Microbiology (2004), 150, 707–713
DOI 10.1099/mic.0.26707-0
Formaldehyde dehydrogenase preparations from
Methylococcus capsulatus (Bath) comprise
methanol dehydrogenase and methylene
tetrahydromethanopterin dehydrogenase
Ekundayo K. Adeosun,13 Thomas J. Smith,14 Anne-Mette Hoberg,11
Giles Velarde,2 Robert Ford2 and Howard Dalton1
1
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK
Correspondence
Howard Dalton
2
Department of Biomolecular Sciences, PO Box 88, UMIST, Manchester M60 1QD, UK
H.Dalton@warwick.ac.uk
Received 13 August 2003
Revised
5 December 2003
Accepted 9 December 2003
In methylotrophic bacteria, formaldehyde is an important but potentially toxic metabolic
intermediate that can be assimilated into biomass or oxidized to yield energy. Previously
reported was the purification of an NAD(P)+-dependent formaldehyde dehydrogenase (FDH)
from the obligate methane-oxidizing methylotroph Methylococcus capsulatus (Bath), presumably
important in formaldehyde oxidation, which required a heat-stable factor (known as the modifin)
for FDH activity. Here, the major protein component of this FDH preparation was shown by
biophysical techniques to comprise subunits of 64 and 8 kDa in an a2b2 arrangement. N-terminal
sequencing of the subunits of FDH, together with enzymological characterization, showed
that the a2b2 tetramer was a quinoprotein methanol dehydrogenase of the type found in other
methylotrophs. The FDH preparations were shown to contain a highly active NAD(P)+-dependent
methylene tetrahydromethanopterin dehydrogenase that was the probable source of the
NAD(P)+-dependent formaldehyde oxidation activity. These results support previous findings
that methylotrophs possess multiple pathways for formaldehyde dissimilation.
INTRODUCTION
Methanotrophic bacteria such as the c-proteobacterium
Methylococcus capsulatus (Bath) grow using methane as
their sole source of carbon and energy (Hanson & Hanson,
1996). Methane is first oxidized to methanol by action of
methane monooxygenase and then to formaldehyde by
methanol dehydrogenase (MDH) (Hanson & Hanson, 1996;
Anthony, 1982). Formaldehyde lies at a metabolic branch
point, where metabolic carbon can either be assimilated
into biomass or oxidized to carbon dioxide to produce
energy (Hanson & Hanson, 1996; Murrell et al., 2000).
Previous studies in our laboratory suggested that Mc.
capsulatus (Bath) could oxidize formaldehyde to formate
3Present address: GlaxoSmithkline Plc, New Frontiers Science Park,
Harlow, Essex CM19 5AW, UK.
4Present address: Biomedical Research Centre, Sheffield Hallam
University, Howard Street, Sheffield S1 1WB, UK.
1Present address: Dept Clinical Pharmacology Q7642, Rigshospitalet,
Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark.
Abbreviations: ES-MS, electrospray-mass spectrometry; FDH, formaldehyde dehydrogenase; H4MPT, tetrahydromethanopterin; HTSE, heattreated soluble extract; MDH, methanol dehydrogenase; methylene
H4MPT-DH, methylene tetrahydromethanopterin dehydrogenase.
0002-6707 G 2004 SGM
using a soluble formaldehyde dehydrogenase (FDH) that
existed as a dimer of 57 kDa subunits, possessed NAD(P)+dependent FDH activity and required a factor (apparently
a low-molecular-mass protein) from heat-treated soluble
extract (HTSE) (Stirling & Dalton, 1978). We conducted a
subsequent investigation into the FDH of Mc. capsulatus
(Bath) which suggested that the protein responsible for
FDH activity was actually a homotetramer of 63 kDa
subunits and that the factor in the HTSE required for
activity with formaldehyde was a heat-stable 8?6 kDa
protein, termed the modifin. The modifin preparation
converted FDH from a general aldehyde dehydrogenase
to a specific FDH and was thus a potential regulator of
formaldehyde metabolism (Tate & Dalton, 1999). Other
reports have also indicated the presence of a membraneassociated FDH (Zahn et al., 2001) and at least some of
the enzyme activities that are necessary for oxidation of
formaldehyde as a conjugate to tetrahydromethanopterin
(H4MPT) (Vorholt et al., 1999).
