Tungsten-containing aldehyde oxidoreductase of Eubacterium acidaminophilum Isolation, characterization and molecular analysis David Rauh, Andrea Graentzdoerffer, Katrin Granderath, Jan R. Andreesen and Andreas Pich* Institut fu ¨ r Mikrobiologie, Martin-Luther-Universita ¨ t Halle-Wittenberg, Halle, Germany Aldehyde oxidoreductase of Eubacterium acidaminophilum was purified to homogeneity under strict anaerobic condi- tions using a four-step procedure. The purified enzyme was present as a monomer with an apparent molecular mass of 67 kDa and contained 6.0 ± 0.1 iron, 1.1 ± 0.2 tungsten, about 0.6 mol pterin cofactor and zinc, but no molybdenum. The enzyme activity was induced if a molar excess of electron donors, such as serine and/or formate, were supplied in the growth medium compared to readily available electron acceptors such as glycine betaine. Many aldehydes served as good substrates, thus enzyme activity obtained with acetal- dehyde, propionaldehyde, butyraldehyde, isovaleraldehyde and benzaldehyde differed by a factor of less than two. Kinetic parameters were determined for all substrates tested. Oligonucleotides deduced from the N-terminal amino acid sequence were used to isolate the encoding aorA gene and adjacent DNA regions. The deduced amino acid sequence of the aldehyde oxidoreductase exhibited high similarities to other tungsten-containing aldehyde oxidoreductases from archaea. Transcription of the aorA gene was monocistronic and started from a r 54 -dependent promoter. Upstream of aorA, the gene aorR is localized whose product is similar to r 54 -dependent transcriptional activator proteins and, thus, AorR is probably involved in the regulation of aorA expression. Keywords: aldehyde oxidoreductase; tungsten; pyranopterin cofactor; transcriptional regulation; Eubacterium acidamino- philum. Tungsten is a rather rare element [1] and the element with the largest mass positively involved in living systems. In recent years, tungsten-containing enzymes have been puri- fied from a wide variety of microorganisms [2–6]. The tungsten-containing aldehyde oxidoreductases (AOR) rep- resent a family within the group of pyranopterin-containing molybdo- and tungstoenzymes. In contrast to enzymes of the dimethyl sulfoxide family, tungsten is always at the enzymatic active site ligated by two pyranopterin cofactors andanoxogroupintheenzymesoftheAORfamily[4]. These features separate the enzymes of the AOR family from those of the aldehyde oxidase-type belonging to the xanthine oxidase (molybdenum hydroxylase) family (containing molybdenum ligated to just one pyranopterin- cofactor and a characteristic sulfido group) as observed for aldehyde oxidase and from sulfate reducers like Desulfovib- rio gigas or milk xanthine oxidase [7]. The tungsten- containing AOR family is subdivided into aldehyde oxidoreductase/aldehyde dehydrogenase (AOR) [8–13], formaldehyde oxidoreductase (FOR) [14,15], glyceralde- hyde-3-phosphate oxidoreductase (GAPOR) [16] and carb- oxylic acid reductase (CAR) [17,18]. These enzymes have mainly been isolated from hyperthermophilic archaea like Pyrococcus furiosus or Thermococcus litoralis and from acetogenic bacteria like Moorella thermoacetica and Clos- tridium formicoaceticum. P. furiosus contains additional tungsten-containing enzymes with high similarities to AOR. Despite these similarities, the purified protein WOR4 exhibited none of the known enzymatic activities with aldehydes [19]. Evident from crystallographic studies of aldehyde oxidoreductase from P. furiosus, tungsten is coordinated by two cis-enedithiolate groups of two moly- bdopterin cofactors [11,15,20]. Besides tungsten, all of these aldehyde oxidoreductases possess also one [4Fe-4S] cluster whichisinvolvedinelectrontransferfromthetungsten-binding pterincofactortoanelectronacceptor,usuallyferredoxin. The anaerobic Gram-positive bacterium Eubacterium acidaminophilum degrades amino acids by Stickland reac- tions [21] and possesses two tungsten-containing formate dehydrogenases that are highly similar in their biochemical characteristics and share more than 65% identity in their primary sequence [6,22]. Formate dehydrogenase-I was partially purified, whereas formate dehydrogenase-II was purified to homogeneity. Tungsten and iron, but no molybdenum, were