

All strains used in this investigation were maintained on solid agar plates containing Aspergillus minimal medium with 50 mM glucose as carbon source and supplements as required. For a list of strains refer to Table 5. Liquid media were generally inoculated with 5 × 106 to 1 × 107 conidia ml−1 (final concentration) and grown at 37°C under vigorous shaking at 220 r.p.m. on a rotary shaker. For phenotypic characterization glucose, acetate, ethanol, propionate and glycerol in different combinations and concentrations were used as described in the single experiments. Plates were incubated at 37°C for at least 48 h and phenotypes were documented by digital imaging (Nikon D 100).

Table 5. Aspergillus nidulans strains used in this study.
RYQ11ΔmcsA::argB, biA1; veA1Zhang and Keller (2004)
RMS011pabaA1, yA2; ΔargB::trpCΔB; veA1, trpC801Stringer et al. (1991)
A26biA1Fungal Genetics Stock Center Kansas City, KS
ΔmcsA1pyrG89, ΔmcsA::argB; ΔargB::trpCΔB; pyroA4; veA1, trpC801Brock et al. (2000)
ΔcoaT 1/12pabaA1, yA2; ΔargB::trpCΔB; ΔcoaT::argB; veA1, trpC801This study
SCF 1.2
(RMS011 × A26)
pabaA1, yA2; veA1This study
(RMS011 × A26)
biA1; ΔargB::trpCΔB; veA1, trpC801This study
SCF 7.1
coaT 1/12 × SCF 3)
pabaA1, yA2; ΔargB::trpCΔB; veA1, trpC801This study
SCF 7.2
coaT 1/12 × SCF 3)
pabaA1, yA2; ΔargB::trpCΔB; veA1, trpC801This study
coaT 1/12 × ΔmcsA1)
pabaA1, yA2; ΔargB::trpCΔB; ΔcoaT::argB; pyroA4; veA1, trpC801This study
(ΔcoaT 1/12 × ΔmcsA1)
pabaA1; ΔmcsA::argB; ΔargB::trpCΔB; pyroA4; veA1, trpC801This study
SCF 11
coaT 1/12 × ΔmcsA1)
pabaA1; ΔmcsA::argB; ΔargB::trpCΔB; pyroA4; ΔcoaT::argB; veA1, trpC801This study
CoaT 1/5pabaA1, yA2; ΔargB::trpCΔB; veA1, trpC801; multiple copies of plasmid pIclCoaTargB1This study
ΔcoaT 1/12 complementedyA2; ΔargB::trpCΔB; ΔcoaT::argB; veA1, trpC801; multiple copies of coaT-pabaAThis study
SCF/lacZ9pabaA1, yA2; ΔargB::trpCΔB; veA1; trpC801; single copy of coaTp:lacZThis study

Enzyme assays

CoA-transferase activities were determined as previously described (Brock and Buckel, 2004). In general the transfer of the CoASH moiety to acetate was tested by use of citrate synthase from pig heart (Roche Diagnostics; Mannheim, Germany) and oxaloacetate in a citrate-forming reaction. Transfer to propionate was determined by use of purified methylcitrate synthase from A. nidulans (Brock et al., 2000) and oxaloacetate in a methylcitrate-forming reaction. Transfer of the CoASH from acetyl-CoA or propionyl-CoA to succinate was tested by an indirect assay with purified enzyme, which based on the significantly higher instability of succinyl-CoA at alkaline pH compared with both acetyl-CoA and propionyl-CoA respectively. Hydrolase activities were determined by measuring the increase of CoASH release from acetyl-CoA, propionyl-CoA and succinyl-CoA after addition of enzyme in the absence of any other alternative acceptor. The release of CoASH was always detected with 5,5′-dithiobis-(2-nitrobenzoic acid) at 412 nm, taking a millimolar extinction coefficient of 13.6 mM−1 cm−1 (Srere, 1963).

