Tài liệu Báo cáo khoa học: The tungsten-containing formate dehydrogenase from Methylobacterium extorquens AM1: Purification and properties docx

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

1
)of107
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
wereidentifiedontheM. 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 (C
1
) metabolism; tungsten; molybdenum.
The conversion of formate to CO
2
is the terminal enzymatic
step in C
1

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-

nopterin (H
4
MPT)-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 H
4
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 vaccae 10 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-

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

suggested 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 aerobic
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. extorquens AM1 was grown in the presence of methanol
(120 m
M
)at30°C in a minimal medium as described
previously [25]. Na
2
WO
4
and (NH
4
)Mo
7
O
21
were added
separately to the culture medium, where specified, to the

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

Tricine/KOH pH 7.0,
30 m
M
sodium formate, 0.5 m
M
NAD
+
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 e
420
¼ 1.02Æm
M
)1
Æcm
)1
); 2,6-dichloro-
phenolindophenol (DCPIP e
600
¼ 16.3Æm
M
)1
Æcm
)1
); FMN

(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

Æcm
)1
). Benzyl viologen was tested under anoxic
conditions. To test the inhibitory effect of sodium azide, the
enzyme was pre-equilibrated with 0.9 m
M
sodium azide for
2 min at 30 °C, then the reaction was started with the
addition of sodium formate.
Protein purification
Frozen cells of M. extorquens AM1 were resuspended in
50 m
M
Mops/KOH pH 7,0 at 4 °C 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
4 °C 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 m

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

0–0.6
M
NaCl, 20 mL 0.6–1
M
NaCl, 50 mL 1–2
M
NaCl.
Active fractions (9 mL) were recovered at  0.6
M
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 m
M
Mops/KOH 0.1
M
NaCl
pH 7.0. Active fractions were loaded on a Resource Q
column, equilibrated with 50 m
M
Mops/KOH pH 7.0.
Purified protein was eluted with an increasing NaCl gradient
(0–1
M
NaCl). The purified enzyme was eluted at  0.4
M
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% 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).

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
326 M. Laukel et al. (Eur. J. Biochem. 270) Ó FEBS 2003
trifluoride) 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 m
M
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 Na
2
S 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 m
M

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-

tive formate dehydrogenase subunits homologous to the
subunits of FDH1 were also identified in the Methylococcus
capsulatus genome 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 l
M
molybdenum, 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)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.

Total protein
(mg)
Total activity
(U)
Specific activity
(UÆmg
)1
)
Purification
(fold)
Recovery
(%)
Soluble fraction 1343 185 0.14 1 100
DEAE–Sephacel 74 152 2.1 15 82
Source 15 Q 8.8 94 10.7 76 51
Superdex 200 1.2 50 42 300 27
Resource Q 0.37 27 73 521 15
Ó FEBS 2003 Tungsten-containing FDH from M. extorquens AM1 (Eur. J. Biochem. 270) 327
the 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
(). The ORF dow-
stream of fdh1B was therefore designated fdh1A.
Apart from the genes for FDH1, the genome of
M. extorquens AM1 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 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

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 FDH
H
of E. coli. The alignment shown
in Fig. 2 indicates that the sequence of FDH1 from
M. extorquens AM1 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. extorquens AM1 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. 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

for FDH1 were determined to be 1.6 m
M
for sodium
formate and 0.07 m
M
for 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 m
M
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).
328 M. Laukel et al. (Eur. J. Biochem. 270) Ó FEBS 2003
The 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 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

)1
, which might indicate that
iron–sulfur clusters have been partially lost upon purifica-
tion of the protein. In addition, 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-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.

