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INTRODUCTION |
Constituting about 25% of the
earth's biomass (18), lignin is a polymer composed of
hydroxylated and methoxylated phenylpropanoid units linked by C-C and
C-O-C bonds that undergoes depolymerization to generate monomeric
methoxylated aromatics (or phenylmethylethers) like vanillate and
syringate. The anaerobic metabolism of phenylmethylethers (11) has been known since 1979 (13). Although
acetogenic bacteria can be selectively isolated by growth on
methoxylated aromatics like syringate (2), they do not
metabolize the aromatic ring itself but use the O-methyl
group as a one-carbon growth substrate (equation 1). Oxidation of one
methyl group to CO2 provides the six electrons required for
conversion of three methyl groups to acetate. Moorella
thermoacetica can metabolize at least 20 different methoxylated aromatics (5). A 22-kDa protein and aromatic
O-demethylase activity are induced when M. thermoacetica is exposed to such compounds (6, 37).
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The aromatic O-demethylases of Acetobacterium
woodii (3), Acetobacterium dehalogenans
(27), and M. thermoacetica (8) require H4folate as the methyl acceptor, producing
CH3-H4folate (8, 15, 27).
Oxidation of one equivalent of CH3-H4folate to
CO2 and H4folate generates six electrons
(equation 2) which drive the synthesis of three equivalents of
acetyl-coenzyme A (acetyl-CoA) by the Wood-Ljungdahl pathway of
acetyl-CoA synthesis (Fig. 1 and reaction
3). This paper describes a three-component aromatic
O-demethylase from M. thermoacetica.
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(2)
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(3)
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FIG. 1.
Scheme describing the conversion of the aromatic
O-methyl group to the methyl group of acetic acid. MtvA, -B,
and -C are the components of the O-demethylase system.
Abbreviations: CFeSP, the corrinoid iron-sulfur protein; MeTr, the
CFeSP methyltransferase; ACS, acetyl-CoA synthase.
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MATERIALS AND METHODS |
Bacterial strains and growth conditions.
M.
thermoacetica was cultivated as described earlier (1)
but included a vitamin mixture (36). Dicamba (2 mM) was
added at an optical density at 600 nm (OD600) of 0.6. Cells
were harvested at an OD600 of 2.0 using a CEPA continuous
centrifuge. The cells were frozen in liquid nitrogen and stored at
80°C.
Analytical methods.
Corrinoid concentrations were measured
by conversion to the dicyano derivative (23) using the
extinction coefficients at 580 and 367 nm of 10.13 × 103 M
1 cm1 and 30.82 × 103 M1 cm1. Metals were
determined by plasma emission spectroscopy (14). Protein
concentrations were determined by the Rose Bengal method (9). Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was done by the method of Laemmli
(20), and the gels were stained as described by Blum et
al. (4).
Electron paramagnetic resonance (EPR) spectra were recorded on a Bruker
ESP 300E spectrometer equipped with an Oxford ITC4 temperature
controller, a model 5340 automatic frequency counter (Hewlett-Packard),
and a Bruker gaussmeter. The molecular mass of MtvC was determined by
electrospray ionization mass spectroscopy using a Micromass Platform II
instrument (Manchester, United Kingdom). N-terminal sequences were
determined by automated N-terminal Edman degradation using an ABI-494
Procise Sequencer after transferring the purified protein (30 nmol)
from the SDS-PAGE gel to a polyvinylidene difluoride membrane. The
protein band was stained with 0.1% Amido Black, excised, and sequenced.
Enzymatic assays. (i) O-demethylase assay.
O-demethylase activity was measured using 3 mM dicamba or 1 mM vanillate as substrate in a 400-µl reaction mixture at 55°C in
the anaerobic chamber. The reaction mixture contained 50 mM 2-(N-morpholino)ethanesulfonate (MES) buffer (pH 6.6), 2 mM
ATP, 3 mM tetrahydrofolate, 4 mM titanium (III) citrate, 5 mM
MgCl2, 5 mM NaCl, and enzyme components. The assay was
quenched after 1 h by adding 0.2 M perchloric acid (final) and
being centrifuged at 10,000 × g for 2 min, and
substrates and products were quantified by thin-layer chromatography
(TLC) and/or reverse-phase high-performance liquid chromatography
(HPLC). For TLC analysis, after being quenched and centrifuged, the
samples were extracted with diethyl ether (1:1 [vol/vol]), the
organic phase was evaporated, and the residue was dissolved in 40 µl
of diethyl ether. This solution was then chromatographed on a silica
gel AL SIL G/UV (Whatman Ltd.) plate using the organic phase of a
toluene-acetic acid-water (6:7:3, vol/vol/vol) mixture as mobile phase.
