Vorholt2 1 Max-Planck-Institut fu¨r terrestrische Mikrobiologie, Marburg, Germany;2Laboratoire de Biologie Mole´culaire des Relations Plantes–Microorganismes, INRA/CNRS, Castanet-Tolosan
Trang 1The tungsten-containing formate dehydrogenase from
Markus Laukel1,2, Ludmila Chistoserdova3, Mary E Lidstrom3,4and Julia A Vorholt2
1
Max-Planck-Institut fu¨r terrestrische Mikrobiologie, Marburg, Germany;2Laboratoire de Biologie Mole´culaire des Relations Plantes–Microorganismes, INRA/CNRS, Castanet-Tolosan, France;3Department of Chemical Engineering
and4Department of Microbiology, University of Washington, Seattle, Washington USA
NAD-dependent formate dehydrogenase (FDH1) was
isolated from the a-proteobacterium Methylobacterium
extorquensAM1 under oxic conditions The enzyme was
found to be a heterodimer of two subunits (a1b1) of 107
and 61 kDa, respectively The purified enzyme contained
per mol enzyme 5 mol nonheme iron and acid-labile
sulfur, 0.6 mol noncovalently bound FMN, and 1.8 mol
tungsten The genes encoding the two subunits of FDH1
were identified on the M extorquens AM1 chromosome
next to each other in the order fdh1B, fdh1A Sequence
comparisons revealed that the a-subunit harbours putative
binding motifs for the molybdopterin cofactor and at least
one iron–sulfur cluster Sequence identity was highest to
the catalytic subunits of the tungsten- and selenocysteine-containing formate dehydrogenases characterized from Eubacterium acidaminophilum and Moorella thermoacetica (Clostridium thermoaceticum) The b-subunit of FDH1 contains putative motifs for binding FMN and NAD, as well as an iron–sulfur cluster binding motif The b-subunit appears to be a fusion protein with its N-terminal domain related to NuoE-like subunits and its C-terminal domain related to NuoF-like subunits of known NADH-ubiqui-none oxidoreductases
Keywords: formate dehydrogenase; methylotrophic bacteria; one-carbon (C1) metabolism; tungsten; molybdenum
The conversion of formate to CO2is the terminal enzymatic
step in C1unit oxidation to CO2in the a-proteobacterium
Methylobacterium extorquens AM1 and other aerobic
methylotrophic bacteria [1,2] M extorquens AM1
posses-ses two separate pathways for conversion of C1-units
between the oxidation levels of formaldehyde and formate
that are essential for growth on methylotrophic substrates,
methanol and methylamine [1,3,4] One of these pathways
involves tetrahydrofolate (H4F)-dependent enzymes [4,5]
Its main function seems to be the provision of methylene–
H4F for the assimilatory serine cycle and the H4F-bound
C1-intermediates at different oxidation levels for various
biosynthetic reactions Formate is an intermediate in this
pathway, a result of the formyl–H4F ligase reaction [6] The
second C1-converting pathway involves
tetrahydrometha-nopterin (H4MPT)-dependent enzymes, and its main
func-tion seems to be in energy metabolism [4,5,7,8] Some of the
enzymes in this latter pathway exhibit sequence identity to
enzymes that are an integral part of the energy metabolism
in methanogenic archaea (methanogenesis) [9] However, in
contrast with the methanogenesis pathway, the H4 MPT-dependent pathway in M extorquens AM1 involves formate as an intermediate which is formed by the formyltransferase/hydrolase complex [2,10]
NAD-dependent formate dehydrogenase (FDH) activity
in cell extracts of M extorquens AM1 has been previously reported, but the enzyme has not been purified to homo-geneity [11] We were interested to learn more about the formate oxidation step in this bacterium, i.e determine whether different FDH enzymes are present, and to which class they belong based on cofactor content and electron acceptor specificity (for a review see [12])
FDHs from a number of methylotrophic bacteria have already been analysed, and it became evident that their occurrence is not uniform Pseudomonas sp 101 and Mycobacterium vaccae10 were shown to express different NAD-linked FDH enzymes dependent on the presence of molybdenum: one devoid of a prosthetic group and one containing molybdenum The latter was suggested to be partially active with tungsten as well [13,14] The cofactor-free homodimeric NAD-dependent FDH from Pseudo-monassp 101 has been studied in detail [15,16] and a similar enzyme was also purified from Moraxella sp C-1 [17] The molybdenum-containing FDH from the methane-oxidizing bacterium Methylosinus trichosporium was studied in detail
as well This FDH was shown to contain iron–sulfur clusters, a flavin and molybdenum It is reported to be composed of either two [18] or four different subunits [19] Methylobacterium sp RXM was reported to exhibit high specific activity of NAD-dependent FDH when either molybdate or tungstate were present in the growth medium
In the absence of molybdate or tungstate, NAD-dependent FDH activity was detected only at low levels [20] It was
Correspondence to J A Vorholt, Laboratoire de Biologie
Mole´culaire des Relations Plantes – Microorganismes,
INRA/CNRS, BP27, 31326 Castanet-Tolosan, France.