found in the final fractions of both enzymes. The presence of a tungsten-dependent aldehyde oxidoreductase activity was induced by high concentrations Correspondence to J. R. Andreesen, Institut fu ¨ r Mikrobiologie, Kurt-Mothes-Str. 3, 06120 Halle, Germany. Fax: + 49 345 5527010, Tel.: + 49 345 5526350, E-mail: j.andreesen@mikrobiologie.uni-halle.de Abbreviations: AOR, aldehyde oxidoreductase; FOR, formaldehyde oxidoreductase; GAPOR, glyceraldehyde-3-phosphate oxidoreductase. *Present address: Institut fu ¨ r Pathologie, MH-Hannover, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Note: The nucleotide sequence data reported here are available in the GenBank database under accession no. AJ318790. (Received 20 August 2003, revised 29 October 2003, accepted 12 November 2003) Eur. J. Biochem. 271, 212–219 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03922.x of serine and formate, potential electron donors [22]. To enable a specific incorporation of tungsten into these enzymes, tungstate is bound very specifically by an extra- cytoplasmic-orientated binding protein TupA, part of an ABC transporter system [23], and a small cytoplasmic Mop protein, of the molbindin family, able to bind tungstate and molybdate [24]. In this paper we describe the isolation and biochemical characterization of the aldehyde oxidoreductase from Eubacterium acidaminophilum as a monomeric iron-sulfur cluster containing tungstoenzyme. The AOR encoding gene aorA and its adjacent DNA regions were cloned and sequenced. Due to the similarity of aorR to a putative regulator, the transcription of aorA and aorR was analyzed. Materials and methods Bacterial strains, phages and plasmids E. acidaminophilum DSM 3953 T was grown anaerobically in a 100 L fermenter in a serine/formate medium (40/30 m M ) as described previously [21]. Other substrate combinations used are indicated under Results. Escherichia coli XL1 blue MRF¢ was used for DNA manipulations and as phage host for Lambda ZAPII, M13 ExAssist helper phage and E. coli SolR for in vivo excision (all Stratagene, Heidelberg, Germany). The E. coli strains were cultivated aerobically at 37 °C in Luria–Bertani broth [25]. If required, ampicillin was added to the medium at a concentration of 125 lgÆmL )1 , tetracycline at 12.5 lgÆmL )1 , isopropyl thio- b- D -thiogalactopyranoside at 40 lgÆmL )1 and 5-bromo- 4-chloro-3-indolyl b- D -galactopyranoside at 48 lgÆmL )1 . For cloning purposes, plasmid pBluescript II SK/KS (Stratagene) was used. Enzyme assay Aldehyde oxidoreductase was measured by a standard procedure [17] at 34 °C under anaerobic conditions by monitoring the acetaldehyde-dependent reduction of benzyl viologen at 578 nm (e ¼ 8.3 m M )1 Æcm )1 ). The reaction mixture contained 50 m M Tris buffer (pH 8.5), 20 m M benzyl viologen, 0.5 m M acetaldehyde and 0.1–10 lLof enzyme. Sodium dithionite (1–5 lL; 50 m M ) was added to the reaction mixture to obtain an extinction of 0.2–0.3, indicating strict anaerobic conditions. Blanks were per- formed first by excluding the substrate, acetaldehyde, before starting the reaction by the anaerobic addition of acetalde- hyde. One unit of aldehyde oxidoreductase activity was defined as the amount of enzyme catalyzing the reduction of 2 lmol of benzyl viologen per minute. For a fast check for the presence of aldehyde oxidoreductase, the acetaldehyde- dependent reduction of methylene blue was followed visually in microtiter plates. The reaction mixture contained 0.2 m M methylene blue, 0.5 m M acetaldehyde and 50 m M Tris, pH 8.5. Enzyme solutions (1–10 lL) were added and the decolorization of methylene blue was observed. Positive reacting fractions were additionally analyzed by using the benzyl viologen assay. The reverse reaction was analyzed under strict anaerobic conditions in a reaction mixture containing 300 m M potassium phosphate buffer (pH 6.0), 0.4 m M methyl viologen, 20 m M semicarbazide, 60 m M sodium acetate and up to 100 lLofenzyme.Methyl viologen was reduced using sodium dithionite (50 m M )to reach e 600 values around 1.0 (e ¼ 13.1 m M )1 Æcm )1 ). Subse- quently, the reaction was started by adding enzyme. Purification of aldehyde oxidoreductase from E. acidaminophilum Extracts containing aldehyde oxidoreductase activity had to be kept under strict anaerobic