Products were visualized with a UV lamp and by being sprayed with 1%
FeCl3. The Rf values of the
aromatics under these conditions were as follows: dicamba, 0.7;
dichlorosalicylate, 0.54; vanillate, 0.72; catechol, 0.3; and
protocatechuate, 0.05. For HPLC, samples were quenched and centrifuged
as above, and then the supernatant was neutralized with NaOH and a
sample (30 to 50 µl) was injected onto a µ-Bondapak C18 3.5- by
150-mm column (Waters, Milford, Mass.). To assay dicamba metabolism,
the column was developed with a linear gradient from 0 to 60% methanol
in 50 mM KPi buffer, pH 7.7. The 280-nm absorbance of
dicamba and the 420-nm fluorescence emission (excitation wavelength, 310 nm) of dichlorosalicylate were monitored. For quantitation of
vanillate and protocatechuate, the column was developed with a linear
gradient from 0 to 60% methanol in 125 mM sodium acetate, pH 4.5 (solvent B), and the elution profile was monitored at 254 nm.
(ii) Vanillate:hydroxocobalamin methyl transfer assay
(MtvB).
The vanillate-dependent methylation of hydroxocobalamin
was performed in the dark at 55°C inside the anaerobic chamber. The reaction mixture contained 0.3 µmol of hydroxocobalamin, 0.4 µmol of vanillate, 0.8 µmol of ATP, 1.6 µmol of titanium (III) citrate, 2 µmol of MgCl2, and 2 µM NaCl in 50 mM MES buffer, pH
6.6 (final volume, 0.4 ml). The reaction was initiated by adding 0.21 nmol of MtvB. The reaction mixture was incubated for 16 h and
quenched and processed for TLC analysis as described above.
(iii) Methylcobalamin:tetrahydrofolate methyl transfer assay
(MtvA).
The methylcobalamin-dependent methylation of
tetrahydrofolate was monitored spectrophotometrically in the dark at
55°C by a modification of a previously described procedure
(12). The reaction was carried out in a 0.4-cm-path-length
cuvette capped with a rubber stopper to maintain an anaerobic
atmosphere. The 0.75-ml reaction mixture contained 15 nmol of
methylcobalamin, 112 nmol of tetrahydrofolate, 3.75 nmol of NaCl, and
0.36 nmol of MtvA in 50 mM MES buffer, pH 6.8. The reaction was
initiated with H4folate and monitored at 525 and 540 nm. In
the absence of MtvA, there was no demethylation of methylcobalamin. The
demethylation rate was calculated using an extinction coefficient for
methylcobalamin at 540 nm of 7.7 mM1 cm1
(19).
(iv) Reduction of the corrinoid protein.
Dithionite stock
solutions (~1.1 M) were prepared in 0.2 M NaOH. The purified
corrinoid protein was reduced chemically by adding 1 mM sodium
dithionite to an argon-purged quartz cuvette containing 0.12 mg of
corrinoid protein per ml in buffer A (25 mM KPi buffer, pH
7.6). The absorption spectrum was recorded 5 min after adding
dithionite to the sample.
Purification of the Mtv O-demethylase components. (i)
Separation of the Mtv components.
Protein purification was
performed at 15°C in an anaerobic chamber (Coy Laboratory Products,
Ann Arbor, Mich.) under an atmosphere of 90% nitrogen and 10%
hydrogen. During purification, the O-demethylase activity
was routinely determined by TLC analysis. Fifty grams (wet weight) of
cells was sonicated in 100 ml of solution containing buffer A, 0.5 mg
of lysozyme per ml, 2 mM dithiothreitol, and 2.5% glycerol. The
sonicate was centrifuged at 30,000 × g for 90 min in a
Type 35 rotor (Beckman Instruments Inc.).
The cell extract was heat treated at 70°C for 20 min in a water bath
and centrifuged at 10,000 × g for 15 min. This step
gave a modest increase in specific activity but appeared to stabilize the enzyme toward further purification. The supernatant was then subjected to ammonium sulfate fractionation at 4°C. The dicamba O-demethylase activity precipitated at 40 to 70%
saturation. After being centrifuged at 10,000 × g for
20 min at 4°C, the 70% pellet was solubilized in 1.2 M ammonium
sulfate in buffer A and fractionated by phenyl Sepharose
chromatography, which resolved the O-demethylase activity
into three components: MtvA, MtvB, and MtvC. To purify each component,
the assays contained two components from the phenyl Sepharose
fractionation step plus column fractions that were being screened for
the third component. The three components were purified to homogeneity
(Fig. 2).