Partial purification of CoA-transferase from methylcitrate synthase mutants

For initial purification of the CoA-transferase the methylcitrate synthase mutant RYQ11 (Table 5) was used. Strain RYQ11 was grown on a medium containing a mixture of 100 mM acetate and 100 mM propionate as carbon sources. The mycelium was harvested over miracloth (Calbiochem; Darmstadt, Germany), ground to a fine powder under liquid nitrogen and re-suspended in 20 mM Tris-HCl buffer pH 8.0 (buffer A). Cell debris was removed by centrifugation at 20 000 g and the supernatant was taken for fractionated ammonium sulphate precipitation. The protein pellet obtained after precipitation between 40% and 70% saturation was dissolved in a minimum amount of buffer A and loaded on a Phenyl-Sepharose column (bed volume 20 ml) previously equilibrated with 20 mM Tris-HCl buffer pH 8.0 containing 1 M ammonium sulphate (buffer B). The column was eluted with a linear ammonium sulphate gradient ranging from 100% buffer B to 100% buffer A. Enzyme-containing fractions were collected and concentrated by the use of centrifugal filter devices (Millipore; Schwalbach, Germany, cut-off 30 kDa). The concentrate was loaded on a Resource-Phenyl column (bed volume 1 ml) equilibrated with buffer B. Elution was performed as described above. Fractions were concentrated and desalted by centrifugal filter devices and loaded on a ResourceQ column, previously equilibrated with buffer A. Enzymes were eluted by a sodium chloride gradient from 0 to 1 M in buffer A. Fractions were checked for CoA-transferase activity and purity was determined either by use of a 15% SDS-polyacrylamide gel (Laemmli, 1970) or by use of NuPage 4–12% Bis/Tris gradient gels (Invitrogen GmbH; Karlsruhe, Germany).

MALDI-TOF analysis

Protein-containing bands were excised from the SDS-polyacrylamide gel and tryptic digestion of proteins was performed as recommended by the manufacturer (Promega; Mannheim, Germany). The resulting peptides were eluted with 0.1% TFA in 50% acetonitrile. Eluates were mixed with the matrix hydroxycinnamic acid for subsequent ionization of peptides and MALDI-TOF analysis was performed on a Bruker Daltonics machine. Peptide masses were used for protein identification via the mascot interface (mascot 2.1.03, Matrix Science, London, UK).

Isolation of RNA, reverse transcription and sequencing of cDNA

Strain RYQ11 was grown for 36 h on a medium containing 50 mM glucose and 100 mM propionate. The mycelium was harvested and frozen in liquid nitrogen and approximately 0.1 g was ground to a fine powder. For RNA extraction, the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) including a DNase treatment was used. An aliquot was taken as a template for first-strand synthesis using the SuperScript First-Strand Synthesis system for RT-PCR (Invitrogen) and the gene-specific oligonucleotide cDNACoAT_down (5′-CAC CCA AGT ATA CCA AAT ATC G-3′). For second-strand synthesis and amplification an aliquot was removed and amplified with oligonucleotides cDNACoAT_up (5′-CTC TGC CGT CCT TTT CGT C-3′) and cDNACoAT_down by use of DynaZyme EXT DNA polymerase (BioCat, Heidelberg, Germany). The resulting PCR product was cloned into the PCR2.1 vector (Invitrogen) and sequenced from both strands by SeqLab (Göttingen, Germany). For the identification of intron regions the sequence was blasted against the genome database from A. nidulans (

Cloning of the CoA-transferase-coding region under the control of the acuD promoter

The promoter region of the acuD gene, coding for isocitrate lyase (Bowyer et al., 1994), was used for overproduction of the CoA-transferase. The promoter was amplified from genomic DNA of A. nidulans RMS011 by use of DynaZyme EXT DNA Polymerase (BioCat, Heidelberg, Germany) with the oligonucleotides IclPromoBglII_up (5′-AGA TCT GTC GTA TTC CAC AAG GAT G-3′; BglII restriction site is shown in bold) and IclPromoBglII_down (5′-AGA TCTCAT GAT GGC AGT ATT CAG-3′; BglII restriction site is shown in bold, reverse complement ATG in italic). The resulting fragment was cloned into the PCR2.1 vector (Invitrogen) and subsequently excised with BglII. The gene AN1547.3 (identified by peptide mass analysis of the partially purified protein to code for the CoA-transferase) was amplified from genomic DNA of strain RMS011 with oligonucleotides BglCoATup (5′-AGA TCT GCT TCC GCC CTG CTC-3′; BglII restriction site is shown in bold) and CoATdown (5′-GCA TAT ATC CGG TAT ACT CAG-3′) with the same polymerase and cloned into the pDrive vector (Qiagen, Hilden, Germany). The resulting vector was linearized with BglII and the acuD promoter was subcloned. Orientation of the promoter fragment was checked by PCR with oligonucleotides IclPromoBglII_up and CoAT_mi_up (5′-GAG TGT GTA CTT GAG CTT GC-3′). As an auxotrophic marker for transformation the argB gene from A. nidulans was used, which was restricted by NotI from plasmid pΔmcsA (Brock et al., 2000) and cloned into the NotI restriction site of the vector containing the picl::coaT fusion. The coding region of the resulting plasmid pIclCoaTargB1 was sequenced from both strands to confirm promoter fusion and reading frame and was used for transformation of A. nidulans RMS011 by standard procedures (Ballance et al., 1983). For overproduction of the CoA-transferase transformants were grown in media containing 20 mM glucose and 100 mM propionate (Brock and Buckel, 2004), which induced the expression of the CoA-transferase gene from the isocitrate lyase promoter.

