Relationships between the ethanol utilization ( alc ) pathway and unrelated catabolic pathways in Aspergillus nidulans Michel Flipphi, Janina Kocialkowska and Be ´ atrice Felenbok Institut de Ge ´ ne ´ tique et Microbiologie, CNRS UMR 8621, Universite ´ Paris-Sud XI, Centre d’Orsay, Orsay, France The ethanol utilization pathway in Aspergillus nidulans is a model system, which has been thoroughly elucidated at the biochemical, genetic and molecular levels. Three main ele- ments are involved: (a) high level expression of the positively autoregulated activator AlcR; (b) the strong promoters of the structural genes for alcohol dehydrogenase (alcA)and aldehyde dehydrogenase (aldA); and (c) powerful activa- tion of AlcR by the physiological inducer, acetaldehyde, produced from growth substrates such as ethanol and L -threonine. We have previously characterized the chemical features of direct inducers of the alc regulon. These studies allowed us to predict which type of carbonyl compounds might induce the system. In this study we have determined that catabolism of different amino acids, such as L -valine, L -isoleucine, L -arginine and L -proline, produces aldehydes that are either not accumulated or fail to induce the alc system. On the other hand, catabolism of D -galacturonic acid and putrescine, during which aldehydes are transiently accumulated, gives rise to induction of the alc genes. We show that the formation of a direct inducer from carboxylic esters does not depend on alcA-encoded alcohol dehydro- genase I or on AlcR, and suggest that a cytochrome P450 might be responsible for the initial formation of a physio- logical aldehyde inducer. Keywords: Aspergillus nidulans; activation of transcription; alc genes; aldehydes; carboxylic esters. The saprophytic hyphal fungus Aspergillus nidulans can utilize a wide range of organic compounds as sole sources of carbon and nitrogen. One such alternative nutrient is ethanol. The pathway-specific transcriptional activator AlcR is essential for the ethanol-induced expression of the two structural genes necessary for the utilization of the alcohol, alcA and aldA (reviewed in [1]). These genes encode, respectively, alcohol dehydrogenase I (ADHI), which oxidizes ethanol into acetaldehyde, and aldehyde dehydrogenase (ALDH), converting acetaldehyde into acetate. Acetate is further metabolized into acetyl-CoA by an acetyl-CoA synthetase encoded by the facA gene [2–4], which is not subject to ethanol-specific control. The molecular means by which transcriptional regulation of ethanol utilization is achieved have been studied exten- sively [1]. The functional characteristics of the AlcR binuclear zinc cluster activator, its DNA-binding specificity, its three-dimensional structure and its nuclear localization sequence, have been elucidated [5–15]. The alcA, alcR and aldA promoters are subject to different powerful mechanisms of transcriptional activation [9,13]. These properties have been exploited in the use of the A. nidulans alc system as a strongly inducible tool for heterologous expression [1,16]. When a rich carbon source such as glucose is present, expression of the alc system is repressed by the action of CreA, the DNA-binding protein mediating carbon catabolite repression in A. nidulans. Several mechanisms account for the direct repression of the alcR and alcA genes while the aldA gene is subject to indirect CreA control via direct repression of alcR [7,12,13,17,18]. A subtle interplay between induction and repression allows A. nidulans to adapt rapidly to changing nutritional conditions in the environment. The degradation pathways of small aliphatic primary alcohols and monoamines as well as that of the amino acid L -threonine, converge on a common catabolic intermediate, an aliphatic aldehyde [1,13,19]. The three principal alc genes, alcA, aldA and alcR, are essential for the use of ethylamine and L -threonine as sole sources of carbon (but not for their utilization as sources of nitrogen). Recently, it has been demonstrated that ethanol, ethylamine and L -threonine do not induce the alc system directly but that these growth substrates have to be converted into acetaldehyde, which is the physiological inducer of the alc system [13,19]. However, the alc system was found to be inducible by a range of carbonyl compounds and, interestingly, some of these provoke substantially higher levels of expression than the maximal level obtainable with acetaldehyde [19]. Fur- thermore, transcription of three genes of yet unknown function, alcO, alcM and alcS (clustered along with the alcR and alcA genes on chromosome VII) is subject to strict AlcR control [20]. Despite being coordinately expressed with the alcR, alcA and aldA genes, these three additional alc genes Correspondence to M. Flipphi, Consejo Superior de Investigaciones Cientı ´ ficas (CSIC), Instituto de Agroquı ´ mica y Tecnologı ´ a de Ali- mentos (IATA), Apartado de Correos 73, 46100 Burjassot, Valencia, Spain. Fax: + 34 96 363 63 01, Tel.: + 34 96 390 00 22, E-mail: flipphi@iata.csic.es Abbreviations: ADHI, alcohol dehydrogenase I; ALDH, aldehyde dehydogenase; GABA, c-aminobutyric acid; P450, cytochrome P450. Enzymes: ADHI, alcohol dehydrogenase I (EC 1.1.1.1); ALDH, aldehyde dehydogenase (EC 1.2.1.5); P450, cytochrome P450 (EC 1.14.14.1). (Received 15 May 2003, revised 26 June 2003, accepted 2 July 2003) Eur. J. Biochem. 270, 3555–3564 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03738.x are dispensable for growth on ethanol, ethylamine and L -threonine (S. Fillinger, M. Flipphi & B. Felenbok, unpublished results). This raises the question as to whether the alc system contributes to the nutritional versatility of A. nidulans beyond providing the fungus with the ability to utilize ethanol, ethylamine and L -threonine as growth substrates. A whole range of structurally diverse alternative carbon and nitrogen sources are catabolized via aldehyde intermediates, some of which might serve as in vivo substrates for ADHI and/or ALDH under physiologically relevant conditions. In this study, we have addressed the possible involvement of the alc genes in the conversion of a number of nutrients, i.e. D -galacturonate, glycerol, six amino acids, putrescine, c-aminobutyric acid (GABA) and small carboxylic esters. Materials and methods Strains, media and growth conditions Aspergillus nidulans strains used in this study are listed in Table 