Fax: +33 5 61 28 50 61, Tel.: +33 5 61 28 54 58,
E-mail: vorholt@toulouse.inra.fr
Abbreviations: FDH, formate dehydrogenase; FDH1,
NAD-dependent formate dehydrogenase; H 4 F, tetrahydrofolate;
H 4 MPT, tetrahydromethanopterin; DCPIP, 2,6-dichlorophenol
indophenol.
(Received 30 July 2002, revised 15 November 2002,
accepted 26 November 2002)
Trang 2suggested that this bacterium contains only one FDH that is
active with both tungsten and molybdenum [20] The enzyme
from Methylobacterium sp RXM grown in
molybdate-containing medium supplemented by methanol was partially
purified [21], and its molecular mass reported to be 75 kDa
While tungsten has been recognized as an important
component in some formate dehydrogenases from
anaer-obic bacteria [22–24], the evidence for its presence in aeranaer-obic
methylotrophic bacteria has been only indirect Here we
describe, for the first time, purification and properties of a
tungsten-containing NAD-dependent FDH from the
aero-bic bacterium, M extorquens AM1
Materials and methods
Organism and growth conditions
M extorquensAM1 was grown in the presence of methanol
(120 mM) at 30C in a minimal medium as described
previously [25] Na2WO4 and (NH4)Mo7O21 were added
separately to the culture medium, where specified, to the
final concentration of 0.3 lM The cells were cultivated in
10-L glass fermenters containing 8 L medium The
fer-menters were stirred at 500 r.p.m and gassed with air
(2 LÆmin)1) The cu ltu res were harvested in the late
exponential phase at a cell density of D578¼ 3.5 Cells
were pelleted by centrifugation at 5000 g and stored at
)20 C Where indicated, cells were grown in 2-L
Erlen-meyer flasks filled with 800 mL medium, shaken at
150 r.p.m
FDH assays
A standard optical assay for FDH activity was performed at
30C, by following the reduction of NAD+ at 340 nm
(e340¼ 6.2ÆmM )1Æcm)1) The reaction mixture contained in
a final volume of 0.72 mL 50 mM Tricine/KOH pH 7.0,
30 mMsodium formate, 0.5 mMNAD+and an appropriate
amount of protein One unit of activity was defined as
the amount of enzyme catalysing the reduction of
1 lmol NAD+ or an alternative electron acceptor
The following alternative electron acceptors were used:
ferricyanide (FeCN e420¼ 1.02ÆmM )1Æcm)1);
2,6-dichloro-phenolindophenol (DCPIP e600¼ 16.3ÆmM )1Æcm)1); FMN
(e445¼ 12.5ÆmM )1Æcm)1); FAD (e450¼ 11.3ÆmM )1Æcm)1);
NADP+ (e340¼ 6.2ÆmM )1Æcm)1); benzyl viologen (e578¼
6.25ÆmM )1Æcm)1) Benzyl viologen was tested under anoxic
conditions To test the inhibitory effect of sodium azide, the
enzyme was pre-equilibrated with 0.9 mMsodium azide for
2 min at 30C, then the reaction was started with the
addition of sodium formate
Protein purification
Frozen cells of M extorquens AM1 were resuspended in
50 mMMops/KOH pH 7,0 at 4C and passed three times
through a French pressure cell at 120 MPa Centrifugation
was performed at 150 000 g for 45 min to remove cell
debris, whole cells and the membrane fraction, which was
shown to contain only 3% of the NAD-dependent FDH
activity and benzyl viologen-dependent FDH activity in
comparison with the cytosolic fraction (both under oxic and
anoxic conditions, see below) Protein was determined by the Bradford assay using Bio-Rad reagent with BSA as a standard [26]
NAD-dependent FDH (FDH1) was purified from
M extorquens AM1 via four chromatographic steps at
4C under oxic conditions All chromatographic materials were from Amersham Pharmacia Biotech The soluble fraction of the cell extract (41 mL) was loaded on to a DEAE–Sephacel column (2.6 cm· 10 cm) equilibrated with 50 mMMops/KOH pH 7.0 Protein was eluted with the following gradients of NaCl in this buffer: 50 mL 0M NaCl, 5 mL 0–0.16MNaCl, 50 mL 0.16MNaCl, 325 mL 0.16–0.6MNaCl, 5 mL 0.6–1MNaCl, 65 mL 1MNaCl,