330 M. Laukel et al. (Eur. J. Biochem. 270) Ó FEBS 2003
Tungsten 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
showntohaveaK
d
value of 0.5 l
M
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
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

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
)in50m
M
Mops/KOH buffer pH 7.0, against a buffer blank.
Ó FEBS 2003 Tungsten-containing FDH from M. extorquens AM1 (Eur. J. Biochem. 270) 331
fermenter 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.
References
1. Vorholt, J.A. (2002) Cofactor-dependent pathways of formalde-
hyde oxidation in methylotrophic bacteria. Arch. Microbiol. 178,
239–249.
2. Pomper, B.K., Saurel, O., Milon, A. & Vorholt, J.A. (2002)
Generation of formate by the formyltransferase/hydrolase com-
plex (fhc) from Methylobacterium extorquens AM1. FEBS Lett.
523, 133–137.
3. Chistoserdova, L., Vorholt, J.A., Thauer, R.K. & Lidstrom, M.E.
(1998) C

1
transfer enzymes and coenzymes linking methylotrophic
bacteria and methanogenic archaea. Science 281, 99–102.
4. Vorholt,J.A.,Chistoserdova,L.,Lidstrom,M.E.&Thauer,R.K.
(1998) The NADP-dependent methylene tetrahydromethano-
pterin dehydrogenase in Methylobacterium extorquens AM1.
J. Bacteriol. 180, 5351–5356.
5. Pomper, B.K., Vorholt, J.A., Chistoserdova, L., Lidstrom, M.E.
& Thauer, R.K. (1999) A methenyl tetrahydromethanopterin
cyclohydrolase and a methenyl tetrahydrofolate cyclohydrolase in
Methylobacterium extorquens AM1. Eur. J. Biochem. 261, 475–
480.
6. Maden, B.E.H. (2000) Tetrahydrofolate and tetrahydrometha-
nopterin compared: functionally distinct carriers in C
1
metabo-
lism. Biochem. J. 350, 609–629.
7. Hagemeier, C.H., Chistoserdova, L., Lidstrom, M.E., Thauer,
R.K. & Vorholt, J.A. (2000) Characterization of a second
methylene tetrahydromethanopterin dehydrogenase from Methy-
lobacterium extorquens AM1. Eur. J. Biochem. 267, 3762–3769.
8. Vorholt, J.A., Marx, C.J., Lidstrom, M.E. & Thauer, R.K. (2000)
Novel formaldehyde-activating enzyme in Methylobacterium
extorquens AM1 required for growth on methanol. J. Bacteriol.
182, 6645–6650.
9. Thauer, R.K. (1998) Biochemistry of methanogenesis: a tribute to
Marjory Stephenson. Microbiology 144, 2377–2406.
10. Pomper, B.K. & Vorholt, J.A. (2001) Characterization of the
formyltransferase from Methylobacterium extorquens AM1. Eur.
J. Biochem. 269, 4769–4775.

11. Johnson, P.A. & Quayle, J.R. (1964) Microbial growth on C
1
compounds. 6. Oxidation of methanol, formaldehyde and formate
by methanol-grown Pseudomonas AM1. Biochem. J. 93, 281–290.
12. Vorholt, J.A. & Thauer, R.K. (2001) Molybdenum and tungsten
enzymes in C
1
metabolism. In Molybdenum and Tungsten. Their
Roles in Biological Processes (Sigel,A.&Sigel,H.,eds),pp.571–
619. M. Dekker, Inc., New York, USA.
13. Karzanov, V.V., Bogatsky, Y.A., Tishkov, V.I. & Egorov, A.M.
(1989) Evidence for the presence of a new NAD
+
-dependent
formate dehydrogenase in Pseudomonas sp. 101 cells grown on a
molybdenum-containing medium. FEMS Microbiol. Lett. 60,
197–200.
14. Karzanov, V.V., Correa, C.M., Bogatsky, Y.G. & Netrusov, A.I.
(1991) Alternative NAD
+
-dependent formate dehydrogenases in
the facultative methylotroph Mycobacterium vaccae 10. FEMS
Microbiol. Lett. 81, 95–100.
15. Lamzin, V.S., Dauter, Z., Popov, V.O., Harutyunyan, E.H. &
Wilson, K.S. (1994) High resolution structures of holo and apo
formate dehydrogenase. J. Mol. Biol. 236, 759–785.
16. Tishkov, V.I., Matorin, A.D., Rojkova, A.M., Fedorchuk, V.V.,
Savitsky, P.A., Dementieva, L.A., Lamzin, V.S., Mezentzev, A.V.
& Popov, V.O. (1996) Site-directed mutagenesis of the formate
dehydrogenase active centre: role of the His