conditions. Due to the high oxygen sensitivity of the enzyme, all buffers and solutions were prepared by boiling for about 10 min and were flushed with N 2 during cooling to room temperature and were stored anaerobically. Buffer A contained 50 m M Tris/HCl, pH 8.0, 2 m M sodium dithionite and 2 m M dithiothreitol. Aldehyde oxidoreductase purification was performed in an anaerobic chamber (Coy Laboratory Products, Michigan, USA) containing an N 2 /H 2 (95/5%) atmosphere. To obtain a crude extract, 13 g of frozen cells were thawed and resuspended in 20 mL of buffer A. The suspension was passed twice through a French pressure cell (SLM Instruments Inc., Silver Spring, USA) at 140 MPa (20 K cell). After centrifugation (30 min, 50 000 g)theclear supernatant was taken as crude extract. A saturated ammonium sulfate solution (in buffer A) was added to the supernatant to obtain an overall saturation of 60%. The mixture was stirred for 30 min and centrifuged at 20 000 g for 15 min. The pellet was discarded, although it contained a high portion of enzyme activity. The concentration of ammonium sulfate in the supernatant was increased to 100% saturation. After another centrifugation, the pellet was resuspended in buffer A, dialyzed against this buffer and was applied to a Q-Sepharose column (20 mL) previously equilibrated with buffer A. After loading of the protein, the column was washed with 40 mL of buffer A, and the proteins were eluted with a gradient from 0.1 to 0.6 M NaCl in 300 mL buffer A. Aldehyde oxidoreductase activity eluted from the column at about 0.3 M NaCl. Fractions containing aldehyde oxidoreductase activity were collected, dialyzed against buffer A, and loaded onto a MonoQ HR5/5 anion exchange column equilibrated with buffer A and connected to an FPLC system (Amersham Pharmacia). The column was washed with 10 mL buffer A and proteins were eluted with an increasing NaCl gradient (35 mL) from 0.2 to 0.8 M in buffer A. Active fractions were combined and concentrated to 200 lL using Microcon C30 concentrators (Amicon, Witten, Germany). The active fraction was passed through a Superdex 200 gel filtration column (Amersham Pharmacia) equilibrated with buffer A containing 200 m M NaCl. Protein and metal determination Protein concentrations were determined by the Bradford method using BSA as standard; SDS/PAGE was per- formed with the Laemmli buffer system as described [6]. Proteins were blotted with a semidry blotter (Biometra, Go ¨ ttingen, Germany) from an SDS gel onto a poly(vinyl- dene difluoride) membrane in 50 m M Na 3 BO 3 buffer (pH 9.0) containing 20% (v/v) methanol at 1.2 mAÆcm )2 membrane for 1–2 h. Edman degradation was performed with blotted protein in a 476 A amino acid sequencer Ó FEBS 2003 Isolation and characterization of aldehyde oxidoreductase (Eur. J. Biochem. 271) 213 (Applied Biosystems, Weiterstadt, Germany), all as des- cribed previously [6]. Metal content of aldehyde oxido- reductase was determined using adsorptive stripping voltammetry [26], and neutron activation [27]. Addition- ally, the iron content was determined by a colorimetric technique as described [28]. Analysis of pterin cofactor The pterin cofactor content was determined as described previously [29]. After oxidation of the cofactor with potassium permanganate, protein was precipitated by the addition of 2 vols of ice-cold ethanol and subsequent centrifugation at 15 000 g for 15 min. The fluorescence of the supernatant was determined. An emission spectrum was obtained using an excitation wavelength of 380 nm and an excitation spectrum was determined using an emission wave length of 450 nm. Pterin-6-carboxylic acid was used as a standard and a commercial available xanthine oxidase (Serva) as positive reference. DNA manipulations and DNA sequence determination Genomic DNA from E. acidaminophilum was isolated as decribed [6]. Plasmid DNA from E. coli XL1 blue MRF¢ and SolR was prepared using Qiagen columns (Qiagen, Hilden, Germany). The molecular procedures were either standard techniques [25] or performed as recommended by the respective manufacturers. Nucleotide sequences were determined by the dideoxy chain termination method using the dRhodamine Terminator Cycle Sequencing Ready Reaction Kit and analyzed using an ABI PRISM 377 DNA Sequencer (Perkin