Here we report analysis of modifin-dependent FDH preparations similar to those which we prepared previously
that show that the principal enzyme activities within these
preparations are a quinoprotein MDH and a methylene
tetrahydromethanopterin
dehydrogenase
(methylene
H4MPT-DH), which is one of the enzymes of the
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Printed in Great Britain
707
E. K. Adeosun and others
H4MPT-dependent formaldehyde
(Vorholt et al., 1998).
oxidation
pathway
METHODS
Coenzymes. NAD+ was purchased from Sigma and H4MPT was
provided by Rudolf K. Thauer. Anoxic solutions of H4MPT were
prepared in 50 mM Tris/HCl (pH 7?0) containing 2 mM DTT.
Methylene H4MPT was generated by spontaneous reaction of
H4MPT with formaldehyde (Vorholt et al., 1998).
Growth of bacteria. Mc. capsulatus (Bath) was cultivated at 45 uC
using methane (15 %, v/v, in air) as the growth substrate, in nitrate
minimal salts medium (Dalton & Whittenbury, 1976) containing
CuSO4.5H2O (0?5 or 1?5 mg l21, as stated for each experiment) as
the sole source of added copper. Cultures were grown in a 100 litre
batch fermentation vessel or continuously, as described previously
(Tate & Dalton, 1999) except that the dilution rate was 0?05 h21.
Cells were harvested according to the published method (Tate &
Dalton, 1999) and stored at 280 uC.
Enzyme assays. All enzyme assays were performed at 45 uC.
Modifin/HTSE-dependent FDH assays were performed by spectrophotometric quantification at 340 nm of the production of NADH,
as described by Tate & Dalton (1999). NAD+-dependent methylene
H4MPT-DH activity was also monitored spectrophotometrically
(Vorholt et al., 1998). Dye-linked oxidation of methanol and
formaldehyde was similarly measured at 600 nm by a modification
of the method of Anthony & Zatman (1967), as follows. The
enzyme sample was incubated in 50 mM Tris/HCl (pH 9?0) containing substrate (10 mM), NH4Cl (15 mM), KCN (5 mM) and
dichloroindophenol (DCIP; 0?1 mM); the reaction was initiated
by adding the mediator phenazine ethosulfate to 1?0 mM and the
activity estimated from the decrease in absorbance at 600 nm due
to reduction of DCIP, presuming an absorption coefficient of
1?916104 M21 cm21.
Preparation of soluble extracts. DNase A (to 20 mg ml21) was
added to the defrosted cell paste and the cells were broken by
passing through a high-pressure cell disrupter (Constant Systems,
Warwick, UK; 172 MPa, 4 uC). The broken cell suspension was
centrifuged (150 000 g, 1?5 h, 4 uC) and the supernatant (the soluble
extract) was removed. HTSE was prepared as described by Tate &
Dalton (1999).
Partial purification of FDH (preparation FDH-1)
Step 1. Ammonium sulfate was added to native soluble extract to
40 % saturation and the proteins that precipitated during a 30 min
incubation on ice were removed by centrifugation (48 500 g,
15 min, 4 uC). The concentration of ammonium sulfate in the supernatant was adjusted to 60 % saturation and, after a further 30 min
on ice, the precipitate, containing the FDH activity, was collected by
centrifugation as above and resuspended in a minimal volume of
buffer A (20 mM potassium phosphate buffer, pH 7?2, containing
1 mM benzamidine). The sample (2–3 ml) was then desalted into 5
column volumes of buffer A using a Pharmacia PD 10 desalt
column equilibrated with the same buffer.
Step 2. The sample was loaded onto a Pharmacia Mono Q anion
exchange column (1?0 cm610 cm) equilibrated with buffer A and
then proteins were eluted with a linear gradient of 0–1 M NaCl in
buffer A. Fractions containing NAD+-dependent FDH activity,
which eluted in the void volume, were pooled and concentrated
using an Amicon PM 30 ultrafiltration membrane.