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FIG. 2.
Polyacrylamide gel electrophoresis of purified MtvA,
MtvB, and MtvC. Proteins were visualized by silver staining. The gels
contained 3 µg of MtvA, 2 µg of MtvB, and 6 µg of MtvC.
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The 40 to 70% ammonium sulfate fraction (above) was applied to a 2.5- by 25-cm phenyl Sepharose CL-6B (Pharmacia LKB) column equilibrated
with the same solution. A linear gradient (1,200 ml) from 1.2 M to 0.3 M ammonium sulfate was run. Then, the column was washed with 5 column
volumes of buffer A and further washed with 3 mM KPi
buffer, pH 7.6. The dicamba O-demethylase activity separated
into three components, MtvA, MtvB, and MtvC. MtvC (450 mg/265 ml)
eluted at 0.94 M ammonium sulfate, MtvA (436 mg/220 ml) eluted at
approximately 0.7 M, and MtvB (484 mg/213 ml) eluted in the final 3 mM
KPi buffer wash. Active fractions were pooled and stored at
4°C.
(ii) MtvA purification.
The MtvA fraction from phenyl
Sepharose was concentrated to 3 ml by ultrafiltration with a YM10
membrane (Amicon Division, Beverly, Mass.) and applied to a 1.5- by
68-cm Sephacryl S-100 (Amersham Pharmacia Biotech AB) gel filtration
column equilibrated with buffer A containing 50 mM KCl. Active MtvA
fractions (282 mg/15 ml) were pooled, applied to a 2.5- by 7-cm
DEAE-Sephacel (Sigma Chemical Co., St. Louis, Mo.) column that was
equilibrated with 50 mM KCl in buffer A and developed with a linear
gradient from 50 to 375 mM KCl in buffer A. MtvA eluted at 130 mM KCl. The pooled fractions (12 mg/42 ml) were diluted threefold with buffer A
and applied to a 2.5- by 3-cm hydroxyapatite fast-performance liquid
chromatography (Calbiochem, La Jolla, Calif.) column. A linear gradient
(60 ml) of 5 to 200 mM KPi buffer, pH 7.6, was applied.
MtvA eluted at approximately 70 mM KPi. Active fractions were pooled (4.6 mg/10 ml) and stored at 4°C.
(iii) MtvC purification.
The phenyl Sepharose fraction
containing MtvC was diluted 10-fold with buffer A containing 125 mM KCl
and loaded on a 2.5- by 7-cm DEAE Sephacel column equilibrated with the
same buffer. A 150-ml linear gradient from 125 to 350 mM KCl was
applied. Fractions with MtvC activity eluted at approximately 270 mM
KCl. The active fractions were pooled (245 mg/53 ml) and concentrated
to 3 ml using a YM10 membrane. This sample was then applied to a 1.5- by 68-cm Sephacryl S-100 gel filtration column equilibrated with buffer
A containing 50 mM KCl. Fractions were collected at a flow rate of 0.2 ml/min. Active MtvC fractions were pooled (8.3 mg/13 ml) and diluted
10-fold with 2 mM KPi buffer, pH 7.6. Active fractions were
pooled and applied to a 2.5- by 3-cm hydroxyapatite high-performance column equilibrated with 5 mM KPi buffer, pH 7.6, which was
developed with a 100-ml linear gradient from 0 to 100 mM
KPi buffer, pH 7.6. MtvC activity eluted at approximately
30 mM KPi. Active MtvC fractions were pooled (3.0 mg/17 ml)
and stored at 4°C.
(iv) MtvB purification.
The MtvB pool from the phenyl
Sepharose column was loaded on a 2.5- by 7-cm DEAE Sephacel column that
had been equilibrated with buffer A. In a linear gradient (400 ml) of 0 to 400 mM KCl, MtvB eluted at 125 mM KCl. The pooled fractions (220 mg/97 ml) were then concentrated with a YM10 ultrafiltration membrane
to 3 ml and loaded on a 1.5- by 68-cm Sephacryl S-100 gel filtration column equilibrated with buffer A containing 50 mM KCl. Active fractions were pooled (55 mg/12 ml) and diluted 10-fold with 20 mM
Tris-HCl buffer, pH 7.6. This sample was loaded on a 2.5- by 3-cm
hydroxyapatite column equilibrated with the same buffer, and a 150-ml
linear gradient from 0 to 200 mM KPi buffer, pH 7.6, was
applied. Active MtvB fractions eluting at 90 mM KPi were
pooled (3.6 mg/12 ml) and stored at 4°C.