Elmer Applied Biosystems, Lan- gen, Germany). The Genome Priming System GPS1 (New England Biolabs) was used for sequence determination. The oligonucleotides were synthesized by Metabion (Martins- ried, Germany). A Lambda ZAPII library was constructed using the Lambda ZAPII Predigested EcoRI/CIAP-Treated Vector Kit (Stratagene). Genomic DNA of E. acidamino- philum was digested with the restriction endonuclease EcoRI and separated in a sucrose density gradient from 10 to 40% (w/v) sucrose for 24 h at 200 000 g. DNA fragments of 5–10 kb mass size range were ligated into the EcoRI site of the Lambda ZAPII vector and subjected to in vitro packaging according to the manufacturer. The resulting phage particles representing a partial library of E. acidami- nophilum were screened by plaque hybridization after infection of E. coli XL1 blue MRF¢. The pBSK phagemid was obtained after in vivo excision of pBSK from the phage DNA using E. coli SOLR cells and M13 ExAssist helper phage as recommended by Stratagene. RNA isolation and Northern hybridization RNA isolation and Northern hybridization was carried out as described recently [6]. Briefly, E. acidaminophilum was grown to mid-exponential phase and harvested by centrif- ugation at 4000 g for 10 min. Total RNA was isolated using the RNeasy Mini Kit (Qiagen). For Northern hybridization experiments, denatured RNA (5 lg per lane) was applied to a formaldehyde agarose gel, separated by electrophoresis, and transferred onto a nylon membrane (Parablot NYamp, Macherey-Nagel, Du ¨ ren, Germany). RNA hybridizations were performed after a modified protocol of Engler-Blum et al. [30] in a high SDS hybridization buffer [0.25 M Na 2 HPO 4 ,pH7.2,1m M EDTA, 20% (v/v) SDS, 0.5% (w/v) blocking reagent) overnight at 68 °C for DNA probes. The membranes were washed three times with 20 m M Na 2 HPO 4 ,pH7.2,1m M EDTA, 1% (v/v) SDS for 20 min at hybridization temperature before detection. Primer extension analysis Primer-extension analysis was performed as described [6]. Two FAM TM -labelled oligonucleotides (5¢-Fam-CTAAG TAGACCTGGAGCGAAG-3¢;5¢-Fam-GGCTTTTCTG ACTTCCCTTCC-3¢) were synthesized and HPLC-purified by Metabion. Total RNA (5 lg) and primer (1 pmol) in a volume of 12 lL were denatured at 70 °C for 10 min and then chilled on ice for 2 min. Then the extension reaction was carried out and the products were analyzed by denaturing PAGE. The size of the extended products was determined using the internal GeneScan-500 (ROX) size standard (Perkin Elmer Applied Biosystems) which was added to the loading buffer. The sizes of the products were analyzed using GENESCAN software (Perkin Elmer Applied Biosystems). Results Isolation of aldehyde oxidoreductase The specific activity of aldehyde oxidoreductase from E. acidaminophilum varieduptoafactorof 12 depending on the substrates and the ratio in which they are present in the growth medium. The highest activity was obtained if cells were grown on serine and formate (40/30 m M ) (Table 1), however, the cell yield was only about 0.5 g Table 1. Specific activities of aldehyde oxidoreductase in crude extracts of cells grown with different carbon and energy sources. Growth substrates (m M ) Activities observed with different substrates Acetaldehyde (UÆmg )1 ) Butyraldehyde (UÆmg )1 ) Benzaldehyde (UÆmg )1 ) Crotonaldehyde (UÆmg )1 ) Formaldehyde (UÆmg )1 ) Glycine (50) 0.05 0.05 0.05 0.03 0.01 Formate/betaine (30/30) 0.24 0.21 0.22 0.11 0.03 Serine/formate/betaine (40/30/30) 0.30 0.29 0.36 0.14 0.06 Serine (40) 0.49 0.45 0.51 0.23 0.08 Serine/formate (40/30) 0.95 0.80 0.99 0.40 0.12 214 D. Rauh et al. (Eur. J. Biochem. 271) Ó FEBS 2003 wet weight per litre in this medium. This lower yield might result from the reduction of acetyl coenzyme A (acetyl- CoA) to ethanol as specially observed for serine-grown cells [21] and the necessity to form additional acetate from C1-compounds (CO 2 or formate) if grown in the presence of a surplus of electron donors [31]. Testing crude extract and benzyl viologen as the electron acceptor, aldehyde oxidoreductase activities were highest with acetaldehyde, butyraldehyde or benzaldehyde as substrate, whereas significantly lower activities were generally obtained with crotonaldehyde and formaldehyde (Table 1). This pattern of substrate specificity