5 mL 1–2M NaCl, 60 mL 2M NaCl NAD-dependent FDH was eluted at 0.4 mM NaCl Combined active fractions (76 mL) were diluted 1 : 2 in 50 mMMops/KOH
pH 7.0, and loaded on to a Source 15Q column (1.6 cm· 10 cm) equilibrated with the same buffer Protein was eluted with the following gradients of NaCl: 250 mL 0–0.6MNaCl, 20 mL 0.6–1MNaCl, 50 mL 1–2MNaCl Active fractions (9 mL) were recovered at 0.6M NaCl These fractions were concentrated using the 30 kDa cut-off Centricon centrifugal filter units (Millipore) to a final volume
of 0.15 mL The protein was loaded on a Superdex 200 column, equilibrated with 50 mMMops/KOH 0.1MNaCl
pH 7.0 Active fractions were loaded on a Resource Q column, equilibrated with 50 mM Mops/KOH pH 7.0 Purified protein was eluted with an increasing NaCl gradient (0–1MNaCl) The purified enzyme was eluted at 0.4M NaCl in 3 mL
For anoxic purification of FDH1, the gas phase of serum bottles containing frozen cells of M extorquens AM1 was replaced by 100% N2 Passing through the French Pressure cell and the centrifugation were performed under N2 atmosphere as well All of the buffers used during the purification were depleted of oxygen by boiling for 5 min followed by cooling under vacuum with stirring, and the addition of 2 mMdithiothreitol All of the chromatographic purification steps were performed in an anaerobic chamber (Coy) under gas atmosphere of 95% N2/5% H2at 15C Elution methods and profiles were similar to those described above
Gel electrophoresis and molecular weight determination
Purified protein was subjected to electrophoresis in a 10% polyacrylamide gel and stained with Coomassie brilliant blue R250 The molecular masses of the subunits of purified FDH1 were also determined by MALDI-TOF analysis using Voyager-DE-RP (Applied Biosystems) The molecu-lar mass of the native enzyme was estimated by gel filtration
on a Superdex 200 column using the following standards: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), ovalbumin (43 kDa), and chymotrypsinogen (25 kDa) Peptide mass finger-printing was performed after trypsin digestion using Voyager-DE-STR (Applied Biosystems) Determination of the N-terminal amino-acid sequence Purified enzyme was separated by electrophoresis in the presence of SDS and electroblotted on to a poly(vinyl
Trang 3trifluoride) membrane (Applied Biosystems) The amino
acid sequence was determinated on a 477-protein/peptide
sequencer (Applied Biosystems)
Analytical methods
For the determination of tungsten, preparations of purified
FDH1 were washed three times with 50 mM Mops/KOH
pH 7.0 using Centricon centrifugal filter units (30-kDa
cut-off, Millipore) Samples were analysed by neutron
activa-tion analysis Molybdenum was determined with an atomic
adsorption spectrometer Zeeman 3030 (Perkin Elmer)
Nonhaem iron was quantified colorimetrically with
neo-cuproin (2,9-dimethyl-1,10-phenanthroline) and ferrozine
[3-(2-pyridyl)-5,6-bis-(4-phenylsulfonate)-1,2,4-triazine by
the method of Fish [27] with Titrisol iron solution (Merck)
as a standard Acid-labile sulfur was determined as
methy-lene blue [28] using Na2S as standard Covalently and
noncovalently bound flavins were determined as described
[29] For pterin cofactor determination, the pH of purified
formate dehydrogenase (0.2 mg in 50 mM Mops/KOH
pH 7.0) was adjusted to pH 2.5 with 2MHCL, then 1% I2/
2% KI was added to the acidified protein at a ratio of 1 : 20
(v/v), and the sample was heated for 30 min in boiling water
bath, followed by cooling and centrifugation at 35 000 g for
10 min The supernatant was filtered through the 30-kDa
cut-off Centricon centrifugal filter units (Millipore) to