Step 3. The concentrated protein was loaded onto a Pharmacia
708
Hi-Trap Blue affinity chromatography column (0?5 cm61 cm)
equilibrated with buffer A. Elution was effected using a linear
gradient of 0–1 M NaCl in buffer A and fractions containing
NAD+-dependent FDH activity, which again eluted in the void
volume, were pooled and concentrated as above.
Step 4. The protein was then subjected to size-exclusion chromatography using a Pharmacia Superdex 200 column (1?6 cm670 cm)
with a flow rate of 0?5 ml min21 of buffer A. The active fractions,
containing the partially purified FDH, were pooled and concentrated
as above. This gave preparation FDH-1, which was drop-frozen in
liquid nitrogen and stored at 280 uC.
General protein characterization. Protein concentration was
determined by using the Bradford assay reagent (Bio-Rad) according
to the manufacturer’s instructions. Bovine serum albumin was used
as the standard. SDS- and native-PAGE were performed using the
discontinuous buffer system of Laemmli (1970). Molecular masses
of polypeptides analysed by SDS-PAGE were estimated by comparison
with Dalton Mk VIIL standards (Sigma). Electroelution of protein
samples from bands excised from polyacrylamide gels was performed
in the gel-running buffer using a model 422 Electroeluter (Bio-Rad).
Samples for N-terminal sequencing were subjected to SDS-PAGE
and then electroblotted onto a PVDF membrane (PharmaciaAmersham) and stained with Coomassie blue. Protein bands of
interest were excised and the peptide sequence was determined using
an Applied Biosystems 476A protein sequencer. Electrospray-mass
spectrometry (ES-MS) was performed using a Quattro II QhQ
tandem mass spectrometer (Micromass, Altrincham, UK) as detailed
by Millar et al. (1998).
Equilibrium sedimentation. Sedimentation equilibrium analysis
of preparation FDH-1 was performed at 4 uC in a Beckman Optima
XL-A analytical ultracentrifuge. Samples were diluted to 0?34, 0?17
and 0?04 mg ml21 using 20 mM potassium phosphate buffer
(pH 7?2). Each dilution (70 ml) was injected into a separate cell in
the centrifuge rotor and the samples were centrifuged to equilibrium
at 6000 and 9000 r.p.m. Scanning absorbance optics were used at
wavelengths of 280 nm (for samples at 0?34 and 0?17 mg ml21) and
223 nm (for samples at 0?04 mg ml21). Sedimentation equilibrium
was considered to have been achieved when a negligible difference
between solute distributions was observed after a time interval of
3 h. After equilibrium at both rotor speeds, the rotor was accelerated
to 45 000 r.p.m. to obtain a reading of non-redistributing absorbance, which was used as baseline reading in the analysis. The partial
specific volume of the proteins and the density of the solvent were
estimated from composition information using the program
SEDNTERP (Laue et al., 1992). Solute distributions were analysed
using non-linear least-squares fitting of exponential equations using
the ORIGIN software package (MicroCal Software, Northampton,
MA, USA). The data obtained were fitted to models that included
association terms, in order to determine the stoichiometry of any
intermolecular interactions.
Electron microscopy and single-particle analysis. Samples
were added to freshly glow-discharged (rendered hydrophilic)
copper/carbon mesh grids for 30 s, the sides of which were then
blotted with Whatman grade 50 filter paper, before negative staining
in 4 % uranyl acetate (Rosenberg et al., 1997). Micrographs of the
grids were taken with a Philips CM10 transmission electron microscope operating at 100 kV and scanned using a leaf microdensitometer (University of Leeds).
Particles from electron micrographs were selected using the SPIDER
software package on an Indigo workstation (Silicon Graphics) and the
densities normalized. Reference-free alignment was carried out and
the results were statistically analysed by sorting particles into groups
for averaging using hierarchical clustering (Holzenburg et al., 1994).