did not vary after growth on different substrates or during purification of the enzyme, indicating the presence of just one aldehyde oxidizing enzyme. The use of benzyl viologen as an artificial electron acceptor gave a higher enzyme activity than the use of methyl viologen, whereas no activity was obtained with NAD(P) as the electron acceptor (data not shown). Thus, aldehyde oxidoreductase was purified from serine/formate- grown cells and was generally assayed using acetaldehyde as a substrate and benzyl viologen as the electron acceptor. A fast purification scheme using strict anaerobic conditions had to be developed due to the very high oxygen sensitivity and instability of aldehyde oxidoreductase. Starting from 10 to 15 g (wet weight) of cells of E. acidaminophilum,the crude extract was fractionated using ammonium sulfate precipitation, Q-Sepharose, MonoQ and Superdex 200 to obtain a homogeneous enzyme preparation using SDS/ PAGE (Table 2, Fig. 1). Aldehyde oxidoreductase was purified 44-fold with an activity yield of 1.4% and a final specific activity of 19.6 UÆmg protein )1 . To obtain a final homogeneous enzyme preparation (Fig. 1), it was necessary to use only the supernatant fraction of 60% saturation during ammonium sulfate fractionation. Consequently, a final lower yield of total activity had to be tolerated by taking only this fraction for further purification. The purified protein should also contain enzymatically inactive species due to the observed high instability of the enzymatic activity and the lower content of metal and pterin constituents determined to be present compared to theor- etic expectation. Characterization of aldehyde oxidoreductase Purified aldehyde oxidoreductase eluted from a gel filtration column at a size of about 65 kDa that correlated well with the 67 kDa mass obtained by SDS/PAGE indicating a monomeric structure of this enzyme in contrast to the dimeric nature of other AORs [20]. The N-terminal amino acid sequence was determined by Edman degradation to be: NH 2 -MYGYXGKVIRIN. Apparent K m values of 50 l M for acetaldehyde and 220 l M for butyraldehyde were determined in crude extracts, whereas 19 l M and 12 l M , respectively, were obtained for the homogeneous enzyme. The specificity of the substrate spectrum and the apparent K m values obtained with a partially purified preparation were quite similar to those of aldehyde oxidoreductase from Thermococcus strain ES1 [12] favoring C2 to C4-aldehydes besides benzaldehyde (Table 3). Formaldehyde and glycer- aldehyde were poor substrates for the enzyme from E. acidaminophilum, thus this enzyme does not function like FOR or GAPOR [16,32]. For some aldehydes, a substrate inhibition was noticed if supplied at a higher (0.5 m M ) concentration. Surprisingly, formate as well as formaldehyde was also oxidized by AOR. Table 2. Purification of aldehyde oxidoreductase from E. acidaminophilum. Activity (U) Protein (mg) Specific activity (UÆmg )1 ) Purification (fold) Yield (%) Crude extract 695 1550 0.45 1.0 100.0 (NH 4 ) 2 SO 4 -precipitation 76 109 0.7 1.5 11.0 Q-Sepharose 32 11 2.9 6.4 4.6 MonoQ 22 2.7 8.1 18 3.2 Gel filtration 9.8 0.5 19.6 44 1.4 Fig. 1. Purification of aldehyde oxidoreductase from E. acidaminophi- lum. Aldehyde oxidoreductase was purified by a four step procedure using ammonium sulfate precipitation, Q-Sepharose, MonoQ and gel filtration on Superdex 200. Proteins of the purification steps were separated in a 12% SDS/PAGE and stained with Coomassie Blue. Lane 1, MonoQ-pool; lanes 2–4, fractions after gel filtration. The size of marker proteins in kDa is indicated on the left; aldehyde oxido- reductaseisdepictedbyanarrow. Ó FEBS 2003 Isolation and characterization of aldehyde oxidoreductase (Eur. J. Biochem. 