The deduced amino acid sequence of the aldehyde oxidoreductase exhibited high similarities to other tungsten-containing aldehyde oxidoreductases from archaea.. The tungsten-containing AO
Trang 1Tungsten-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, propionalacetal-dehyde, butyralacetal-dehyde, 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 r54-dependent promoter Upstream of aorA, the gene aorR is localized whose product is similar to
r54-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
and an oxo group in the enzymes of the AOR family [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 acidaminophilumdegrades 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)
Trang 2of 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 acidaminophilumas 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 acidaminophilumDSM 3953Twas grown anaerobically in
a 100 L fermenter in a serine/formate medium (40/30 mM)
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 37C 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 34C under anaerobic conditions by
monitoring the acetaldehyde-dependent reduction of benzyl
viologen at 578 nm (e¼ 8.3 mM )1Æcm)1) The reaction
mixture contained 50mM Tris buffer (pH 8.5), 20mM
benzyl viologen, 0.5 mM acetaldehyde and 0.1–10 lL of
enzyme Sodium dithionite (1–5 lL; 50mM) 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 mMmethylene blue, 0.5 mM acetaldehyde and 50mM
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 mM potassium phosphate buffer (pH 6.0),
0.4 m methyl viologen, 20m semicarbazide, 60m
sodium acetate and up to 100 lL of enzyme Methyl viologen was reduced using sodium dithionite (50mM) to reach e600values around 1.0(e¼ 13.1 mM )1Æcm)1) Subse-quently, the reaction was started by adding enzyme
Purification of aldehyde oxidoreductase fromE 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 10min and were flushed with N2 during cooling to room temperature and were stored anaerobically Buffer A contained 50mMTris/HCl,
pH 8.0, 2 mM sodium dithionite and 2 mM dithiothreitol Aldehyde oxidoreductase purification was performed in an anaerobic chamber (Coy Laboratory Products, Michigan, USA) containing an N2/H2(95/5%) atmosphere
To obtain a crude extract, 13 g of frozen cells were thawed and resuspended in 20mL of buffer A The suspension was passed twice through a French pressure cell (SLM Instruments Inc., Silver Spring, USA) at 140MPa (20K cell) After centrifugation (30min, 50000g) the clear 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 30min and centrifuged at 20000g 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 (20mL) previously equilibrated with buffer A After loading of the protein, the column was washed with 40mL of buffer A, and the proteins were eluted with a gradient from 0.1 to 0.6MNaCl in 300 mL buffer A Aldehyde oxidoreductase activity eluted from the column at about 0.3M 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 10mL buffer A and proteins were eluted with an increasing NaCl gradient (35 mL) from 0.2 to 0.8Min 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 mMNaCl
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 50mM Na3BO3 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
Trang 3(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 380nm and an
excitation spectrum was determined using an emission wave
length of 450nm 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-philumwas digested with the restriction endonuclease EcoRI
and separated in a sucrose density gradient from 10to 40%
(w/v) sucrose for 24 h at 200 000 g DNA fragments of
5–10kb 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 10min 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 [0.25M
Na2HPO4, pH 7.2, 1 mM EDTA, 20% (v/v) SDS, 0.5% (w/v) blocking reagent) overnight at 68C for DNA probes The membranes were washed three times with 20mM
Na2HPO4, pH 7.2, 1 mM EDTA, 1% (v/v) SDS for 20min at hybridization temperature before detection Primer extension analysis
Primer-extension analysis was performed as described [6] Two FAMTM-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 70C for 10min 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 usingGENESCANsoftware (Perkin Elmer Applied Biosystems)
Results Isolation of aldehyde oxidoreductase The specific activity of aldehyde oxidoreductase from
E acidaminophilumvaried up to a factor of 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 mM) (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)
Serine/formate/betaine (40/30/30) 0.30 0.29 0.36 0.14 0.06
Trang 4wet 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 (CO2or 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
10to 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:
NH2-MYGYXGKVIRIN Apparent Kmvalues of 50 lM
for acetaldehyde and 220 lM for butyraldehyde were determined in crude extracts, whereas 19 lM and 12 lM, respectively, were obtained for the homogeneous enzyme The specificity of the substrate spectrum and the apparent
Km values obtained with a partially purified preparation were quite similar to those of aldehyde oxidoreductase from Thermococcusstrain 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 mM) 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 (%)
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-reductase is depicted by an arrow.
Trang 5The 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 theaor-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 ± 1.059 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
Glutaraldehyde 1.0 ± 0.1 19 2.8 ± 0.5 44 2.6 ± 0.4 84 ± 44 0.5
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
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 r54-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.
Trang 6fragment upstream of the 4.2 kb fragment was isolated
using a PCR HindIII digested chromosomal DNA of
E acidaminophilumwas 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
furiosusand 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
r54-dependent transcriptional activator proteins [33] In
addition, the intermediate gene region contained a
consen-sus sequence of a r54-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 (180amino 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 theaor 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 r54
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 r70 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-nophilumif 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.
Trang 7aldehyde 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
CO2to 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 furiosusenzyme (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
r54-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 r54-like promotor No indication for a constitutive transcription by a r70-dependent promotor was obtained The protein AorR encoded upstream of aorA might act as a positive regulator of the aldehyde oxidoreductase due to the high similarities to r54-dependent transcriptional activators
of its deduced protein sequence [34] The N-terminal domain
of AorR is most likely the sensor domain to measure, for example, the aldehyde concentrations within the cell Acknowledgements
This work was supported by a grant from the Land Sachsen-Anhalt and by the Fonds der Chemischen Industrie We thank Dr P.L Hagedoorn from Delft University of Technology, Delft, the Nether-lands, for the electroanalytical determination of tungsten, and Dr
D Alber from the Hahn-Meitner-Institut, Berlin, Germany, for the neutron activation analysis.
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