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Formaldehyde metabolism in Methylococcus capsulatus
Table 1. Fractionation of cell-free extract from Mc. capsulatus (Bath) resulting in preparation FDH-1
Purification step
Cell-free extract
Ammonium sulfate precipitation
Ion exchange (Mono Q)
Affinity chromatography (Hi-Trap Blue)
(Gel filtration) Superdex 200; preparation FDH-1
Total protein
(mg)
Total activity
(nmol min”1)
Specific activity
[nmol min”1 (mg protein)”1]
Yield
(%)
Purification
factor
3450
554
174
86
42
17 300
1 110
9 740
8 940
7 010
5
20
56
104
167
100
64
56
52
41
1?0
4?0
11?2
20?8
33?4
Fourier ring correlation between two subaverages was used to assess
the resolution. The point at which the Fourier ring correlation values
dropped to 0?5, indicating higher resolution shells did not correlate
significantly, was determined by fitting the data with a four-parameter
Boltzman function (using ORIGIN). A threshold for each class that gave
the best averages in terms of resolution was thus determined.
The major protein components of preparation
FDH-1 are identical to the components of FDH
isolated previously
RESULTS
Initial preparation of the components of FDH:
preparation FDH-1 and the modifin
In order to obtain sufficient of the modifier- and NAD+dependent FDH from Mc. capsulatus (Bath) for detailed
characterization, we repeated the cell growth and purification procedures described by Tate & Dalton (1999). Despite
repeated attempts, these yielded material with a mean
specific activity of only 55 nmol min21 (mg protein)21,
which was less than a quarter of the 234 nmol min21 (mg
protein)21 obtained previously (Tate & Dalton, 1999)
[NB: the FDH and modifin activities of Tate & Dalton
(1999) that are presented here have been reduced by 1000fold to compensate for a typographical error on the part
of the authors]. However, by modifying the purification
protocol to that described in Methods and by increasing
the concentration of copper in the growth medium (from
0?1 to 1?5 mg l21 of CuSO4.5H2O), higher NAD+dependent FDH activities were obtained (Table 1). The
resulting preparations, which will be referred to as preparation FDH-1, consistently contained the 64 kDa polypeptide at a purity of about 80 %. The major contaminant
was an 8 kDa protein (Fig. 1) that was previously attributed to modifin co-purifying with the 64 kDa moiety. The
specific activity of preparation FDH-1 [167 nmol min21
(mg protein)21] was 71 % of that observed for the FDH of
Tate & Dalton (1999) and considerably lower than the
4278 nmol min21 (mg protein)21 obtained in an earlier
study (Stirling & Dalton, 1978). In a separate experiment
the modifin was prepared from HTSE using the procedure
described by Tate & Dalton (1999) (data not shown).
The apparent specific activity of the purified modifin
[26 nmol min21 (mg protein)21] was considerably less than
that found previously (Tate & Dalton, 1999).
The low specific activity of preparation FDH-1 and the
modifin, compared to the previous studies, suggested that
inactivation or inhibition of the enzyme was occurring
http://mic.sgmjournals.org
during the purification procedures. Alternatively, the preparations may have lacked a component, other than the
64 and 8 kDa polypeptides, that was necessary for NAD+linked FDH activity.
The major polypeptides of preparation FDH-1 were subjected to N-terminal sequencing and accurate mass analysis by means of ES-MS in order to investigate whether
degradation of the proteins has occurred that might account
for the low FDH activity. The N-terminal sequences of the
64 and 8 kDa species from preparation FDH-1 (Fig. 2a, b)
were very similar to those found previously (Tate & Dalton,
1999). The positions of the N termini in the two proteins
were unchanged, which suggested that N-terminal degradation was not the cause of inactivation, although in the case
of the 8 kDa moiety there was a discrepancy in the identification of the N-terminal residue. The molecular masses
of the two major polypeptides in preparation FDH-1 were
63 615 and 8212?5 Da, which were identical to the masses
Fig. 1. SDS-PAGE of preparation FDH-1 (lane 2) showing the
64 and 8 kDa polypeptides and minor contaminants. Molecular
masses of standards (lane 1) are indicated in kDa.