Purification of CoA-transferases from the overproducing strains CoaT 1/5

The overproducing strain CoaT 1/5, which contained several copies of the CoA-transferase-coding region under the control of the isocitrate lyase promoter (Table 5; Southern analysis in Fig. S1), was used for homogenous purification of the CoA-transferase. The strain was grown for 42 h in a medium containing 20 mM glucose and 100 mM propionate as carbon sources. Mycelium was harvested, ground under liquid N2 and fractionated by (NH4)2SO4 precipitation as described above. After chromatography on Phenyl-Sepharose enzyme-containing fractions were concentrated, desalted and diluted in buffer C [50 mM Tris-HCl pH 8.0 with 20% (v/v) glycerol]. The fraction was loaded on a Cibacron-Blue 3GA column (bed volume 2 ml), previously equilibrated with buffer C. Elution was performed with buffer D (buffer C with 2 M NaCl) and enzyme-containing fractions were concentrated and desalted as described above. For storage the glycerol content was brought to 50% (v/v) and the enzyme was frozen at −20°C without significant loss of activity.

Determination of Km values

The apparent Km values for the different substrates propionyl-CoA, succinyl-CoA, acetate and propionate were determined by varying the concentration of one substrate, whereas that of the other was kept constant. The Km value was calculated from Lineweaver–Burk plots using at least six independent substrate concentrations for each determination.

Temperature and pH stability and dependency

For all biochemical characterizations with respect to temperature and pH, the CoA-transferase reaction with the substrate couple propionyl-CoA and acetate was utilized. Temperature stability was measured by incubating the CoA-transferase for different points of time in 50 mM Tris-HCl buffer (pH 8.0) at 4°C, 23°C, 40°C, 50°C and 60°C. For determination of pH stability the enzyme was incubated at room temperature for distinct time periods in a coupled buffer system containing 0.1 M boric acid, 0.1 M acetic acid and 0.1 M phosphoric acid, whereby the pH was adjusted with 10 M NaOH in increments of 0.5 to pH 5−11.

The temperature dependency of the CoA-transferase activity was measured in a range of 9−67°C in 50 mM Tris-HCl buffer (pH 8.0) by adjusting the temperature of the assay mixture within the spectrophotometer by a connected water bath. The pH dependency was measured in a range of pH 5−11 by exchanging the Tris-HCl buffer in the standard assay with the coupled buffer system described above.

Effectors of CoA-transferase activity

In order to determine the effect of different putative inhibitors or activators of the CoA-transferase, CuSO4, MgCl2, MgSO4, ZnSO4, FeSO4, (NH4)2SO4, EDTA, NAD, NADP, NADH, NADPH, ATP and 3-nitropropionate were added in varying concentrations (between 4 μM and 10 mM). All effectors were added to the standard assay mixture before the assay was started by the addition of acetate. As a reference for 100% of activity, the standard assay without the addition of one of the effectors was used.

Effect of hydroxylamine and borohydride on CoA-transferase activity

In order to classify the CoA-transferase to one of the three described families, two types of inactivation experiments were performed. (i) Inhibition by hydroxylamine: Purified enzyme (204 μg of protein; 3.6 nmol) was loaded with the CoA donor by pre-incubation for 30 min at room temperature in 100 μl of 50 mM Tris-HCl buffer (pH 8.0) with 20% glycerol and 1.7 μmol of propionyl-CoA. After pre-incubation 44 μmol of hydroxylamine was added, whereas in a control reaction an equal amount of water was applied. After different points of time aliquots were taken and the CoA-transferase activity was measured in comparison with the control reaction. (ii) Inhibition by sodium borohydride: Purified enzyme (627 μg; 11.1 nmol) was mixed in 100 μl of a 50 mM Tris-HCl buffer (pH 8.0) with 20% glycerol and 2 μmol of propionyl-CoA and incubated for 30 min at room temperature. Thereafter, 3 μmol of sodium borohydride (or water in the control reaction) was added. Aliquots were taken after different incubation times and enzyme activity in comparison with the control reaction was measured in the standard assay.