1. The references refer to the mutations relevant to this work; all other markers are in standard use [21]. Media composition, supplements and basic growth conditions at 37 °C were as described by Cove [22], using diammonium tartrate (5 m M ) as nitrogen source with the carbon source present at 1% (w/v or v/v), unless stated otherwise. Mycelia for transcript analysis and esterase expression were grown for 20–24 h in minimal medium with lactose (3% w/v) as the carbon source and urea (5 m M )asthe nitrogen source. Figure 1 shows that this growth medium is perfectly neutral with respect to induction by ethanol. alc gene transcript levels in lactose-grown and ethanol-induced mycelia are essentially the same as those observed in ethanol-grown biomass and notably 10- to 20-fold higher than those obtained in ethanol-induced mycelia grown on 0.1% (w/v) D -fructose. Regardless of the growth substrate, the gratuitous inducer 2-butanone always elicites more powerful induction than the convertible compound ethanol. Furthermore, the response of the acetyl-CoA synthetase gene (facA) showed that inducing amounts of acetate had been formed from the ethanol applied to lactose-grown biomass during the induction period as well as in the ethanol-grown mycelia. The use of lactose as the growth substrate allows accurate analysis of alc gene transcription upon addition of different compounds to principally nonrepressed and noninduced mycelia in all genetic back- grounds, and even monitoring catabolism of certain effector compounds beyond the first aldehyde intermediate. Induction was achieved by addition of the effector compounds to 50 m M (final concentration), unless stated otherwise. Cultures were harvested after a further 2.5 h of incubation (inducing conditions) for Northern analysis and after 4–6 h for esterase expression. Where necessary, the effector compound was added from a concentrated solution Table 1. A. nidulans strains and transformants used in this study. Strain Genotype References for characterized mutation or strain BF054 yA2 pabaA1 BF064 yA2 pabaA1; alc500 [13,20] BF107 yA2 pabaA1; aldA67 [13] BF129 yA2 pabaA1; alcR125; aldA67 [13,55] M F. Cochet & B. Felenbok (unpublished results) G277 biA1; punA11 [47] H1269–12.1 yA2 biA1;(riboB2) D alcC::riboB a [43] TgpdA::alcR yA2;(argB2); (alcR125) pantoB100 TargB/gpdA::alcR b [13] TgpdA::aldA pabaA1;(argB2); (aldA67)TargB/gpdA::aldA b [13] alc500 TalcR yA2 pabaA1;(argB2); alc500 TargB/alcR c This work (see Materials and methods) Corresponding phenotype: a riboflavin prototroph; b arginine prototroph, ethanol utilizing; c arginine prototroph, ethanol nonutilizing. Fig. 1. Lactose is a neutral growth substrate with respect to induction of the alc genes by ethanol. Wild type mycelia were grown on either D -fructose or lactose (noninduced conditions, NI) and cultures were induced by addition of ethanol (E) or the gratuitous inducer 2-butanone (2B) to a final concentration of 50 m M , as detailed in Materials and method. Ethanol-grown biomass is considered to be induced from the start of cultivation (E*). Culture growth conditions, RNA isolation andNorthernblotswereasdescribedinMaterialsandmethods. Membranes were hybridized with 32 P-labelled probes specific for the alcA, alcR, facA, facB and c-actin (acnA) genes. Hybridization with a probe specific for the 18S rRNA species provided a control of the quantity of total RNA in each lane that correlates well with the con- comitant expression of the regulatory facB gene (encodes a transacti- vator of acetate catabolism [2,29]), under the various growth conditions. Note that transcription of the c-actin gene was much higher in D -fructose-grown mycelia than in biomass generated on lactose or ethanol. The c-actin gene cannot therefore be used as an internal control to normalize the amounts of mRNA when comparing D -fructose-grown mycelia with lactose- or ethanol-grown mycelia. However, this gene still provided a reliable control among different cultures grown on one particular carbon source, and also for com- paring lactose- with ethanol-grown cultures. 3556 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 of pH 6.8. In those cases where the effector compound constitutes a poor nitrogen source for the fungus mycelia were also grown in the presence of the effector (25 m M )as the sole nitrogen source. Such cultures were supplemented with D -biotin to enhance uptake of these compounds [23]. Chemicals were purchased either from Sigma-Aldrich or Merck-Eurolab. Generation of a mutant strain lacking the ADHI-encoding alcA gene The best characterized alc deletion mutant isolated upon selection for resistance to allyl alcohol is alc500 [24,25]. This mutant lacks all five AlcR-controlled genes comprizing the alc gene cluster [20] (J. Kocialkowska, B. Felenbok & M. Flipphi, unpublished results). The unlinked aldA gene is no longer inducible but is constitutively expressed at a low level [13]. To monitor pathway-specific induction of the aldA gene in the absence of ADHI-activity, a single, functional copy of the alcR gene was introduced into an L -arginine-auxotroph argB2 alc500 double mutant strain by cotransformation with the plasmids pBSalcRSal (carrying the alcR gene [26]) and pFB39 (carrying the A. nidulans argB gene [27]), essentially as described by Flipphi et al.[13]. L -Arginine-prototrophs were screened for the presence of the alcR gene by dot-blot analysis. A single copy cotransformant was selected by Southern blot analysis (not shown). This transformant was back-crossed with an alc500 mutant and offspring carrying a functional alcR gene were screened via their ability to be crossfed by a leaky aldA15 mutant strain, which accumulates and secretes acetaldehyde on plates containing 1% ethanol as sole carbon source and 10 m M sodium nitrate as the nitrogen source [13]. The alcR-complemented alc500 strains behave as alcA loss-of-function mutants. In one of the crossfed strains, alc500 TalcR, proper inducibility of alcR and aldA was demonstrated by Northern blot analysis. Isolation of RNA and Northern blot analysis Transcript analysis was carried out as described by Flipphi et al. [13] using 32 P-labelled probes corresponding to fragments of the cloned A. nidulans genes alcA [9], alcR [26], aldA [28], facA [4], facB [29], c-actin (acnA)[30]and prnD (GenBankÒ AJ223459). To monitor gabA transcrip- tion, a probe was made from gabA cDNA [31]. 