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RESULTS AND DISCUSSION |
Dicamba demethylation by M. thermoacetica.
M. thermoacetica cells were grown anaerobically with glucose
and dicamba to yield approximately 55 g (wet weight) of cells. In the
absence of glucose, the cells grew slowly, with a low yield. Glucose
did not appear to repress the O-demethylase activity. Dicamba conversion to dichlorosalicylate was monitored by TLC and HPLC,
and cells were harvested anaerobically when ~85% of the dicamba was metabolized.
Besides metabolizing dicamba, extracts from cells grown on dicamba
could demethylate vanillate to protocatechuate and further decarboxylate protocatechuate to catechol under standard assay conditions. Similarly, extracts of cells grown in the presence of
vanillate could demethylate dicamba to dichlorosalicylate as well as
convert vanillate to protocatechuate and catechol (not shown). Daniel
et al. also found that M. thermoacetica cells grown on
different methoxylated aromatic compounds catalyze the
O-demethylation of the same particular methoxylated
compound, suggesting the presence of a broad specificity of
O-demethylase (6). As shown below, the enzyme
from dicamba-grown cells utilizes vanillate much more efficiently than
dicamba. To conform to the nomenclature used by Krzycki and Thauer to
describe the components of the methanogenic methyltransferases involved
in demethylation of methylamines, methylsulfides, and pterins (reviewed
in reference 32), we designate this as the Mtv system,
i.e., methyltransferase for vanillate. The reaction catalyzed by the
Mtv system of M. thermoacetica is most similar to that of
the four-component system from A. dehalogenans (17).
Enzymatic properties of the O-demethylase. (i) Overall
reaction requirement of MtvA, MtvB, and MtvC.
The three components
required for O-demethylation of dicamba and vanillate were
resolved and purified to homogeneity. Omission of MtvA, -B, or -C from
the O-demethylase reaction mixture results in complete loss
of O-demethylase activity; thus, all three protein components are required for dicamba O-demethylase activity.
Cell extracts that were bubbled with air for 0.5 h and then with
nitrogen for 15 min (so that O2 was not introduced into the
assay mixture) retained full O-demethylase activity.
Therefore, the reaction, which involves Co(I), is O2
sensitive, not the enzyme. The demethylation reaction requires a
reductant. Although dithionite can reduce Co(II) to Co(I), it inhibits
the O-demethylase. On the other hand, Ti(III) citrate
reduces Co(II) and drives the O-demethylase reaction. The
optimal temperature is 55°C and the optimal pH is 6.6. As described
above, H4folate is required as the acceptor of the methyl group from vanillate. The reaction rate is linear for up to 2 h at
55°C.
(ii) Steady-state kinetics.
The apparent
Km values for dicamba and vanillate are 9.0 mM
and 85 µM, respectively, and the Vmax values
with dicamba and vanillate as the methyl donors are 276 and 900 nmol
mg1 h1, respectively (Fig.
3). The 350-fold (based on relative
V/K values) preference for vanillate is striking,
since the cells were grown on dicamba. These results prompted us to
name the enzyme vanillate, instead of dicamba,
O-demethylase. The Km value for
H4folate is 0.23 mM.
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FIG. 3.
Dependence of the O-demethylase reaction on
dicamba and vanillate. The assay contained MtvA (3.8 µg), MtvC (2.24 µg), and MtvB (9.6 µg) in a reaction volume of 0.1 ml. The
reactions were performed for 1 h under standard assay conditions,
and the products formed were analyzed by reverse-phase HPLC. See
Materials and Methods for details.
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As has been found for some other methyltransferases,
hydroxocobalamin or methylcobalamin could substitute for
MtvC, which allowed dissection of the roles of the individual
components of the Mtv system. In the first partial reaction, MtvB is
the only protein required to catalyze the vanillate- or
dicamba-dependent methylation of cob(I)alamin; in the second partial
reaction, MtvA is the only enzyme required for the
methylcobalamin-dependent methylation of H4folate.
MtrB, MtvB, a vanillate:cobalamin methyltransferase.
The
function of MtvB was established by studying an
O-demethylase partial reaction:transfer of the
O-methyl group of vanillate to the Co(I) state of
B12 to form methylcobalamin. MtvA and H4folate were not required for the demethylation of vanillate; B12
was absolutely required (in the absence of MtvC). TLC analysis of the
partial reaction showed protocatechuic acid formation as the concentration of vanillate decreased, demonstrating that MtvB participates in the first part of the overall O-demethylase
reaction, transferring the methyl group from the methoxylated aromatic
substrate to cobalamin. The enzymatic function of MtvB is similar to
that of the 36-kDa component B from A. dehalogenans
(15, 17).