remove any precipitate As a positive control, milk xanthine
oxidase (Sigma-Aldrich) was treated in the same way As
negative controls, FMN and NADH were used
Fluores-cence spectra were recorded in a Carian Eclipse
spectroflu-orometer (Varian) at a fixed excitation wavelength of
380 nm and emission wavelengths of 380–700 nm
Sequence analysis
The genes encoding the subunits of FDH1 purified in this
study were identified viaBLASTsearch against the genomic
database of M extorquens AM1 (http://vixen.microbiol
washington.edu/), using the N-terminal amino acid
sequence of the b-subunit as a query The sequence of
4741 bp containing fdh1A and fdh1B has been deposited
with GenBank under the accession number AF489516 The
amino acid sequences translated from fdh1A and fdh1B
were used as queries to search the nonredundant database
(http://www.ncbi.nlm.nih.gov) The sequences for the
puta-tive formate dehydrogenase subunits homologous to the
subunits of FDH1 were also identified in the Methylococcus
capsulatusgenome whose sequence has been released before publishing by the Institute for Genomic Research (http:// tigrblast.tigr.org/ufmg/)
Results and discussion
Purification of NAD-dependent formate dehydrogenase and identification of encoding genes
Cell extracts of M extorquens AM1 grown in the presence
of methanol in a fermenter under standard conditions (0.3 lMmolybdenum, no addition of tungsten) contained NAD-dependent FDH activity of about 0.1–0.2 UÆmg)1 and benzyl viologen-dependent FDH activity in the same range Purification of FDH was attempted under both oxic and anoxic conditions Both conditions resulted in similar protein yields, specific activity and stability of the enzyme, therefore only results of purification under oxic conditions are shown in Table 1 One major FDH activity peak was detected via NAD- or benzyl viologen-dependent activity determination upon each purification step After four chromatographic steps, FDH was enriched 520-fold with
a yield of 15% and a specific activity of 73 UÆmg)1 SDS/PAGE analysis revealed the presence of two poly-peptides, of molecular masses of 105 and 60 kDa, respectively (Fig 1) MALDI-TOF analysis also showed the presence of two molecules, of 107 and 61 kDa The N-terminal amino acid sequence of the smaller subunit was determined to be: SEASGTV?SFAHPG?G?NVA?AV-PKG?QVDP It was, however, not possible to determine the N-terminal amino acid sequence of the larger subunit using a number of different preparations of the protein The gene encoding the smaller subunit (fdh1B) was identified
in the unfinished genome database of M extorquens AM1 (L Chistoserdova and M E Lidstrom, unpublished data), viaBLASTsearch with the N-terminal amino acid sequence shown above The 26 amino acid residues identified by Edman degradation were identical to the respective N-ter-minal 26 amino acid residues in the polypeptide translated from fdh1B (see Fig 3) Fdh1B has a predicted molecular mass of 62 kDa, which is in agreement with the experi-mentally determined mass for the b-subunit (61 kDa) The gene located 56 nucleotides downstream of fdh1B poten-tially encodes a polypeptide with a predicted molecular mass
of 107 kDa, which is in perfect agreement with the determined molecular mass of the larger subunit The identity of this ORF as the larger subunit of FDH1 was confirmed by peptide mass finger-printing analysis All of
Table 1 Purification of NAD-dependent formate dehydrogenase (FDH1) from M extorquens AM 1 Enzyme activity was determined at 30 C u nder standard assay conditions Cells were cultivated in 8 L -fermenters in the presence of methanol in minimal medium supplemented with 0.3 l M
molybdenum.