18S ribosomal RNA was detected with a probe for horseradish 18S rDNA [32]. Autoradiographs were exposed for various times to avoid saturation of the film. In lactose-grown mycelia the c-actin gene is constitutively expressed and was used as an internal control for the amounts of mRNA loaded. All induction experiments were repeated at least once. Qualitative zymogram analysis of carboxyl-/ acetylesterase activity For the analysis of intracellular esterase activity cell-free extracts were prepared from about 250 mg mycelial powder, obtained by grinding freshly harvested mycelia in liquid nitrogen. The mycelial powder was quickly suspended in 500 lL of ice-cold extraction buffer (10 m M sodium phos- phate pH 6.5, 2 m M dithiothreitol). The suspension was subsequently centrifuged in an Eppendorf centrifuge for 5min at 10 000 g at 4 °C and the supernatant was recovered and put on ice. For the analysis of extracellular esterase activity, media samples ( pH 6.8) were taken directly from cultures. After centrifugation as above, the supernatant was recovered and put on ice. Protein concen- tration was determined with Bradford’s method using bovine serum albumin as the standard. Proteins were separated in native 7.5% polyacrylamide (29 : 1 acrylamide/bisacrylamide, v/v) minigels buffered with 10 m M Tris, 76 m M glycine, pH 8.5. Electrophoresis was performed at 4 °C. For cell-free extracts, samples containing 25 lg protein were mixed with 1/5 volume of loading buffer (10 m M sodium phosphate pH 7.0, 50% (v/v) glycerol, 2 per thousand (w/v) bromophenol blue). For extracellular samples, 20 lL of culture medium (containing 3–5 lgproteinÆmL )1 ) were applied to gels. The samples were allowed to migrate into the gel at 1 VÆcm )1 ,afterwhich the gel was run at 4 VÆcm )1 for 3–4 h. After electrophoresis, the gel was immersed in 100 mL of assay buffer (25 m M potassium phosphate pH 7.0, 1 m M EDTA, 1 per thousand (v/v) 2-mercaptoethanol) at room temperature for 2 min. 4-Methylumbelliferyl acetate was gradually added from a 1 M solution in dimethylsulfoxide to a final concentration of 500 l M . The fluorogenic ester is a substrate for both carboxyl- and acetylesterases (EC 3.1.1.1/3.1.1.6). Forma- tion of 4-methylumbelliferone could be detected within minutes upon illumination with UV light (312 nm). Results and discussion D -Galacturonic acid provokes an induction response but glycerol does not Induction of the alc system in lactose-grown mycelia by D -galacturonic acid is very strong at 50 m M (Fig. 2A, left panel) but hardly significant at 10 m M (not shown). D -Galacturonic acid is a good growth substrate for A. nidu- lans that is catabolized into pyruvate and D -glyceraldehyde [33], and the observed induction should be attributable to the latter compound. D -Glyceraldehyde appears to be a relatively weak inducer when added to pregrown mycelia (Fig. 2B) but this could be due to inefficient uptake. D -Glyceraldehyde is converted into the glycolytic inter- mediate D -glyceraldehyde-3-phosphate via glycerol and glycerol-3-phosphate [34]. Glycerol is noninducing (Fig. 2B) even in the strongly derepressed mutant creA d 30 or in a strain carrying a derepressed alcA gene in addition to a constitutively expressed alcR gene (results not shown). Dihydroxyacetone, a noninducing double hydroxyl- substituted a-ketone [19], is also catabolized via glycerol. In A. nidulans, reduction of D -glyceraldehyde is achieved by NADPH-dependent reductases, one of which is constitu- tively expressed but substrate-specific [35] while the second is specifically induced on D -galacturonate [36]. It is therefore not surprising that mutants in alc grow normally on both D -galacturonic acid and glycerol [34]. The alc gene induction observed upon addition of the higher concentration of the uronic acid is probably due to a transient accumulation of D -glyceraldehyde, which ceases upon full expression of the inducible reductase. In mycelia grown on D -galacturonic acid, expression of the alc genes is repressed (Fig. 2A, right Ó FEBS 2003 Activation of alc genes in Aspergillus nidulans (Eur. J. Biochem. 270) 3557 panel). However, induction does not cease in mycelia that are grown on ethanol, or on lactose with either ethylamine or L -threonine as the nitrogen source (results not shown). Catabolism of L -proline and L -arginine does not lead to induction of the alc genes L -Proline and L -arginine can serve as sole carbon and nitrogen sources for A. nidulans and both are catabolized via L -D 1 -pyrroline-5-carboxylate, the internal Schiff base of L -glutamic semialdehyde with which it is in continuous equilibrium [37,38]. The Schiff base intermediate can be reduced by PrnD ( L -D 1 -pyrroline-5-carboxylate reductase/ L -proline oxidase EC 1.5.1.2) to yield L -proline, and oxi- dized by PrnC ( L -D 1 -pyrroline-5-carboxylate dehydrogenase EC 1.5.1.12) to produce L -glutamate. These three amino acids do not induce the alc genes while, as expected, L -proline and L -arginine do induce the prnD gene. This was also the case in a strain constitutively overexpressing the alcR gene, TgpdA::alcR (Fig. 3). In this genetic background, alcA transcription is more sensitive to minor inducing signals because the expression of its activator, AlcR, is no longer limiting [13]. Thus, and in agreement with previous predictions [19], the semialdehyde does not induce the alc genes as it carries a carboxy substituent. Loss-of-function aldA mutants are reported to grow more slowly on L -proline than wild type strains [37]. This could be a pleiotropic effect due to accumulation of acetaldehyde [13] in addition to that of L -glutamic semialdehyde in these mutants. However, we were unable to detect a phenotype characteristic for the aldA67 mutation on plates with L -proline or L -arginine as sole carbon and nitrogen sources, distinguishing it from alc mutations (results not shown). Catabolism of L -valine, L -isoleucine and L -tryptophan does not induce the alc system The branched-chain aliphatic amino acids L -valine, L -leucine and L -isoleucine can serve as (poor) growth substrates for A. nidulans [39,40]. The first catabolic step is a transamination to yield the corresponding 2-oxo acids. In Saccharomyces cerevisiae these latter compounds enter the Ehrlich pathway where they undergo decarboxylation to form the corresponding aldehydes, which are subsequently detoxified by constitutive alcohol dehydrogenase activity [41]. 