Construction of a CoA-transferase::lacZ reporter strain

To study the promoter activity of the CoA-transferase gene during growth on various carbon sources, the promoter region of coaT (1012 bp including ATG) was amplified with oligonucleotides BamH_PromCoAT_dow (5′-GGA TCC CAT TAT GAG AAG AAT TGA CGA AAA GG-3′) and BamHI_PromCoAT_for (5′-GGA TCC CTG CCG GTC CTA AAA ATG-3′) and ligated into the pCR4 sequencing vector (Invitrogen). The promoter region of coaT was cut from the vector by BamHI restriction and ligated with the previously BamHI-restricted and dephosphorylated vector pAN923-41B (van Gorcom et al., 1986), which contains the lacZ gene from E. coli in frame with the BamHI site. In addition, the vector contains the argB gene from A. nidulans, which was used as selection marker in the subsequent transformation of the A. nidulans strain RMS011. Transformants were checked by Southern blot analysis and a single-copy transformant was used for determination of the β-galactosidase activity, which was measured in a continuous assay as described earlier (Ebel et al., 2006).

Generation of a CoA-transferase::eGFP fusion strain

For studying the subcellular localization of the CoA-transferase a 772 bp promoter fragment and the 5′ coding region, which spanned a 234 bp fragment including the putative mitochondrial targeting signal and the first intron, was amplified by PCR from genomic DNA of A. nidulans strain SCF 1.2 using the oligonucleotides Nco_lead_coaT_rev (5′CCA TGG CAT TCT TCT CGA CGT GGT CAG-3′; NcoI site is shown in bold) and BamUpCoATup (see above). The fragment was cloned into the pDrive cloning vector (Qiagen) and transferred to E. coli TOP10 cells. The resulting plasmid was linearized with NcoI and NotI and the eGFP gene, which was excised from plasmid p123 by restriction with the same nucleases (Basse et al., 2000), was ligated in frame with the 5′ coding region of the coaT gene. The resulting plasmid was transferred to E. coli TOP10 cells, re-isolated, restricted with NotI and dephosphorylated for subcloning of the auxotrophic marker argB (see above). The resulting plasmid was used for transformation of A. nidulans SCF 1.2. Transformants were pre-screened for eGFP fluorescence. Positive clones were selected and cultivated overnight in 100 mM acetate medium. MitoTracker Red 580 (MolecularProbes, Invitrogen) was added (10 nM) 30 min prior to harvesting of the mycelium. Mycelium was collected by centrifugation and washed twice with pre-warmed medium before fluorescence was monitored by fluorescence microscopy (Leica Microsystems; Type: Leica DM 4500 B). Pictures were taken with a digital camera (Leica DFC 480) at a 40-fold magnification.

Deletion of the CoA-transferase-coding region

The method for deletion of the CoA-transferase was based on a procedure described by Higuchi et al. (1988) and modified as described in Brock et al. (2007). For that purpose the coding sequence of the CoA-transferase was replaced by a NotI restriction site. Oligonucleotides BamUpCoATup (5′-GGA TCC GTG TGG CCG AAC AGG-3′; BamHI restriction site is shown in bold) and BamDownCoAT_down (5′-GGA TCC CCC GAT ATA TCG GAT TGT G-3′; BamHI restriction site is shown in bold) bind to the flanking regions of the CoA-transferase. Oligonucleotides NotUpCoATdown (5′-GGG ATTGCG GCC GCC GAC ATT ATG AGA AG-3′) and NotDownCoATup (5′-GTC GGC GGC CGCAAT CCC ACT AG-3′) are partially complementary to each other (shown in italic) and contain a full NotI restriction site (shown in bold). PCR with the proofreading polymerase Accuzyme (Bioline, Luckenwalde, Germany) on genomic DNA of strain RMS011 with the two sets of oligonucleotide pairs BamUpCoATup/NotUpCoATdown and NotDownCoATup/BamDownCoAT_down yielded the upstream and downstream flanking regions, excluding the coaT-coding region. Mixing of both fragments, followed by denaturation, annealing and elongation by DynaZyme EXT Polymerase (BioCat; Heidelberg, Germany), led to linking of both fragments and introduction of a NotI restriction site. The fragment was subsequently amplified by the addition of oligonucleotides BamUpCoATup and BamDownCoAT_down. The amplified product was cloned into the PCR2.1 vector (Invitrogen), excised with BamHI and subcloned into a BamHI-restricted pUC18 vector (MBI Fermentas; St. Leon-Rot). The resulting plasmid was linearized by NotI restriction and the NotI-restricted argB gene (see above) was ligated. Two independent plasmids, which contained the gene in opposite directions, were used for transformation of A. nidulans RMS011. For this purpose the deletion fragment was excised from the vector by BamHI restriction and gel purified. For determination of homologous integration and deletion of the CoA-transferase-coding region genomic DNA of transformants was isolated by using the MasterPure Yeast DNA Purification Kit (Epicentre, Madison, USA) and restricted with EcoRV. DNA was blotted to a nylon membrane and Southern analysis was performed by use of a digoxygenin-labelled probe hybridizing to the downstream region of the CoA-transferase.