332
-Gln
313
pair in
enzyme catalysis. FEBS Lett. 390, 104–108.
17. Asano, Y., Sekigawa, T., Inukai, H. & Nakazawa, A. (1988)
Purification and properties of formate dehydrogenase from Mor-
axella sp. strain C-1. J. Bacteriol. 170, 3189–3193.
18. Yoch, D.C., Chen, Y P. & Hardin, M.G. (1990) Formate dehy-
drogenase from the methane oxidizer Methylosinus trichosporium
OB3b. J. Bacteriol. 172, 4456–4463.
19. Jollie, D.R. & Lipscomb, J.D. (1991) Formate dehydrogenase
from Methylosinus trichosporium OB3b. Purification and spec-
troscopic characterization of the cofactors. J. Biol. Chem. 266,
21853–21863.
20. Girio, F.M., Amaral-Collaco, M.T. & Attwood, M. (1994) The
effect of molybdate and tungstate ions on the metabolic rates and
enzyme activities in methanol-grown Methylobacterium sp. RXM.
Appl. Microbiol. Biotechnol. 40, 898–903.
21. Duarte, R.O., Reis, A.R., Girio, F., Moura, I., Moura, J.J.G. &
Collaco, T.A. (1997) The formate dehydrogenase isolated from the
aerobe Methylobacterium sp. RXM is a molybdenum-containing
protein. Biochem. Biophys. Res. Com. 230, 30–34.
22. Kletzin, A. & Adams, M.W.W. (1996) Tungsten in biological
systems. FEMS Microbiol. Rev. 18, 5–63.
23. Ljungdahl, L.G. & Andreesen, J.R. (1975) Tungsten, a component
of active formate dehydrogenase from Clostridium thermo-
aceticum. FEBS Lett. 54, 279–282.
24. Yamamoto, I., Saiki, T., Liu, S M. & Ljungdahl, L.G. (1983)
Purification and properties of NADP-dependent formate dehy-

drogenase from Clostridium thermoaceticum, a tungsten-selenium-
iron protein. J. Biol. Chem. 258, 1826–1832.
25. Fulton, G.L., Nunn, D.N. & Lidstrom, M.E. (1984) Molecular
cloningofamalylcoenzymeAlyasegenefromPseudomonas sp.
strain AM1, a facultative methylotroph. J. Bacteriol. 160, 718–
723.
26. Bradford, M.M. (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal. Biochem. 72, 248–254.
27. Fish, W.W. (1988) Rapid colorimetric micromethod for the
quantitation of complexed iron in biological samples. Methods
Enzymol. 158, 357–364.
28. Hedderich, R., Berkessel, A. & Thauer, R.K. (1990) Purification
and properties of heterodisulfide reductase from Methanobacter-
ium thermoautotrophicum (strain Marburg). Eur. J. Biochem. 193,
255–261.
29. Heim, S., Ku
¨
nkel, A., Thauer, R.K. & Hedderich, R. (1998)
Thiol:fumarate reductase (Tfr) from Methanobacterium thermo-
autotrophicum. Identification of the catalytic sites for
332 M. Laukel et al. (Eur. J. Biochem. 270) Ó FEBS 2003
fumarate reduction and thiol oxidation. Eur. J. Biochem. 253,292–
299.
30. Oh, J I. & Bowien, B. (1998) Structural analysis of the fds operon
encoding the NAD
+
-linked formate dehydrogenase of Ralstonia
eutropha. J. Biol. Chem. 273, 26349–26360.
31. Bokranz, M., Gutmann, M., Kortner, C., Kojro, E., Fahrenholz,