2-Methylbutyraldehyde emerged previously as the most effective alc gene inducer [19]. In A. nidulans,thisaldehyde could be formed as a physiological intermediate when L -isoleucine is catabolized via the Ehrlich pathway. However, transcript analyses in wild type (Fig. 4) and an alcA-deletion strain (not shown) grown on either of the branched-chain aliphatic amino acids as sole nitrogen source showed that the alc system was not induced. Therefore, the expected aldehyde intermediates are either not accumulated or not formed at all. This result can be anticipated for L -leucine as this amino acid is not catabo- lized via the Ehrlich pathway in A. nidulans [40]. Even if L -isoleucine and L -valine are degraded in A. nidulans as they are in yeast, then the formation and conversion of the Fig. 2. D -Galacturonic acid provokes induction of the alc genes. (A) Northern blot analysis of induction upon addition of D -galact- uronic acid ( D -GAA) to lactose-grown wildtype mycelia (left panel) andupongrowthon D -galacturonic acid ( D -GAA*)(rightpanel). (B) Northern blot analysis of the response of the alc genes to addition of D -glyceraldehyde and glycerol. Ethanol served as the reference for induction. Induction was achieved by adding effector compounds to uninduced cultures to 50 m M (final concentration) except for D -glyceraldehyde (*), which was added to 4 m M .Whentheeffector compound served as the carbon source for growth, its initial concen- tration in culture was 50 m M . Experimental details and abbreviations were as described in the legend to Fig. 1. Fig. 3. The amino acids L -proline and L -arginine do not induce the alc system. Northern blot analysis was done in gpdA::alcR,astraincon- stitutively overexpressing the alcR gene (alcR*). The prnD ( L -proline oxidase) and alcA genes were used to monitor the responses to the two amino acids. Experimental details and abbreviations were as described in the legends to Figs 1 and 2. 3558 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 aldehyde intermediates clearly does not require activation of the ethanol utilization pathway. Interestingly both L -isoleu- cine and L -valine modestly induce the alc genes in a strain deleted for another alcohol dehydrogenase, ADHIII, pre- sumably due to some aldehyde accumulation, while L -leucine does not (results not shown). Expression of the ADHIII-encoding alcC gene is not under the control of AlcR and is irrelevant for ethanol catabolism [1,42,43]. L -Tryptophan can also serve as a nitrogen source for A. nidulans [39]. Multiple routes are known in bacteria, fungi and plants to convert this amino acid into indole- 3-acetic acid, an important plant hormone (in bacteria, reviewed by [44]; in fungi [45,46]). The pathways via indole- 3-pyruvate and tryptamine produce indole-3-acetaldehyde as an intermediate, which is further oxidized by an alcohol- inducible ALDH in the fungus Ustilago maydis [45]. In A. nidulans it has been observed that tryptamine is inducing for the alc genes at relatively low (nontoxic) concentrations (M. Flipphi, J. Kocialkowska & B. Felenbok, unpublished results), probably by conversion into indole-3-acetaldehyde. This reaction would be similar to that of the deamination of unbranched aliphatic monoamines; these latter compounds have been shown previously to be inducers of the alc system [19]. However, L -tryptophan itself is noninducing even in mycelia grown on lactose with this amino acid as sole nitrogen source (results not shown). Therefore indole-3- acetaldehyde is apparently not accumulated upon L -trypto- phan degradation in A. nidulans. Putrescine provokes an induction of the alc system but GABA does not A. nidulans is capable of using putrescine (1,4-diamino- butane) as a sole source of nitrogen but not as a carbon source [47]. Breakdown of this diamine into the tricarboxylic acid-cycle intermediate succinate (Fig. 5) involves two different aldehyde intermediates, c-aminobutyraldehyde and succinic semialdehyde [37,48–50]. Both have a back- bone of four carbon atoms, the optimal length for the aliphatic tail of an alc-inducing aldehyde. Succinic semi- aldehyde is predicted to be noninducing due to its terminal carboxy group [19] but c-aminobutyraldehyde permits analysis of the effect of an amino substituent on the inductive capacity of a physiologically relevant aldehyde. Transcript analysis shows that putrescine is indeed able to moderately induce the alc genes when added to pregrown mycelia (Fig. 6A). As expected, no induction is observed upon addition of c-aminobutyric acid (GABA). The forma- tion of GABA from putrescine can be monitored with the expression of the GABA permease gene (gabA) [31,49]. Using the leaky punA11 mutation, which partially impairs the utilization of putrescine [47], we could confirm that the diamine needs to be converted into the corresponding aldehyde to induce the alc genes; putrescine does not induce the alc system in this background, while the gabA gene is only very modestly expressed (Fig. 6B). Moreover, induction by putrescine is titratable in vivo with semicarbazide, an aldehyde scavenger (see References [13,19]; results not shown). Thus, it can be concluded that putrescinebreakdown results in the accumulation of a weakly inducing compound, c-aminobutyraldehyde, and that an amino substituent strongly limits the inducing capacity of an aldehyde. As is the case for D -galacturonic acid, no alc gene induction could be observed in mycelia grown on lactose/ putrescine although the gabA gene is expressed under these growth conditions (results not shown). Expression of the alc gene is thus likely to respond to a transient accumulation of c-aminobutyraldehyde when putrescine is added to a non- induced culture. Interestingly, GABA is also produced from putrescine in the stringent loss-of-function aldA67 mutant as well as in an aldA67 alcR125 double mutant, which is unable to induce the alc genes (Fig. 6A). This indicates that Fig. 4. The branched-chain aliphatic amino acids L -valine and L -isoleu- cine do not induce the alc system. Transcript analysis was performed in a wild-type strain. Induced mycelia were grown in the presence of either amino acid (25 m M ) as the sole nitrogen source instead of urea. The aldehyde that was presumed to be formed upon L -isoleucine turnover, 2-methylbutyraldehyde (2MB), served as an additional control of induction and was added to urea-grown mycelia at 2 m M [19]. Further experimental details and abbreviations were as described in the legends to Figs 1 and 2. Similar results were obtained for L -leucine (results not shown). Fig. 5. Catabolism of putrescine and GABA in A. nidulans. Putrescine is first deaminated to form c-aminobutyraldehyde, which is further oxidized by an unknown ALDH to yield c-amino butyric acid (GABA). The second amino group is liberated by GABA amino transferase (gatA gene [48]) to produce succinic semialdehyde, which is finally converted into succinate by a substrate-specific ALDH specified by the ssuA locus [37,49,50]. Ó FEBS 2003 Activation of alc genes in Aspergillus nidulans (Eur. J. Biochem. 