Total protein (mg)
Total activity (U)
Specific activity (UÆmg)1)
Purification (fold)
Recovery (%)
Trang 4the seven most intense mass peaks obtained after trypsin
digestion of the larger subunit of FDH1 fitted within a
20-p.p.m range of the predicted masses calculated using
PEPTIDEMASS (http://www.expasy.org) The ORF
dow-stream of fdh1B was therefore designated fdh1A
Apart from the genes for FDH1, the genome of
M extorquensAM1 contains two additional gene clusters
potentially encoding formate dehydrogenases (L
Chisto-serdova, M Laukel, J A Vorholt & M E Lidstrom,
unpublished data), one similar to the soluble
NAD-depend-ent FDH characterized from R eutropha [30] and one
similar to the membrane-bound FDH from Wolinella
succinogenes[31] The presence of multiple FDH enzymes
in M extorquens AM1 lead us to use a nonstandard gene
nomenclature (fdh1AB), which will aid in the future in
discriminating between the three different enzymes (FDH1,
FDH2 and FDH3)
Sequence analysis
Analysis of the amino acid sequence translated from fdh1A
revealed similarity to the molybdopterin binding family of
FDHs Fdh1A shares 40% of identical amino acid
residues with the catalytic a subunits of the two tungsten
and selenocysteine-containing FDHs from Eubacterium
acidaminophilum[32] and with the a-subunit of FDH from
the thermophilic acetogenic bacterium Moorella
thermoace-tica(Clostridium thermoaceticum) (Acc No U73807), and it
shows about 35% identity with FDHHfrom Escherichia coli
[33] (Fig 2) The highest sequence identity, however, was
found with the polypeptides translated from, respectively,
the genomic sequence of Methylococcus capsulatus (63%),
another aerobic methylotrophic bacterium (http://tigrblast
tigr.org/ufmg/) and a DNA region sequenced in the course
of Leishmania major genome sequencing project that is
believed to belong to an unknown bacterium (64%, Acc
No AC091510; data not shown)
FDHHfrom E coli was studied in detail biochemically and crystal structures of the enzyme are known [34] The enzyme contains selenocysteine, molybdenum and two molybdopterin guanine dinucleotide cofactors, and a [4Fe)4S] cluster Twelve conserved amino acid residues have been identified that coordinate directly the two molybdopterin cofactors and are conserved among the known sequences of molybdopterin-containing FDH enzymes [34] The molybdopterin cofactors each provide two sulfur atoms for the ligation of the central Mo/W atom Besides, the Mo/W is coordinated by the selenium atom of selenocysteine and cysteine, respectively, that corresponds
to position 140 of FDHHof E coli The alignment shown
in Fig 2 indicates that the sequence of FDH1 from
M extorquensAM1 fits well in the family of molybdopterin-containing FDH All of the amino acid residues that have been shown to participate in the coordination of the central Mo/W atom via two pterin cofactors are also identified in the sequence of FDH1
The primary sequence of the b-subunit of FDH1 from
M extorquensAM1 indicates that the protein is composed
of two regions: an N-terminal region (amino acid residues 1–185) and a C-terminal region (amino acid residues 186– 572) (Fig 3) The latter exhibits sequence identities of