F., Lauterbach, F. & Kro
¨
ger, A. (1991) Cloning and nucleotide
sequence of the structural genes encoding the formate dehydro-
genase of Wolinella succinogenes. Arch. Microbiol. 156, 119–128.
32. Graentzdoerffer, A. (2000) Formiat-Stoffwechsel in Eubacterium
acidaminophilum: Molekulare und biochemische Charakterisie-
rung der Wolfram- und Selen-haltigen Formiat-Dehydrogenasen
sowie einer Eisen-Hydrogenase. Dissertation, Martin-Luther-
Universita
¨
t, Halle-Wittenberg, Germany.
33. Zinoni, F., Birkman, A., Stadtman, T.C. & Bo
¨
ck, A. (1986)
Nucleotide sequence and expression of the selenocysteine-con-
taining popypeptide of formate dehydrogenase (formate-hydro-
gen-lyase-linked) from Escherichia coli. Proc.Natl.Acad.Sci.USA
83, 4650–4654.
34. Boyington, J.C., Gladyshev, V.N., Khangulov, S.V., Stadtman,
T.C. & Sun, P.D. (1997) Crystal structure of formate dehydro-
genase H: catalysis involving Mo, molybdopterin, selenocysteine,
andanFe
4
S
4
cluster. Science 275, 1305–1308.
35. Appel, J. & Schulz, R. (1996) Sequence analysis of an operon of a
NAD(P)-reducing nickel hydrogenase from the cyanobacterium
Synechocystis sp. PCC 6803 gives additional evidence for direct

coupling of the enzyme to NAD(P)H-dehydrogenase (complex I).
Biochim. Biophys. Acta 1298, 141–147.
36. Schmitz, O., Boison, G., Hilscher, R., Hundeshagen, B., Zimmer,
W., Lottspeich, F. & Bothe, H. (1995) Molecular biological ana-
lysis of a bidirectional hydrogenase from cyanobacteria. Eur. J.
Biochem. 233, 266–276.
37. Deckert, G., Warren, P.V., Gaasterland, T., Young, W.G., Lenox,
A.L.,Graham,D.E.,Overbeek,R.,Snead,M.A.,Keller,M.,
Aujay, M., Huber, R., Feldman, R.A., Short, J.M., Olsen, G.J. &
Swanson, R.V. (1998) The complete genome of the
hyperthermophilic bacterium Aquifex aeolicus. Nature 392, 353–
358.
38. Boison, G., Schmitz, O., Schmitz, B. & Bothe, H. (1998) Unusual
gene arrangement of the bidirectional hydrogenase and functional
analysis of its diaphorase subunit HoxU in respiration of the
unicellular cyanobacterium Anacystis nidulans. Curr. Microbiol.
36, 253–258.
39. Blanchard, J.S. & Cleland, W.W. (1980) Kinetic and chemical
mechanisms of yeast formate dehydrogenase. Biochemistry 19,
3543–3550.
40. Friedebold,J.&Bowien,B.(1993)Physiologicalandbiochemical
characterization of the soluble formate dehydrogenase, a
molybdoenzyme from Alcaligenes eutrophus. J. Bacteriol. 175,
4719–4728.
41. Scherer, P.A. & Thauer, R.K. (1978) Purification and properties of
reduced ferredoxin: CO
2
oxidoreductase from Clostridium pas-
teurianum, a molybdenum iron-sulfur-protein. Eur. J. Biochem. 85,
125–135.

42. Hille, R., Re
´
tey, J., Bartlewski-Hof, U., Reichenbecher, W. &
Schink, B. (1999) Mechanistic aspects of molybdenum-containing
enzymes. FEMS Microbiol. Rev. 22, 489–501.
43. Ferry, J.G. (1990) Formate dehydrogenase. FEMS Microbiol.
Rev. 87, 377–382.
44. Burgdorf, T., Bo
¨
mmer, D. & Bowien, B. (2001) Involvement of
an unusual mol operon in molybdopterin cofactor biosynthesis in
Ralstonia eutropha. J. Mol. Microbiol. Biotechnol. 3, 619–629.
45. Makdessi, K., Andreesen, J.R. & Pich, A. (2001) Tungstate uptake
by a highly specific ABC transporter in Eubacterium acid-
aminophilum. J. Biol. Chem. 276, 24557–24564.
46. Zindel, U., Freudenberg, W., Reith, M., Andreesen, J.R., Schnell,
J. & Widdel, F. (1988) Eubacterium acidaminophilum new species.
A versatile amino acid-degrading anaerobe producing or utilizing
hydrogen or formate. Description and enzymatic studies. Arch.
Microbiol. 150, 254–266.
Ó FEBS 2003 Tungsten-containing FDH from M. extorquens AM1 (Eur. J. Biochem. 270) 333