270) 3559 c-aminobutyraldehyde is oxidized by another ALDH enzyme not dependent upon AlcR for its expression. In mammals, a specialized ALDH is involved in the conversion of this aminoaldehyde [51]. However, its expression does not seem to be as rapid as that of the alc genes. Interestingly, constitutive ALDH overexpression in the TgpdA::aldA transformant not only decreases alc gene induction by putrescine but also results in reduced gabA expression (Fig. 6A). Overall, the data suggest that c-aminobutyralde- hyde can serve as a (poor) in vivo substrate of both aldA- encoded ALDH and alcA-encoded ADHI. Moreover, it appears that expression of the putrescine pathway-specific ALDH is inducible by its aminoaldehyde substrate. Interestingly, the level of pseudo-constitutive expression of the alc genes in the loss-of-function aldA67 mutant [13] is reduced upon addition of either putrescine or GABA (Fig. 6A). This could be explained on the basis that the accumulated aldehyde is partly neutralized as an inducer in the presence of these primary amines, suggesting that Schiff base interactions between aldehydes and primary amines occur in vivo. The formation of a Schiff base is a possible mechanism by which the DNA-binding regulator AlcR is activated by direct binding of the inducer compound [19]. An alternative hypothesis is that the aldehydes formed from putrescine and GABA (c-aminobutyraldehyde and succinic semialdehyde, respectively) are able to compete in some way with the aldehyde accumulated from general metabolism in aldA67, leading to decreased pseudo-constitutive alc gene expression. This second hypothesis implies that a non- inducing aldehyde, succinic semialdehyde, competes with the inducer for binding to AlcR, although it is unable to effect activation. Some small carboxylic esters induce the alc system but lactones do not Carboxylic esters are interesting compounds with respect to alc gene induction. They are structurally related to ketones, one class of direct inducers of the alc system, and their hydrolysis results in alcohols that upon oxidation yield aldehydes, a second class of direct inducers. We have shown previously that the small methyl ketones 2-butanone and 2-pentanone induce the alc genes [19]. Circular ketones such as cyclopentanone and cyclohexanone also induce but the smallest linear b-ketone, 3-pentanone, does not. We have therefore investigated whether or not the esters and lactones (intramolecular esters) corresponding to the above ketones induce the alc genes. Transcript analyses show that whereas the ester ethyl- acetate (which corresponds to the inducer 2-pentanone) indeed provokes a strong induction of the alc genes, methylpropionate (which corresponds to the inert ketone 3-pentanone) fails to induce (Fig. 7A). By contrast, none of the lactones tested (c-butyrolactone, d-valerolactone and e-caprolactone) provoked induction (Fig. 7B). Moreover, although 2-butanone is the most powerful of all the ketone inducers, its structurally related ester methylacetate elicits only a marginal induction of alcA while alcR and aldA expression do not exceed their basal, noninduced levels (results not shown). These data thus show that there is no correlation between the inducing capabilities of a ketone and the structurally related ester or lactone. It rather appears that esters induce the alc genes when their degradation results in the produc- tion of an inducing aldehyde derived from the ester’s alcohol moiety. Acetaldehyde would therefore be responsible for the induction observed on ethylacetate. Methylpropionate does not induce as formaldehyde is inert while hydrolysis of the lactones tested would principally yield noninducing carb- oxy-substituted alcohols and aldehydes, in agreement with previous predictions [19]. We have verified these deductions using the unbranched aliphatic esters propylacetate and ethylpropionate. Upon hydrolysis, ethylpropionate would yield acetaldehyde while propylacetate would yield propionaldehyde, both direct Fig. 6. Putrescine catabolism provokes induction of the alc system. (A) Transcript analysis of the effect of putrescine (Putr) addition to pregrown mycelia of a wildtype strain (wt) and three strains mutant in ethanol catabolism: aldA67, gpdA::aldA and aldA67 alcR125.ThegabA gene encodes GABA permease and is inducible by x-amino acids [31]. The aldA67 mutant exhibits pseudo-constitutive expression of the alcA and alcR genes (NI *) [13]. (B) Putrescine-induced transcription of the alcA and gabA genes in the leaky putrescine utilization mutant punA11 compared to that in a wild type strain. Putrescine (1,4-diaminobutane) was added to uninduced cultures to 25 m M . Further experimental de- tails and abbreviations were as described in the legends to Figs 1 and 2. 