Complementation of the CoA-transferase deletion strain

The phenotype of the CoA-transferase mutant was confirmed by two independent methods. One method included sexual crossing of a deletion strain with an arginine auxotrophic wild-type strain (with respect to the CoA-transferase) (SCF 3, Table 5) using the argB gene as a segregation marker for the coaT gene. Sexual crossing was enforced on agar plates lacking supplements required for growth of the single mating partners. Conidial suspensions of both strains were mixed and streaked out. Plates were sealed and incubated for 10 days at 37°C. Ascospores were plated on different media to check for the phenotypes of the offsprings.

In a second approach the CoA-transferase gene was re-introduced into the deletion strain by transformation using the pabaA gene as a selection marker. The CoA-transferase gene including upstream and downstream regions was amplified from genomic DNA of strain RMS011 with oligonucleotides BamUpCoaTup and CoaT_down (5′-GCA TAT ATC CGG TAT CTC CAG-3′) and cloned into plasmid pCRII-TOPO (Invitrogen). After transformation of E. coli DH5α plasmid DNA was isolated and sequenced. The pabaA gene from A. nidulans was restricted from pTOPOpabaAnid (Tüncher et al., 2005) with KpnI and ligated to the KpnI-restricted CoA-transferase-containing plasmid. The plasmid was used for transformation of CoA-transferase deletion strains.

Generation of a CoA-transferase and methylcitrate synthase double-deletion strain

For generation of a double mutant strain carrying the deletion of the coaT gene and of the methylcitrate synthase gene (mcsA), sexual crossing was performed as described above. Conidia of the CoA-transferase mutant ΔcoaT 1/12 (Table 5) were mixed with conidia from strain ΔmcsA1 (Table 5) and incubated on selective media. Ascospores were plated on non-selective media and conidia of offsprings were tested for their ability to utilize propionate as sole source of carbon and energy. Strains, which were unable to grow on propionate, as a marker for methylcitrate synthase deficiency, were further tested by PCR for the deletion of the CoA-transferase gene with a mixture of the oligonucleotides up_coat_for (5′-CCG AGT GAC GTC AGT CTA TCC-3′) (binding to the upstream region of the CoA-transferase), argB_atg (5′-CCA ATG GCA TCC CTT CGC TC-3′) (binding to the argB gene) and coaT_rev (5′ ATG ACC TTG TCC GCC ATC TGC-3′) (binding to the CoA-transferase-coding region) as well as by Southern blot analysis with probes on the downstream region of the coaT gene and upstream region of the mcsA region (Fig. S5). Probes were amplified from genomic DNA of the A. nidulans wild-type strain RMS011 with oligonucleotides NotDownCoaTup (5′-GTC GGC GGC CGC AAT CCC ACT AG-3′)/BamDownCoaTdown (5′-GGA TCC CCC GAT ATA TCG GAT TGT G-3′) for coaT and McsPromoBam.up (5′-GCG GAT CCT TAA GTT AAT GGC C-3′)/McsPromoBam.down (5′-GGA TCC CAT CTC GAG AAG AAT CTG-3′) for mcsA and directly labelled with digoxygenin dUTP.


We thank A.A. Brakhage and W. Buckel for helpful discussion and D. Wilson for proofreading of the manuscript. This work was supported by grants of the Deutsche Forschungsgemeinschaft (DFG) and of the Leibniz Institute for Natural Product Research and Infection Biology e.V. (Hans Knoell Institute).