30% to the subunit HoxF of pyridine-nucleotide-redu-cing nickel hydrogenases [35,36], the subunit NuoF of NADH-ubiquinone oxidoreductases from various organ-isms, e.g Aquifex aeolicus [37], and to the b-subunit of formate dehydrogenase of Ralstonia eutropha [30] (Fig 3) All of these sequences contain putative motifs for flavin, NAD, and iron–sulfur cluster binding sites The N-terminal part exhibits sequence identities to the subunit HoxE of nickel hydrogenases, e.g from Synechococcus sp [38], to the subunit NuoE of NADH-ubiquinone oxidoreductases and the c-subunit of formate dehydrogenase from R eutropha [30] All of these sequences contain four conserved cysteines, which might be involved in iron–sulfur cluster binding Polypeptides showing the highest identities with Fdh1B (both at 58%) are translated from the chromosomes of
M capsulatusand of the unknown bacterial contaminant of
L majorDNA (see above) In these two latter cases, the polypeptides also reveal the two-domain nature described above for Fdh1B
Properties The optimum pH for formate oxidation with NAD+was determined to be between pH 8.0 and 8.5 in 120 mM potassium phosphate buffer
Purified FDH1 could reduce the artificial electron acceptors DCPIP and benzyl viologen However, none of the natu ral electron acceptors, i.e FAD, FMN, or NADP+, could replace NAD+ The apparent Kmvalues for FDH1 were determined to be 1.6 mM for sodium formate and 0.07 mMfor NAD+ Sodium azide is known
as a transition state analogue of formate and therefore a general inhibitor of FDHs [39] A 50% inhibitory effect was observed in the presence of 0.9 mM sodium azide in standard assay conditions A stabilizing effect of sodium azide and potassium nitrate on some FDHs upon enzyme purification was reported [21,40,41] This effect was not observed for FDH1 from M extorquens AM1
Fig 1 SDS/PAGE analysis of purified FDH1 of M extorquens AM1.
Proteins were separated in a 10% polyacrylamide gel and stained with
Coomassie brilliant blue R250 Lane A, molecular mass standards:
phosphorylase B (97 kDa), albumin (66 kDa), ovalbumin (45 kDa),
carbonic anhydrase (30 kDa); lane B, 5 lg purified FDH1 The use of
a higher percentage polyacrylamide gel (14%) did not indicate the
presence of smaller polypeptides (data not shown).
Trang 5The UV/visible spectrum of the purified enzyme (Fig 4),
as well as its brownish/yellowish colour were indicative of
the presence of cofactors, and this finding is in agreement
with the primary sequence analysis data (see above) The
spectrum shows shoulders at about 362, 375, 415, 455, and
570 nm The absorption peaks at 375 and 455 might originate form a flavin (see below), other peaks might be due
to FeS centres in the enzyme The enzyme could be partially
Fig 2 Alignment of amino acid sequences for Fdh1A from M extorquens AM1, FdhAI and FdhAII from Eubacterium acidaminophilum [32], FdhA from Moorella thermoacetica (U73807), and FdhH from E coli [33] The alignment was performed using the CLUSTAL W method ( DNASTAR ) Identical residues present in at least four of the sequences are marked by grey boxes Conserved regions for the 4Fe )4S iron–sulfur cluster binding are shown in blue Amino acids coordinating the two molybdopterin guanine dinucletoide cofactors of Fdh H of E coli (MGD 801 and MGD 802 ) that were found to be invariant among molybdopterin-containing FDH [34] are shown in red Amino acids coordinating MGD801 and MGD802 that are less well conserved among the family of molybdopterin-containing FDH [34] but are conserved in the sequences shown here are marked in pink The selenocysteine at position 140 of Fdh H that is a direct ligand of the Mo [34] is shown in dark-red.