3560 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 inducers of the alc system. Figure 7A clearly shows that both these esters are inducing despite the fact that the carbonyl function in ethylpropionate resides in the b-position, as is the case for the inert compounds methyl- propionate and 3-pentanone. That the induction provoked by ethylacetate, apparently the most effective ester, is considerably less in a strain constitutively overexpressing ALDH, TgpdA::aldA (data not shown), is in accord with the formation of acetaldehyde from this ester. Furthermore, induction of the acetyl-CoA synthetase gene (facA) upon addition of ethylacetate, propylacetate and ethylpropionate (Fig. 7A), actually suggests that all esters are hydrolyzed into acetate during the induction period. In this regard it is worth noting that wild type facA transcription responds to acetate and its precursor ethanol (see Fig. 1) but not to propionate or n-propanol (M. Flipphi & B. Felenbok, unpublished results) explaining why this gene is not induced in the presence of methylpropionate. In conclusion, the results obtained are in agreement with our previous data and show that aliphatic and cyclic ketones directly induce the alc genes. There is no correlation between the inductive capacities of ketones and their structurally related ester counterparts. In addition, small aliphatic carboxylic esters induce the alc genes indirectly via the aldehyde formed from their ethanol or n-propanol moieties. Acetylester breakdown yields inducing compounds independently of the alc system The induction of the facA gene by all esters tested except methylpropionate suggests that carboxylic esters are degraded during the induction period of 2.5 h. Strong indications that the hydrolysis of esters is a catalyzed process come from zymogram analyses of cell-free extracts and culture medium samples, which evidenced the presence of multiple intra- and extracellular carboxyl-/acetylesterase activities. As shown for the intracellular activity in Fig. 8A, these esterase activities are constitutively produced by the fungus when grown on lactose and urea. Interestingly, the esterase spectrum did not change upon addition of typical inducers of the alc gene system, i.e. L -threonine and 2-butanone, nor by the supply of the inducing ester ethylacetate. Only addition of 2-methylbutyraldehyde to a lactose-grown culture resulted in induction of an intracel- lular esterase. However, even this inducible activity does not depend on AlcR for its expression as the esterase spectrum in the alc500 deletion mutant was identical to that in a wild type strain (Fig. 8A). In line with our previous studies [19], it is to be expected that the alcohol resulting from ester hydrolysis is oxidized to an inducing aldehyde. Transcript analysis in a strain completely lacking the ADHI-encoding alcA gene (alc500- TalcR) showed that the two AlcR-controlled genes present in this background, alcR and aldA, are induced by ethylacetate and ethylpropionate (Fig. 8B). These genes are also induced in the presence of ethanol despite the fact that this strain cannot use the latter as a growth substrate. The acetyl-CoA synthetase gene facA is not ethanol- inducible in the deletion mutant (M. Flipphi & B. Felenbok, unpublished results), also indicating that not much acetate is formed from ethanol. These observations suggest that inducing amounts of the physiological aldehyde inducer are Fig. 7. Analysis of the induction of the alc genes by carboxylic esters and lactones. (A) Transcript analysis of the effect of four carboxylic esters on the expression of the alcA, alcR and facA genes in a wild type strain. The inducing ketone 2-pentanone, which is structurally related to ethylacetate, and the inert ketone 3-pentanone, structurally related to methylpropionate, provided the controls. The structures of these latter four compounds are shown below. (B) Northern analysis of wild type mycelia supplemented with three different lactones. The inducing ketone cyclohexanone served as the control for induction. The struc- tures of the circular ketone and its lactone counterpart, d-valero- lactone, are shown below. Experimental details and abbreviations were asdescribedinthelegendstoFigs1and2. Ó FEBS 2003 Activation of alc genes in Aspergillus nidulans (Eur. J. Biochem. 270) 3561 produced from ethanol as well as from ethylesters by other enzymes in alcA loss-of-function mutants. However, the limited conversion capacity is clearly not sufficient to support growth on ethanol. It was surprising to find that ethylacetate can serve as a sole source of carbon for alc loss- of-function mutants (not shown). Despite being highly induced in its presence, the alc genes are dispensable for growth on this acetylester, strongly suggesting that the acetate moiety from the ester is preferentially consumed. From the above analysis it can be questioned whether the limited, AlcR-independent ethanol conversion capacity in the alcA deletion mutant is sufficient to produce and maintain an inducing quantity of acetaldehyde in the presence of ethylacetate. Induction of the alc system in an alcA-independent manner could be explained presuming that acetaldehyde is produced directly from the ethylester by an AlcR-independent mechanism (Fig. 9). Some mamma- lian cytochrome P450 isozymes produce carbonyl com- pounds (aldehydes or ketones) from carboxylic esters in vitro by oxidative cleavage [52,53]. Interestingly, the ethanol- and acetone-inducible P450 2E1 from mammals is also capable of catalyzing conversion of ethanol into acetate via two subsequent oxidation reactions [54]. Although the main product is acetate, acetaldehyde is also produced by this enzyme because the intermediate product/second substrate is only loosely bound to the catalytic site. Preliminary results indicate that one putative P450-encoding gene is weakly but constitutively transcribed in A. nidulans (M. Flipphi & B. Felenbok, unpublished results). We therefore propose that the fungus constitutively produces at least one (but possibly more) P450 oxidase able to form the physiological aldehyde inducer from ethanol and other alcohols as well as from inducing carboxylic esters. Initial, AlcR-independent production of acetaldehyde by P450 could play a crucial role in triggering ethanol catabolism in A. nidulans. Acknowledgements We thank John Clutterbuck and Heather Sealy-Lewis for providing us with mutant A. nidulans strains, Michael Hynes for plasmids harboring the facA and facB genes, Irene Garcia for the prnD probe and Joan Tilburn for the gabA cDNA clone. We are grateful to Sabine Fillinger Fig. 8. Formation of the physiological aldehyde inducer from acetyl esters does not depend on the alc system. (A) Zymogram analysis of intracellular carboxyl-/acetylesterase activity in wild type compared to that in an alc deletion mutant, alc500. Cell-free extracts from mycelia subjected to noninduced or induced growth conditions for the alc genes were prepared as decribed in Materials and methods. Induction was achieved by adding L -threonine ( L -Thr) (to 50 m M )or2-methyl- butyraldehyde (2MB) (to 2 m M ) to lactose-grown mycelia. Protein was separated in a native polyacrylamide gel at pH 8.5. Carboxyl-/acetyl- esterase activity was resolved directly in the gel as described in Mate- rials and methods. The zymograms are presented as the negatives. (B) Northern analysis of the induction of aldA and alcR in the pres- ence of ethanol or acetylesters in alc500 TalcR, an absolute alcA deletion mutant (D alcA) (see Materials and methods). Further experimental details and abbreviations were as described in the legends to Figs 1 and 2. Fig. 9. A role for cytochrome P450 in the initial formation of physio- logical aldehyde inducers from alcohols and carboxylic esters in A. ni dulans. Constitutively expressed cytochrome P450 isozymes could oxidize ethanol and ethylesters to yield directly acetaldehyde, trigger- ing a cascade reaction of coupled expression of alcA-encoded ADHI and accelerated formation of the physiological inducer. Esterases are responsible for the energetically more favorable hydrolysis of carb- oxylic esters to alcohols and carboxylic acids. 