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709
E. K. Adeosun and others
The 64 and 8 kDa polypeptides form a
complex with the same a2b2 stoichiometry
as known MDHs and similar molecular
topology
Fig. 2. N-terminal sequence comparisons. Comparisons of (a)
the 64 kDa component of preparation FDH-1 with sequence of
the similar polypeptide obtained by Tate & Dalton (1999);
(b) the 8 kDa component of FDH-1 with the modifin of Tate
& Dalton (1999); (c) the 64 kDa component of preparation
FDH-1 with the large subunit of MDH from Methylobacter
albus (Chistoserdova et al., 1994); (d) the 8 kDa component of
preparation FDH-1 with the b-subunit of MDH from Hyphomicrobium methylovorum (Tanaka et al., 1997). Numbers refer
to the amino acids of the heterologous MDHs, which were the
highest scoring matches obtained from BLAST searches
(Altschul et al., 1997).
of the major protein components of FDH and the modifin,
respectively, determined previously (Tate & Dalton, 1999).
Consequently, the minor discrepancies in the N-terminal
sequences could be put down to sequencing inaccuracies
and degradation of the 64 and 8 kDa polypeptides could
be discounted as a cause of the low NAD+-dependent
FDH activity in preparation FDH-1. The mass spectrum
of preparation FDH-1 also revealed a number of minor
contaminants, including a polypeptide of 34 525 Da (data
not shown).
The major components of preparation FDH-1
were similar to the subunits of known
quinoprotein MDHs
The N-terminal sequences of the 64 and 8 kDa components
of preparation FDH-1 (Fig. 2a, b) were longer than those
obtained previously (Tate & Dalton, 1999). By performing
sequence database searches using these extended sequences
it was possible to identify significant similarities between
the N termini of the 64 and 8 kDa polypeptides and
equivalent regions of the a and b subunits, respectively,
of quinoprotein MDHs from methylotrophic bacteria
(Fig. 2c, d). The subunits of such MDHs have masses of
66 and 8?5 kDa (Anthony, 1992), further suggesting that
preparation FDH-1 contained such an enzyme.
710
Previously the native molecular mass of the major protein
complex in FDH preparations was estimated at 250 kDa,
based on gel filtration chromatography (Tate & Dalton,
1999). In order to obtain an accurate value for the native
molecular mass that could be used to determine the
stoichiometry of the complex, the molecular mass(es) of
the protein complex(es) in preparation FDH-1 were determined in solution under native conditions by sedimentation equilibrium analysis. Analysis of residual plots after
fitting of the experimental data from FDH-1 samples to
specific models of association indicated that the data were
best accounted for by a single species of molecular mass
140 600±1200 Da. Models which included self-association
terms and/or non-interacting species did not describe the
data well (data not shown). Assuming that this species
is composed only of the 8 and 64 kDa polypeptides, the
complex must have an a2b2 stoichiometry, with a molecular
mass of 143 655 Da as calculated from the masses of the
individual polypeptides determined by ES-MS. The slight
discrepancy between sedimentation equilibrium results
and the mass derived from the ES-MS data was attributed
to inaccuracies in the estimation of solute partial specific
volume and solvent density.
The topology of the major macromolecular constituents
in the FDH preparations was investigated in electron
micrographs of negatively stained particles of FDH by
means of single-particle analysis. Two distinct species were
identified by visual inspection of the micrographs. The
first (species A) was small, thin and tubular; the second
(species B) was broader and appeared to be a sandwich of
the first. Particles of each species were separately selected
by size into subpopulations before performing rotational
and translational alignment. This process used an interactive procedure of cross-correlation and auto-correlation
of images which were thereby (Frank, 1990) averaged to
produce a refined image. Hierarchical cluster analysis of
each aligned particle dataset (corresponding to species A
and B, respectively) resulted in final averages from both
datasets with two very different structures. The resolution
of the species A image extends about 26 Å, and that of
species B to 35 Å. Enlarged versions of the best averages
of each species were compared with the 1?9 Å resolution
crystal structure of the a2b2 tetramer of MDH from Methylophilus W3A1 (Xia et al., 1999) (Fig. 3). Species A was
similar in size and shape to the MDH crystal structure,
consistent with the proposal that the 64 and 8 kDa FDH
components, like the subunits of MDH from Methylophilus
W3A1, existed in an a2b2 configuration. Species B resembled a sandwich of two MDH heterotetramers and suggested
that, under the conditions used for sample preparation,
further aggregation of the a2b2 complex in preparation
FDH-1 was possible.
