The tungsten-containing formate dehydrogenase from
Methylobacterium extorquens
AM1: Purification and properties
Markus Laukel
1,2
, Ludmila Chistoserdova
3
, Mary E. Lidstrom
3,4
and Julia A. Vorholt
2
1
Max-Planck-Institut fu
¨
r terrestrische Mikrobiologie, Marburg, Germany;
2
Laboratoire de Biologie Mole
´
culaire des Relations
Plantes–Microorganismes, INRA/CNRS, Castanet-Tolosan, France;
3
Department of Chemical Engineering
and
4
Department of Microbiology, University of Washington, Seattle, Washington USA
NAD-dependent formate dehydrogenase (FDH1) was
isolated from the a-proteobacterium Methylobacterium
extorquens AM1 under oxic conditions. The enzyme was
found to be a heterodimer of two subunits (a
1
b

unit oxidation to CO
2
in the a-proteobacterium
Methylobacterium extorquens AM1 and other aerobic
methylotrophic bacteria [1,2]. M. extorquens AM1 posses-
ses two separate pathways for conversion of C
1
-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 (H
4
F)-dependent enzymes [4,5].
Its main function seems to be the provision of methylene–
H
4
F for the assimilatory serine cycle and the H
4
F-bound
C
1
-intermediates at different oxidation levels for various
biosynthetic reactions. Formate is an intermediate in this
pathway, a result of the formyl–H
4
F ligase reaction [6]. The
second C
1
-converting pathway involves tetrahydrometha-

monas sp. 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:
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)
Eur. J. Biochem. 270, 325–333 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03391.x

final concentration of 0.3 l
M
. 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
).Thecultureswereharvestedinthelate
exponential phase at a cell density of D
578
¼ 3.5. Cells
were pelleted by centrifugation at 5000 g andstoredat
)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
30 °C, by following the reduction of NAD
+
at 340 nm
(e
340
¼ 6.2Æm
M
)1
Æcm
)1
). The reaction mixture contained in
a final volume of 0.72 mL 50 m
M

(e
445
¼ 12.5Æm
M
)1Æ
cm
)1
); FAD (e
450
¼ 11.3Æm
M
)1
Æcm
)1
);
NADP
+
(e
340
¼ 6.2Æm
M
)1
Æcm
)1
); benzyl viologen (e
578
¼
6.25Æm
M
)1

M
Mops/KOH pH 7.0. Protein was eluted with
the following gradients of NaCl in this buffer: 50 mL 0
M
NaCl, 5 mL 0–0.16
M
NaCl, 50 mL 0.16
M
NaCl, 325 mL
0.16–0.6
M
NaCl, 5 mL 0.6–1
M
NaCl, 65 mL 1
M
NaCl,
5mL 1–2
M
NaCl, 60 mL 2
M
NaCl. NAD-dependent
FDH was eluted at  0.4 m
M
NaCl. Combined active
fractions (76 mL) were diluted 1 : 2 in 50 m
M
Mops/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

replaced by 100% N
2
. Passing through the French Pressure
cell and the centrifugation were performed under N
2
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 m
M
dithiothreitol. All of the chromatographic
purification steps were performed in an anaerobic chamber
(Coy) under gas atmosphere of 95% N
2
/5% H
2
at 15 °C.
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).

Mops/KOH
pH 7.0) was adjusted to pH 2.5 with 2
M
HCL, then 1% I
2
/
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 via
BLAST
search against the genomic
database of M. extorquens AM1 (robiol.
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
(). The sequences for the puta-

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)wasidentified
in the unfinished genome database of M. extorquens AM1
(L. Chistoserdova and M. E. Lidstrom, unpublished data),
via
BLAST
search 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. e xtorquens AM 1. Enzyme activity was determined at 30 °Cunder
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.

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 FDH
H
from 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).
FDH
H
from E. coli was studied in detail biochemically
and crystal structures of the enzyme are known [34]. The

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. capsulatus and of the unknown bacterial contaminant of
L. major DNA (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 m
M
potassium phosphate buffer.
Purified FDH1 could reduce the artificial electron
acceptors DCPIP and benzyl viologen. However, none of
thenaturalelectronacceptors,i.e.FAD,FMN,or
NADP
+
, could replace NAD
+
. The apparent K
m
values

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 Eubacte rium 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.
Ó FEBS 2003 Tungsten-containing FDH from M. extorquens AM1 (Eur. J. Biochem. 270) 329
reduced 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
)1
and the acid-labile sulfur content was determined
to be at  4.7 molÆmol enzyme

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-subunitofFDHfromRalstonia eutropha [30], and NuoF from A. aeolicus [37]. Identical residues are shown by dark-grey boxes.
Conserved regions for binding FMN (according to
EXPASY
, ), 4Fe)4S iron–sulfur cluster, and NAD (NCBI, http://
www.ncbi.nlm.nih.gov/BLAST/) are underlined.

sequence identity to TupA from Eubacterium acidamino-
philum. Gene clusters similar to the one in Eubacterium
acidaminophilum containing 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 of
M. 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

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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