3562 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 who selected the initial alcR transformant in the alc500 deletion mutant background, Andrew MacCabe for correcting the English, Miguel Pen ˜ alva for helpful suggestions and Herb Arst for critical reading of the manuscript. This work was supported by the Centre National de la Recherche Scientifique, UMR 8621, the Universite ´ Paris-Sud XI and the European Community, grant QLK3-CT99-00729. References 1. Felenbok, B., Flipphi, M. & Nikolaev, I. (2001) Ethanol catabo- lism in Aspergillus nidulans: a model system for studying gene regulation. Prog. Nucleic Acid Res. Mol. Biol. 69, 149–204. 2. Apirion, D. (1965) The two-way selection of mutants and rever- tants in respect of acetate utilization and resistance to fluoro- acetate in Aspergillus nidulans. Genet. Res. 6, 317–329. 3. Armitt, S., McCullough, W. & Roberts, C.F. (1976) Analysis of acetate non-utilizing (acu)mutantsinAspergillus nidulans. J. Gen. Microbiol. 92, 263–282. 4. Sandeman, R.A. & Hynes, M.J. (1989) Isolation of the facA (acetyl-Coenzyme A synthetase) and acuE (malate synthase) genes of Aspergillus nidulans. Mol. Gen. Genet. 218, 87–92. 5.Kulmburg,P.,Sequeval,D.,Lenouvel,F.,Mathieu,M.& Felenbok, B. (1992) Identification of the promoter region involved in the autoregulation of the transcriptional activator ALCR in Aspergillus nidulans. Mol. Cell. Biol. 12, 1932–1939. 6. Kulmburg, P., Judewicz, N., Mathieu, M., Lenouvel, F., Sequeval, D. & Felenbok, B. (1992) Specific binding sites for the activator protein, ALCR, in the alcA promoter of the ethanol regulon of Aspergillus nidulans. J. Biol. Chem. 267, 21146–21153. 7. Mathieu, M. & Felenbok, B. (1994) The Aspergillus nidulans CREA protein mediates glucose repression of the ethanol regulon at various levels through competition with the ALCR-specific transactivator. EMBO J. 13, 4022–4027. 8. Lenouvel, F., Nikolaev, I. & Felenbok, B. (1997) In vitro recognition DNA targets by AlcR, a zinc binuclear cluster acti- vator different from the other proteins of this class. J. Biol. Chem. 272, 15521–15526. 9. Panozzo, C., Capuano, V., Fillinger, S. & Felenbok, B. (1997) The zinc binuclear cluster activator AlcR is able to bind to single sites, but requires multiple repeated sites for synergistic activation of the alcA gene in Aspergillus nidulans. J. Biol. Chem. 272, 22859– 22865. 10. Nikolaev,I.,Lenouvel,F.&Felenbok,B.(1999)UniqueDNA binding specificity of the binuclear zinc AlcR activator of the ethanol utilization pathway in Aspergillus nidulans. J. Biol. Chem. 274, 9795–9802. 11. Nikolaev, I., Cochet, M F., Lenouvel, F. & Felenbok, B. (1999) A single amino acid, outside the AlcR zinc binuclear cluster, is involved in DNA binding and in transcriptional regulation of the alc genes in Aspergillus nidulans. Mol. Microbiol. 31, 1115–1124. 12. Mathieu, M., Fillinger, S. & Felenbok, B. (2000) In vivo studies of upstream regulatory cis-acting elements of the alcR gene encoding the transactivator of the ethanol regulon in Aspergillus nidulans. Mol. Microbiol. 36, 123–131. 13. Flipphi, M., Mathieu, M., Cirpus, I., Panozzo, C. & Felenbok, B. (2001) Regulation of the aldehyde dehydrogenase gene (aldA)and its role in the control of the coinducer level necessary for induction of the ethanol utilization pathway in Aspergillus nidulans. J. Biol. Chem. 276, 6950–6958. 14. Cahuzac, B., Cerdan, R., Felenbok, B. & Guittet, E. (2001) The solution structure of an AlcR-DNA complex sheds light onto the unique tight and monomeric DNA binding of a Zn 2 Cys 6 protein. Structure 9, 827–836. 15. Nikolaev, I., Cochet, M F. & Felenbok, B. (2003) Nuclear import of zinc binuclear cluster proteins proceeds through multiple, overlapping transport pathways. Eukaryot. Cell 2, 209–221. 16. Nikolaev, I., Mathieu, M., van de Vondervoort, P.J.I., Visser, J. & Felenbok, B. (2002) Heterologous expression of the Aspergillus nidulans alcR–alcA system in Aspergillus niger. Fungal Genet. Biol. 37, 89–97. 17. Kulmburg, P., Mathieu, M., Dowzer, C., Kelly, J. & Felenbok, B. (1993) Specific binding sites in the alcR and alcA promoters of the ethanol regulon for the CREA repressor mediating carbon cata- bolite repression in Aspergillus nidulans. Mol. Microbiol. 7, 847–857. 18. Panozzo, C., Cornillot, E. & Felenbok, B. (1998) The CreA repressor is the sole DNA-binding protein responsible for carbon catabolite repression of the alcA gene in Aspergillus nidu- lans via its binding to a couple of specific sites. J. Biol. Chem. 273, 6367–6372. 19. Flipphi, M., Kocialkowska, J. & Felenbok, B. (2002) Character- istics of physiological inducers of the ethanol utilization (alc) pathway in Aspergillus nidulans. Biochem. J. 364, 25–31. 20. Fillinger, S. & Felenbok, B. (1996) A newly identified gene cluster in Aspergillus nidulans comprises five novel genes localized in the alc region that are controlled both by the specific transactivator AlcR and the general carbon-catabolite repressor CreA. Mol. Microbiol. 20, 475–488. 21. Clutterbuck, A.J. (1993) Aspergillus nidulans.InGenetics Maps. Locus Maps of Complex Genomes (O’Brien, S.J., ed.), pp. 3.71– 3.84. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 22. Cove, D.J. (1966) The induction and repression of nitrate reduc- tase in the fungus Aspergillus nidulans. Biochim. Biophys. Acta 113, 51–56. 23. Arst, H.N. Jr & Page, M.M. (1973) Mutants of Aspergillus nidu- lans altered in the transport of methylammonium and ammonium. Mol. Gen. Genet. 121, 239–245. 24. Pateman,J.A.,Doy,C.H.,Olsen,J.E.,Norris,U.,Creaser,E.H.