271) 215 The metal content of the purified enzyme was first analyzed by neutron activation. Significant amounts of tungsten, iron and zinc were determined but no molyb- denum or selenium was identified (data not shown). Using adsorptive stripping voltammetry, 1.1 ± 0.2 g atom tung- sten per subunit was detected. Iron was determined by a colorimetric method to give 6.0 ± 0.1 g atom per subunit. The enzyme contained a pterin cofactor as identified by the fluorescence spectrum of the oxidized pterin and 0.6 mol pterin per subunit was recovered after oxidation. However, a higher pterin content could be calculated by comparison with the commercial xanthine oxidase reference. The reverse reaction, the reduction of acetate to acetaldehyde was not catalyzed by this aldehyde oxidoreductase, even at low pH values reported to facilitate the reverse direction [17,18]. Cloning and analysis of the aor -operon The primer, AORW2 (5¢-ATGTAYGGHTAYTGGGG HAARGTIATH-3¢) was deduced from the N-terminal sequence of the aldehyde oxidoreductase. From the known sequences of tungsten-containing aldehyde oxidoreductases [32], conserved motifs were identified and used to deduce primer AORW1r (5¢-TCDCCIGCIGGDCCDAT-3¢). With this primer combination, a 600 bp long DNA fragment was amplified from genomic DNA of E. acidaminophilum that was used as a probe to identify a 4.2 kb EcoRI fragment from a lambda ZAPII gene library of E. acidaminophilum. The insert of the resulting plasmid (pDR2) was sequenced and encodes exactly as determined for the N-terminal sequence of the aldehyde oxidoreductase (Fig. 2). The DNA Table 3. Kinetic parameters of aldehyde oxidoreductase from E. acidaminophilum. In the first columns, specific and relative rates (100% for acetaldehyde) of aldehyde oxidoreductase for different aldehydes at concentrations of 50 l M and 500 l M are given. In the last three columns, the specific activities, apparent K m values and a ratio for the most active aldehydes are shown. Benzyl viologen was the electron acceptor; a partially purified enzyme was used. Aldehyde AOR-activity at Specific activity (UÆmg )1 ) Apparent K m (l M ) Specific activity/K m (gÆs )1 ) 50 l M aldehyde (UÆmg )1 )(%) 500 l M aldehyde (UÆmg )1 ) (%) Acetaldehyde 5.1 ± 1.5 100 6.2 ± 1.4 100 6.0 ± 0.6 19 ± 8.6 5.3 Benzaldehyde 4.9 ± 1.3 96 3.6 ± 1.0 59 4.9 ± 0.6 7.3 ± 4.7 11.2 Butyraldehyde 5.2 ± 0.4 101 5.8 ± 0.9 93 5.7 ± 0.5 12 ± 5.8 7.7 Crotonaldehyde 2.4 ± 0.4 47 2.1 ± 1.3 34 2.9 ± 0.4 16 ± 7.7 3.0 DL -Glyceraldehyde 0.5 ± 0.2 9 0.2 ± 0.2 3 – – – Formaldehyde 0.1 ± 0.2 2 0.5 ± 0.3 7 – – – Formate 0.2 ± 0.0 5 0.4 ± 0.4 6 – – – Glutaraldehyde 1.0 ± 0.1 19 2.8 ± 0.5 44 2.6 ± 0.4 84 ± 44 0.5 Glyoxylate 0.3 ± 0.2 6 0.4 ± 0.3 6 – – – Isovaleraldehyde 3.4 ± 1.3 66 4.3 ± 2.5 69 5.9 ± 0.2 33 ± 5.0 3.0 Phenylacetaldehyde 1.0 ± 0.3 20 3.8 ± 2.0 61 5.6 ± 1.6 67 ± 47 1.3 Propionaldehyde 6.1 ± 0.1 119 6.7 ± 1.0 109 7.2 ± 0.6 17 ± 6.2 7.0 Salicylaldehyde 0.2 ± 0.3 4 0.2 ± 0.1 4 – – – Fig. 2. Genomic organization and transcription of the aor operon of E. acidaminophilum. (A) Genes encoding aldehyde oxidoreductase and its putative regulator AorR are indicated as grey arrows. The localization of the isolated plasmid pDR2 is shown (Top) and also the length of the identified mRNA (below). The identified r 54 -dependent promotor structure (P), the putative enhancer like element (ELE), and the loop structure downstream of aorA are indicated. (B) Sequence of the r 54 -dependent promotor of the aorA gene. The conserved promotor sequence, the putative Shine Dalgano sequence (SD) and the start codon are highlighted by grey boxes. The mRNA start is indicated by an arrow. 216 D. Rauh et al. (Eur. J. Biochem. 271) Ó FEBS 2003 fragment upstream of the 4.2 kb fragment was isolated using a PCR. HindIII digested chromosomal DNA of E. acidaminophilum was ligated into pBluescript SK+ and the ligation mixture was used as a template in a PCR with primer P1 (5¢-ATTCTATGGCGATGCGTTCAG-3¢)and the standard sequencing primer T7. A 0.5 kb DNA fragment that covered about 0.3 kb of the upstream DNA-sequence was generated and directly sequenced. Thus, a stretch of 4.5 kb of this DNA gene region was sequenced. Three open reading frames were detected: aorA encoded the aldehyde oxidoreductase consisting of 608 amino acids with a calculated molecular mass of 66.4 kDa. The amino acid sequence exhibited highest similarities (57% identity) to the aldehyde oxidoreductase of Pyrococcus furiosus and lower similarities to other aldehyde oxidizing enzymes mainly from archaeal sources. As the three- dimensional structure of the P. furiosus aldehyde oxido- reductase is available [11,20], similar functions might be assumed for several conserved amino acids of the aldehyde oxidoreductase of E. acidaminophilum (Fig. 3). Upstream of aorA, an open reading frame encoding 518 amino acids