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Microbiology 150
Formaldehyde metabolism in Methylococcus capsulatus
Fig. 3. Single-particle analysis of the a2b2
complex from preparation FDH-1, showing
average images of the tubular species A
and the broader species B. A space-filling
molecular model derived from the X-ray
crystal structure MDH from Methylophilus
W3A1 (Xia et al., 1999) is shown on the
same scale (top left) for reference.
The a2b2 heterotetramer has dye-linked
dehydrogenase activity
Previously described pyrolloquinoline quinone (PQQ)dependent dehydrogenases, including MDHs, do not use
NAD(P)+ as the electron acceptor (Anthony, 1992) and
so it was a surprise to find an apparently PQQ-dependent
enzyme complex as the major constituent of a preparation
that exhibited NAD+-linked dehydrogenase activity. The
natural electron acceptor of quinoprotein MDHs is cytochrome cL (Anthony, 1992), but such enzymes can also
utilize the artificial electron acceptor phenazine ethosulfate,
which can be coupled to the reduction and concomitant
decolourization of the dye 2,6-dichloroindophenol (DCIP)
to produce a dye-linked spectrophotometric assay (Anthony
& Zatman, 1967). The dehydrogenase activity of the a2b2
heterotetramer was therefore assayed using the phenazine
ethosulfate/DCIP electron acceptor system in order to
determine whether it could be activated in the same manner
as previously described quinoprotein MDHs. To exclude
the possibility that any dye-linked dehydrogenase activity
http://mic.sgmjournals.org
might be due to minor contaminating protein(s) and
not to the 144 kDa a2b2 heterotetramer, the tetramer was
further purified by preparative native-PAGE followed by
electroelution under non-denaturing conditions. The
electroeluted a2b2 heterotetramer showed a high level of
dye-linked dehydrogenase activity with methanol (kcat=
17?8 s21; Km=0?48 mM) and formaldehyde (kcat=16?2 s21;
Km=0?77 mM). The kcat values were similar to those
obtained with the quinoprotein MDH from Methylophilus
methylotrophus. In comparison to the MDH from Mp.
methylotrophus (Ghosh & Quayle, 1981), the Km values
exhibited by the FDH-associated a2b2 heterotetramer were
elevated (by 24-fold in the case of methanol), probably
because of the presence of KCN in the assays described here.
KCN, which competitively inhibits MDHs with respect to
the alcohol substrate, was included because it inhibits the
endogenous reduction of phenazine ethosulfate that otherwise occurs even in the absence of substrate (Anthony &
Zatman, 1964; Duine & Frank, 1980; Harris & Davidson,
1993).
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711
E. K. Adeosun and others
The FDH-1 preparations contain a methylene
H4MPT-DH that may be involved in
NAD+-dependent FDH activity
The observation that the a2b2 heterotetramer alone showed
no NAD+-dependent FDH activity suggested that the
enzyme(s) responsible for the NAD+-linked FDH activity
must be among the minor contaminants in preparation
FDH-1. The previous observation of enzyme activities
associated with the H4MPT-dependent formaldehyde dissimilation pathway in cell-free extracts of Mc. capsulatus
(Bath) (Vorholt et al., 1999) suggested one possible
explanation. In M. extorquens, in which this pathway is
well characterized, the formation of methylene H4MPT is
catalysed by a specific formaldehyde-activating enzyme
(Vorholt et al., 2000); however, formaldehyde and H4MPT
also react spontaneously to form methylene H4MPT.
Methylene H4MPT is the substrate for the NAD(P)+dependent enzyme methylene H4MPT-DH (Chistoserdova
et al., 1998) and so, in the presence of H4MPT, methylene
H4MPT-DH would oxidize formaldehyde in an NAD+dependent manner. Moreover, since H4MPT is heat-stable
(Romesser & Wolfe, 1982) it, rather than the 8 kDa polypeptide, could be the active component of HTSE and the
modifin that stimulates oxidation of formaldehyde. Consistent with this hypothesis, it was found that the NAD+linked FDH activity of preparation FDH-1 increased from
167 nmol min21 (mg protein)21 to 23 000 nmol min21
(mg protein)21 when the HTSE in the assay was replaced
by H4MPT (to 35 mM). Thus the FDH preparations
contained an NAD+-dependent methylene H4MPT-DH
and it was likely that H4MPT was at least one of the
components of the modifin preparation that stimulated
the NAD+-linked FDH activity.