Trang 6reduced with either NADH or dithionite leading to a
decreased absorption in the spectral range of interest
The iron content was determined to be at 5.4 molÆmol
enzyme)1and the acid-labile sulfur content was determined
to be at 4.7 molÆmol enzyme)1, which might indicate that
iron–sulfur clusters have been partially lost upon
purifica-tion of the protein In addipurifica-tion, the enzyme was found to
contain 0.6 molÆmol)1 FMN while FAD could not be
detected Upon iodine oxidation, a fluorescent compound
was liberated from the enzyme that exhibited an emission
maximum at 470 nm and is supposed to be the pterin
cofactor (Fig 5) For comparison, a spectrum of identically
treated milk xanthine oxidase is given The amounts of
purified FDH1 used for isolation of the pterin cofactor were
not sufficient to allow quantification or the identification of
the exact nature of the molybdopterin cofactor However,
the primary sequence analysis of FDH1 clearly indicates
that all of the amino acid residues required for coordination
of the two molecules of molybdopterin cofactor are present
(Fig 2) It therefore seems very likely that FDH1 contains
two molybdopterin cofactors per active site as is generally
found for molybdopterin-containing prokaryotic
oxotrans-ferases [42]
No molybdenum was detected in the enzyme by neutron
activation analysis, or by atomic adsorption spectrometry
Instead, tungsten could clearly be detected by neutron activation analysis in the purified FDH1 The stoichiomet-ric calculation indicated a ratio of about 1.8 mol tung-stenÆmol enzyme)1 This value is probably somewhat overestimated as the coordination of more than one tungsten in the active site could not be expected Thus, FDH1 from M extorquens AM1 is a tungsten-containing enzyme Even though the sequence of FDH1 indicates a closer relatedness to known tungsten-containing FDHs than to known molybdenum-containing FDHs, this finding
is still very surprising Untill now, FDHs of aerobic bacteria were generally believed to be molybdenum-dependent enzymes or enzymes devoid of prosthetic groups [12,43] The presence of a tungsten-containing formate dehydrogenase in a strictly aerobic bacterium may indicate that tungsten-containing enzymes are not restricted to anaerobic organisms and are probably more widespread than previously believed For example, M capsulatus, another aerobic methylotroph, also contains genes poten-tially encoding an enzyme very similar to FDH1 (see above), and a membrane-bound tungsten FDH has been detected in R eutropha [44] Another surprising property of FDH1 is the lack of oxygen sensitivity, while all of the previously characterized tungsten-containing FDHs were reported to be extremely oxygen-sensitive [22]
Fig 3 Alignment of amino acid sequences for Fdh1B from M extorquens AM1 with subunits of NAD(P)-dependent nickel-hydrogenases, subunits of the soluble FDH from Ralstonia eutropha and subunits of NADH-ubiquinone oxidoreductases The N-terminal part of Fdh1B from M extorquens AM1 (amino acid 1–185) is aligned with HoxE from Synechococcus sp [38], the c-subunit of FDH from Ralstonia eutropha [30], and NuoE from Aquifex aeolicus [37] The C-terminal part of Fdh1B from M extorquens AM1 (amino acid 186–578) is aligned with HoxF from Anabaena variabilis [36], the b-su bu nit of FDH from Ralstonia eutropha [30], and NuoF from A aeolicus [37] Identical residues are shown by dark-grey boxes Conserved regions for binding FMN (according to EXPASY , http://www.expasy.ch), 4Fe )4S iron–sulfur cluster, and NAD (NCBI, http:// www.ncbi.nlm.nih.gov/BLAST/) are underlined.