& Hynes, M. (1983) Regulation of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (AldDH) in Aspergillus nidulans. Proc. R. Soc. Lond. B. Biol. Sci. 217, 243–264. 25. Lockington, R.A., Sealy-Lewis, H.M., Scazzocchio, C. & Davies, R.W. (1985) Cloning and characterization of the ethanol utiliza- tion regulon in Aspergillus nidulans. Gene 33, 137–149. 26. Felenbok, B., Sequeval, D., Mathieu, M., Sibley, S., Gwynne, D.I. & Davies, R.W. (1988) The ethanol regulon in Aspergillus nidu- lans: characterization and sequence of the positive regulatory gene alcR. Gene 73, 385–396. 27. Upshall, A., Gilbert, T., Saari, G., O’Hara, P.J., Weglenski, P., Berse,B.,Miller,K.&Timberlake,W.E.(1986)Molecularana- lysis of the argB gene of Aspergillus nidulans. Mol. Gen. Genet. 204, 349–354. 28. Pickett, M., Gwynne, D.I., Buxton, F.P., Elliot, R., Davies, R.W., Lockington, R.A., Scazzocchio, C. & Sealy-Lewis, H.M. (1987) Cloning and characterization of the aldA gene of Aspergillus nidulans. Gene 51, 217–226. 29. Katz, M.E. & Hynes, M.J. (1989) Isolation and analysis of the acetate regulatory gene, facB,fromAspergillus nidulans. Mol. Cell. Biol. 9, 5696–5701. 30. Fidel, S., Doonan, J.H. & Morris, N.R. (1988) Aspergillus nidulans contains a single actin gene which has unique intron locations and encodes a c-actin. Gene 70, 283–293. 31. Hutchings, H., Stahmann, K P., Roels, S., Espeso, E.A., Tim- berlake, W.E., Arst, H.N. Jr & Tilburn, J. (1999) The multiply- regulated gabA gene encoding the GABA permease of Aspergillus nidulans:ascoreofexons.Mol. Microbiol. 32, 557–568. 32. Delcasso-Tremousaygue, D., Grellet, F., Panabieres, F., Ananiev, E.D. & Delseny, M. (1988) Structural and trans- criptional characterization of the external spacer of a ribosomal RNA nuclear gene from a higher plant. Eur. J. Biochem. 172, 767–776. Ó FEBS 2003 Activation of alc genes in Aspergillus nidulans (Eur. J. Biochem. 270) 3563 33. Uitzetter, J.H.A.A., Bos, C.J. & Visser, J. (1986) Characterization of Aspergillus nidulans mutants in carbon metabolism isolated after D -galacturonate enrichment. J. Gen. Micriobiol. 132, 1167– 1172. 34. Hondmann, D.H.A., Busink, R., Witteveen, C.F.B. & Visser, J. (1991) Glycerol catabolism in Aspergillus nidulans. J. Gen. Microbiol. 137, 629–636. 35. Schuurink, R., Busink, R., Hondmann, D.H.A., Witteveen, C.F.B. & Visser, J. (1990) Purification and properties of NADP + - dependent glycerol dehydrogenases from Aspergillus nidulans and A. niger. J. Gen. Microbiol. 136, 1043–1050. 36. Sealy-Lewis, H.M. & Fairhurst, V. (1992) An NADP + -dependent glycerol dehydrogenase in Aspergillus nidulans is inducible by D -galacturonate. Curr. Genet. 22, 293–296. 37. Arst, H.N. Jr, Jones, S.A. & Bailey, C.R. (1981) A method for the selection of deletion mutations in the L -proline catabolism gene cluster of Aspergillus nidulans. Genet. Res. 38, 171–195. 38. Gavrias, V., Cubero, B., Cazelle, B., Sophianopoulou, V. & Scazzocchio, C. (1994) The proline utilisation gene cluster of Aspergillus nidulans.InThe Genus Aspergillus. From Taxonomy and Genetics to Industrial Application (Powell,K.A.,Renwick,A. & Peberdy, J.F., eds). FEMS Symposium Series 69, pp. 225–232. Plenum Press, New York. 39. Hynes, M.J. (1973) Pleiotropic mutants affecting the control of nitrogen metabolism in Aspergillus nidulans. Mol. Gen. Genet. 125, 99–107. 40. Gallardo, M.E., Desviat, L.R., Rodrı ´ guez, J.M., Esparza- Gordillo, J., Pe ´ rez-Cerda ´ ,C.,Pe ´ rez, B., Rodrı ´ guez-Pombo, P., Criado, O., Sanz, R., Morton, D.H. et al. (2001) The molecular basis of 3-methylcrotonylglycinuria, a disorder of leucine cata- bolism. Am.J.Hum.Genet.68, 334–346. 41. ter Schure, E.G., Flikweert, M.T., van Dijken, J.P., Pronk, J.T. & Verrips, C.T. (1998) Pyruvate decarboxylase catalyzes decarboxy- lation of branched-chain 2-oxo acids but is not essential for fusel alcohol production by Saccharomyces cerevisiae. Appl. Environ. Microbiol. 64, 1303–1307. 42. McKnight, G.L., Kato, H., Upshall, A., Parker, M.D., Saari, G. & O’Hara, P.J. (1985) Identification and molecular analysis of a third Aspergillus nidulans alcohol dehydrogenase gene. EMBO J. 4, 2093–2099. 43. Jones, I.G. & Sealy-Lewis, H.M. (1989) Chromosomal mapping and gene disruption of the ADHIII gene in Aspergillus nidulans. Curr. Genet. 15, 135–142. 44. Patten, C.L. & Glick, B.R. (1996) Bacterial biosynthesis of indole- 3-acetic acid. Can. J. Microbiol. 42, 207–220. 45. Basse, C.W., Lottspeich, F., Steglich, W. & Kahmann, R. (1996) Two potential indole-3-acetaldehyde dehydrogenases in the phytopathogenic fungus Ustilago maydis. Eur. J. Biochem. 242, 648–656. 46. Robinson, M., Riov, J. & Sharon, A. (1998) Indole-3-acetic acid biosynthesis in Colletotrichum gloeosporioides f. sp. Aeschynomene. Appl. Environ. Microbiol. 64, 5030–5032. 47. Spathas, D.H., Clutterbuck, A.J. & Pateman, J.A. (1983) Putrescine as a nitrogen source for wild type and mutants of Aspergillus nidulans. FEMS Microbiol. Lett. 17, 345–348. 48. Richardson, I.B., Hurley, S.K. & Hynes, M.J. (1989) Cloning and molecular characterisation of the amdR controlled gatA gene of Aspergillus nidulans. Mol. Gen. Genet. 217, 118–125. 49. Arst, H.N. Jr (1976) Integrator gene in Aspergillus nidulans. Nature 262, 231–234. 50. Arst, H.N. Jr (1977) Some genetical aspects of ornithine meta- bolism in Aspergillus nidulans. Mol. Gen. Genet. 151, 105–110. 51. Ambroziak, W. & Pietruszko, R. (1993) Metabolic role of alde- hyde dehydrogenase. Adv. Exp. Med. Biol. 328, 5–15. 52. Guengerich, F.P. (1987) Oxidative cleavage of carboxylic esters by cytochrome P-450. J. Biol. Chem. 262, 8459–8462. 53. Peng, H M., Raner, G.M., Vaz, A.D.N. & Coon, M.J. (1995) Oxidative cleavage of esters and amides to carbonyl products by cytochrome P450. Arch. Biochem. Biophys. 318, 333–339. 54. Bell-Parikh, L.C. & Guengerich, F.P. (1999) Kinetics of cyto- chrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde. J. Biol. Chem. 274, 23833–23840. 55. Roberts, T., Martinelli, S. & Scazzocchio, C. (1979) Allele specific, gene unspecific suppressors in Aspergillus nidulans. Mol. Gen. Genet. 177, 57–64. 3564 M. Flipphi et al.(Eur. J. Biochem. 270) Ó FEBS 2003 . Relationships between the ethanol utilization ( alc ) pathway and unrelated catabolic pathways in Aspergillus nidulans Michel Flipphi, Janina Kocialkowska. (199 9) A single amino acid, outside the AlcR zinc binuclear cluster, is involved in DNA binding and in transcriptional regulation of the alc genes in Aspergillus