DISCUSSION
The data presented here show that the major component
of the FDH preparation of Tate & Dalton (1999), which
was previously described as a homotetramer of 64 kDa
subunits, is in fact a quinoprotein MDH with an a2b2
arrangement of 64 and 8 kDa subunits, which has similar
properties to quinoprotein MDHs from other methylotrophic bacteria (Anthony, 1992; Chistoserdova et al., 1994;
Tanaka et al., 1997). The MDH from Mc. capsulatus (Bath)
has similar catalytic properties to the dye-linked methanol
(or primary alcohol) dehydrogenase of the closely related
methanotroph Mc. capsulatus (Texas), which is a quinoprotein of comparable native molecular mass (Patel et al.,
1972; Wadzinski & Ribbons, 1975). The conclusion that
the NAD+-linked FDH activity must be due to a minor
protein in preparation FDH-1 also explained our difficulty
in purifying active ‘FDH’, because the NAD+-dependent
dehydrogenase activity that was being assayed did not
reside in the polypeptide that was previously believed to
contain it.
712
Our observation that the FDH preparation FDH-1, described here, contained a methylene H4MPT-DH showed
that such an activity might be responsible for the NAD+dependent FDH activity of preparation FDH-1 and offered
a possible explanation for the FDH-stimulating activity of
the HTSE and modifin preparations, by providing the heatstable coenzyme H4MPT. These results confirm the previous observation of the activities of enzymes of the H4MPT
pathway in Mc. capsulatus (Bath) (Vorholt et al., 1999) and
demonstrate the importance of H4MPT as a C1-carrier
during oxidation of formaldehyde by this methanotrophic
bacterium.
The NAD+-dependent FDH activity observed by Tate &
Dalton (1999), within a preparation that also contained
principally the quinoprotein MDH, must likewise have been
due to a minor enzyme in the preparation. It is possible
that this was the same methylene H4MPT-DH observed
during the present study; however, the complex kinetics,
the effect of the modifin of inhibiting oxidation of aldehydes larger than formaldehyde and the effect of NAD+ in
changing the spectral properties of the preparation cannot
be explained by the model presented here. If H4MPT is
indeed the active component of HTSE and the modifin
preparations, the FDH activities in the presence of modifin
and HTSE reported here and by Tate & Dalton (1999) are
overestimated by about 4?4-fold, because of the increase in
A340 due to oxidation of methylene H4MPT to methenyl
H4MPT (Vorholt et al., 1998) that was not taken into
account during calculation of these activities.
The available evidence suggests at least two routes via
which formaldehyde can be oxidized in Mc. capsulatus
(Bath): (1) via the H4MPT-dependent pathway; (2) by
oxidation to formate by the membrane-associated FDH
described by Zahn et al. (2001). It is also intriguing that
the soluble FDH purified from Mc. capsulatus (Bath) by
Stirling & Dalton (1978) was clearly distinct from the
enzymes identified in preparation FDH-1 because it used
NAD+ as its electron acceptor but was dependent on a
factor from HTSE which, since it was sensitive to trypsin,
is unlikely to have been H4MPT. Thus, although the preparation that Tate & Dalton (1999) classified as a soluble
FDH is clearly principally composed of an MDH, the
existence of a soluble NAD(P)+-dependent FDH in this
organism cannot currently be excluded.
ACKNOWLEDGEMENTS
We thank Arthur Moir (University of Sheffield) for performing
N-terminal sequencing, Neil Errington (National Centre for Macromolecular Hydrodynamics, University of Nottingham) for performing
equilibrium sedimentation analysis, Rudolf K. Thauer (Max-PlanckInstitute for Terrestrial Microbiology, Marburg, Germany) for the gift
of a sample of tetrahydromethanopterin, Susan Slade for technical
assistance during protein purification and Keith Jennings for advice
about mass spectrometry. This work was supported by the BBSRC
through a studentship to E. K. A.
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