was detected that exhibited high similarities to r 54 -dependent transcriptional activator proteins [33]. In addition, the intermediate gene region contained a consen- sus sequence of a r 54 -dependent promoter [34] (Fig. 2). However, only one enhancer-like element (ELE) and no binding site for an integration host factor was found in the cloned DNA region. Nevertheless, this open reading frame was termed AorR to illustrate its potential regulatory function. Downstream of aorA, orfX¢ was located which had an opposite orientation to aorA. The deduced amino acid sequence (180 amino acids) of the truncated protein exhibited similarities to the C-terminal part of putative efflux pumps from Neisseria gonorrhoeae (Ntrf, EMBL acc. No. AF176821) and Pasteurella multocida (EMBL acc. No. AE006151). At least, four transmembrane helices as calcu- lated with the dense alignment surface method [35] can be identified in the C-terminal part of the deduced amino acid sequence of orfX. Most likely, this protein has no functional connection to the aldehyde oxidoreductase. Transcriptional analysis of the aor operon Northern blot analysis shows that aorA is transcribed in a monocistronic manner by an mRNA of 2.2 kb (Fig. 2). Transcription was detected during growth on serine and serine/formate. However, if an electron acceptor like betaine or sarcosine was also added to the growth medium, no mRNA signal was found (data not shown). Primer exten- sion analyses were performed with two different primers and revealed the same transcriptional start point at a r 54 promotor consensus sequence. The mRNA started at a guanine base at position 1774. The determined size of 2.2 kb of the mRNA matched perfectly with this promotor as well as a loop structure which was identified in the downstream region of aorA (Fig. 2) that exhibited similarities to rho- independent transcription termination signals. No other start point was determined and no sequence similar to the consensus sequence of r 70 promoters was identified upstream of aorA. No transcript was detected by Northern analysis for the aorR gene, probably due to its low expression. Discussion As with all other known tungsten-containing aldehyde oxidoreductases [2,4,20], the purified enzyme of E. acidami- nophilum contained tungsten (but no molybdenum), iron and a pterin cofactor, was very oxygen labile and used viologen dyes as artificial electron acceptors. The enzyme catalyzed the oxidation of several aldehydes to the corres- ponding acids obtaining the highest catalytic efficiencies with acetaldehyde, propionaldehyde, butyraldehyde, and benzaldehyde. This substrate spectrum correlated well with those of other AORs but excluded FOR or GAPOR-like functions [12,14,16]. Acetaldehyde might be one of the main substrates in vivo, because E. acidaminophilum expressed the highest specific activity of aldehyde oxidoreductase under growth conditions when high amounts of serine and formate were present in the medium. Pyruvate ferredoxin oxidoreductase of P. furiosus exhibited an increased side reaction that decarboxylates pyruvate to acetaldehyde if the ratio of reduced to oxidized ferredoxin increases [36]. A similar decarboxylase activity might be assumed for the unstable pyruvate ferredoxin oxidoreductase of E. acidami- nophilum if high concentrations of serine – deaminated to pyruvate [21] – are provided in the presence of formate. Thus, aldehyde oxidoreductase should be induced under these conditions, as was observed. This would also correlate to the observed production of the reduced products ethanol and low amounts of hydrogen that were actually formed during growth on serine as only substrate [21]. Most probably, a ferredoxin might constitute the natural electron acceptor of the enzyme from an anaerobic organism like E. acidaminophilum as happens for the closely related enzymes of P. furiosus and Thermococcus ES1[9,12].The Fig. 3. Alignment of the deduced primary sequences aldehyde oxidoreductase from E. acidaminophilum and P. furiosus. Amino acids with known functions [20] are indicated in both proteins. The GenBank accession numbers are XY1234 for Ea and XY5678 for Pf. Ó FEBS 2003 Isolation and characterization of aldehyde oxidoreductase (Eur. J. Biochem. 