Trang 7Tungsten was determined in FDH1 preparations from
M extorquens AM1 even if no tungstate was added to
the growth medium, when cultivated in fermenters We
assume that in these cases tungsten must have been
leached from the steel of the fermenters as the fermenters
used in this study have been routinely used for cultivating
methanogenic archaea, and respective media have been
supplemented with tungstate Since tungsten was clearly
preferred over molybdenum for incorporation into
FDH1, even in the presence of excess of molybdate in
the growth medium, M extorquens AM1 must possess a
specific high-efficiency tungstate transporter Such a
transporter belonging to the ABC transporter group
was recently identified in Eubacterium acidaminophilum
[45] Its existence was initially predicted based on the fact
that tungstate was present in FDH preparations isolated
from cells grown in the absense of tungstate [46] TupA,
the substrate binding subunit of this transporter was
shown to have a Kd value of 0.5 lM for tungstate,
whereas molybdate and sulfate were bound weakly when
added at a more than 1000-fold molar excess [45] The
analysis of the genomic database of M extorquens AM1
(L Chistoserdova and M E Lidstrom et al unpublished
data) indicates the presence of a putative ABC
transpor-ter whose putative substrate-binding protein shows 46%
sequence identity to TupA from Eubacterium
acidamino-philum Gene clusters similar to the one in Eubacterium
acidaminophilumcontaining tupA are also found in Vibrio
cholerae, Campylobacter jejuni, Haloferax volcanii and
Methanothermobacter thermautotrophicus, and a function
was suggested for these genes in the specific uptake of
tungstate [45] It is very likely that the TupA orthologue
in M extorquens AM1 serves such a function
Effect of molybdate and tungstate on methylotrophic growth ofM extorquens AM1 and NAD-dependent FDH activity
To test the effect of the addition of molybdate and tungstate on methylotrophic growth of M extorquens AM1 and FDH activity, we performed batch culture experiments using Erlenmeyer flasks No significant effect
on growth of the wild type M extorquens AM1 was observed when molybdate or tungstate or none of these trace elements were added to the methanol-containing growth medium However, the activity of NAD-dependent FDH varied depending on the presence of these trace elements Extracts of cells grown in the medium to which tungstate and no molybdate were added exhibited a specific activity of approximately 0.2 UÆmg)1 About half
of this specific activity was found in extracts of cells grown
in the medium containing molybdate and no tungstate In the absence of either of the trace elements, FDH activity was only 0.003 UÆmg)1 This low activity is probably an indication that no tungsten contamination was present in the flasks The activity of NAD-dependent FDH found in these conditions in the presence of molybdate might be due to an alternative, molybdenum-containing enzyme that we have so far been unable to detect biochemically in
Fig 5 Emission fluorescence spectra of purified FDH1 and milk xan-thine oxidase after iodine treatment (I) Fluorescence spectrum of FDH1 after oxidation with KI/I 2 ; (II) the same spectrum corrected for FMN and Raman; (III) fluorescence spectrum of milk xanthine oxidase The emission fluorescence spectra were measured at
pH < 2.5 The emission spectra were taken at an excitation wave-length of 380 nm The peak at 470 nm indicates the presence of a pterin cofactor in FDH1.
Fig 4 UV/visible absorption spectra of purified FDH1 Spectra shown
are as isolated in an air-oxidized state (black line), reduced by the
addition of crystalline NADH (dark grey line) or dithionite (pale grey
line) Spectra were recorded with the enzyme (0.14 mgÆmL)1) in 50 m M
Mops/KOH buffer pH 7.0, against a buffer blank.
Trang 8fermenter grown cultures, possibly due to tungstate
inhibition This alternative enzyme might be encoded by
a cluster of four genes homologous to the genes encoding
the soluble FDH of R eutropha [31] (see above) A third
gene cluster is present in the M extorquens AM1 genome,
potentially encoding a membrane-bound FDH similar to
the one characterized from W succinogenes [32] Work is
in progress focusing on the roles of the three different
FDHs in M extorquens AM1 and their expression pattern
under different growth conditions
Acknowledgements
This work was supported by the Max-Planck-Gesellschaft, the Centre
National de la Recherche Scientifique, the Deutsche
Forschungsgeme-inschaft, and the Public Health Service National Institutes of Health
(GM58933) We thank D Alber (Hahn-Meitner-Institut, Berlin,
Germany) for the determination of tungsten, A Pierik (University of
Marburg, Germany) for helpful discussions, M Rossignol (UMR 5546
CNRS/Universite´ P Sabatier, Castanet-Tolosan, France) for the
peptide mass finger-printing analysis and D Linder (University of
Giessen, Germany), for determination of the N-terminal amino-acid
sequence of the purified FDH1.
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