271) 217 aldehyde oxidoreductase of E. acidaminophilum contained a sequence motif for a [4Fe-4S] iron-sulfur cluster that might be involved in an electron transfer from the tungsten- ligating pterin to ferredoxin. The formaldehyde oxido- reductase of P. furiosus was cocrystallized with ferredoxin and Cys287 was identified to be important for the inter- action of ferredoxin and formaldehyde oxidoreductase [15]. Only artificial acceptors with a redox potential similar to ferredoxin were suitable to interact with aldehyde oxido- reductase of E. acidaminophilum. Reduced ferredoxin might be reoxidized in E. acidaminophilum by one of the two hydrogenases or the two formate dehydrogenases present in this organism. The latter enzymes catalyze a reduction of CO 2 to formate [6,22] which might become further reduced to acetate [31]. Aldehyde oxidoreductase from E. acidaminophilum did not catalyze the reverse reaction under the conditions tested. Only the carboxylic acid reductases (CAR) as a special group of enzymes of the AOR family are also able to catalyze the reduction of acetate to acetaldehyde, e.g. the reduction of a nonactivated but probably protonated carboxylic acid [17,18]. On the basis of the few known N-terminal sequences, only a low similarity of the aldehyde oxidoreductase from E. acidaminophilum to these enzymes was determined . The sequence of the gene encoding one of these carboxylic acid reducing enzymes is not known so far. Aldehyde oxidoreductase from E. acidaminophilum con- tained tungsten, iron and zinc but no molybdenum or selenium. The determined tungsten content of 1 atom per subunit fits perfectly. Tungsten is bound to the dithiolene groups of two pyranopterin cofactor molecules as has been identified in the aldehyde oxidoreductase of P. furiosus [11] but only 0.6 mol of pterin moiety (instead of two) was calculated to be present in the homogeneous aldehyde oxidoreductase of E. acidaminophilum. However, the amino acids known to be responsible for binding two pterin cofactors per subunit in the P. furiosus enzyme [20] were highly conserved in E. acidaminophilum except that Asp338 was exchanged to Asn (Fig. 3). The low amount of identified pterin cofactor might also be responsible for some of the loss in activity during enzyme purification. About 6 mol iron were identified in the aldehyde oxido- reductase of E. acidaminophilum – a high number consid- ering that only one iron-sulfur cluster of the [4Fe-4S] type should be formed – as observed in all known tungsten- containing aldehyde oxidoreductases [1,4,32]. However, the enzyme of P. furiosus was reported to contain 7 iron atoms for every tungsten atom [9]. In contrast, the molybdenum- containing aldehyde oxidoreductases contain two [2Fe-2S] clusters and belong to the xanthine oxidase family [7]. As noted for the pterin, the amino acids binding this putative [4Fe-4S] cluster are highly conserved in the aldehyde oxidoreductase of E. acidaminophilum (Fig. 3). The pro- posed mononuclear iron bridging the homodimeric subunits of aldehyde oxidoreductase in P. furiosus [11] might add to the iron content and should not be present in the enzyme of E. acidaminophilum due to its monomeric structure. The corresponding amino acids involved in binding in the P. furiosus enzyme (Glu332, His383) are both exchanged to uncharged Val in the case of the E. acidaminophilum enzyme. It was speculated that this mononuclear iron might bridge both subunits and would be responsible to some extent for the thermostability of the P. furiosus enzyme [11]. This feature is not relevant for the enzyme from the mesophile E. acidaminophilum that does not need such stabilization and has obviously no dimeric structure. A mutant of the E. acidaminophilum aldehyde oxidoreductase containing a Val/Glu and a Val/His exchange would be of interest concerning such a possible structure. Aldehyde oxidoreductase might be regulated by r 54 -dependent transcription due to its obvious involvement in amino acid metabolism as anticipated for E. acidamino- philum [33]. Only one transcript was detected starting at the r 54 -like promotor. 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Transcription of the. amount of enzyme catalyzing the reduction of 2 lmol of benzyl viologen per minute. For a fast check for the presence of aldehyde oxidoreductase, the acetaldehyde- dependent