Purified MtvB is a homodimer with an apparent monomeric molecular mass
of 48 kDa. Like MtvA, MtvB is colorless and exhibits the typical
UV-visible spectrum of a protein lacking chromophoric prosthetic
groups. Metals, including zinc and iron, were found in less than
stoichiometric amounts (<0.1 g-atm/mol). To assess the possibility
that this is a Zn protein, we determined the effect of Zn addition on
the demethylation of vanillate. Even at concentrations as high as 4 mM,
there was no stimulation of activity. Either this is not a zinc protein
or reconstitution requires different conditions. In methionine synthase
and the methyltransferases that use coenzyme M, zinc is involved in
coordinating and thus activating the thiol substrate (reviewed in
references 25 and 32). Zinc is also found in the
methyltransferases involved in the transfer of the methyl group of
methanol to cobalamin (MtaB) (31).
MtvC, a corrinoid protein.
Homogeneous preparations of MtvC
contain a single subunit with an apparent molecular mass of 27 kDa
based on SDS analysis and 22 kDa based on mass spectrophotometric
analysis. Presumably, this is the 22-kDa protein identified by Daniel
et al. to be induced when M. thermoacetica cells are exposed
to syringate (6). Functionally, this enzyme is most
similar to the 26-kDa corrinoid protein of A. dehalogenans
called component A (15, 17). A. dehalogenans also contains a fourth component (called component C), which is an
activating protein that couples the ATP-dependent reduction of Co(II)
to Co(I) (17). The M. thermoacetica
O-demethylase system was assayed using an artificial electron
donor [Ti(III) citrate]; as was found with A. dehalogenans, with this assay the reductive activation system was
not required. The N-terminal sequence of the protein,
MLTDTL(S)KAMAELEEEQ(V)LA, is not significantly similar to
the sequence of the A. dehalogenans corrinoid protein (component A) (16).
The UV-visible spectrum (Fig. 4) of the
reddish brown as-isolated protein is similar to that of Co(II)
corrinoids, with a major absorption peak at 474 nm (22).
MtvC was calculated to contain 0.8 mol of cobalamin per mol of protein.
Cobalt is the only metal found in stoichiometric amounts. There were
variable amounts of zinc and iron at levels of <0.2 g-atm per mol
of protein.
Reduction of MtvC with 1 mM Ti(III) citrate or dithionite bleaches the
474-nm peak of cob(II)alamin as an absorption peak forms at 388 nm,
which is characteristic of cob(I)alamin. However, the Co(I) state of
MtvC, when generated with dithionite, was unable to demethylate dicamba
or vanillate in the presence of MtvA or MtvB. Dithionite also inhibited
the demethylation reaction in the standard assay. Therefore, Ti(III)
citrate (4 mM) concentration was routinely used in the demethylation assays.
The EPR spectrum of purified as-isolated MtvC (Fig.
5) is characteristic of a Co(II)
corrinoid (29). The g
resonance is split
into eight lines by hyperfine interactions between the unpaired
electrons and the cobalt nucleus, which has a nuclear spin,
I, of 7/2. The strength of the interaction is described by
the hyperfine coupling constant, AZ (Co) 
105 G. Each of these hyperfine lines is split into a triplet superhyperfine
splitting pattern [AZ (N) 20 G] (Fig. 5, inset)
that is characteristic of corrinoid proteins containing a coordinated
axial nitrogen ligand. This ligand could be a histidine residue from
the protein, as in methionine synthase, methanogenic corrinoid proteins
involved in methyl transfer, and some adenosylcobalamin enzymes
(reviewed in reference 24). Proteins that have the
histidine ligand generally contain a conserved motif,
DxHxG-(41-42)-gxSxL-(21/22)-GG (24), although there are
exceptions (32). Component A of the A. dehalogenans O-demethylase contains this "his" binding motif
(16). Alternatively, the ligand could be from the
benzimidazole group appended to the corrin ring, as in ribonucleotide
reductase (21) and diol dehydrase (35).
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FIG. 5.
EPR spectra of the Co(II) in as-isolated MtvC. The
spectrum of 90 µM MtvC in 25 mM KPi buffer, pH 7.6, was
recorded using the following EPR conditions: temperature, 100 K; gain,
104; power, 100 mW; modulation amplitude, 10 G; microwave
frequency, 9.47 GHz; scans, 16. The coupling constants calculated were
Az(Co) = 105 G and Az(N) = 20 G. The
inset was recorded under identical conditions, except that the
temperature was 85 K.
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Properties of MtvA, a methylcobalamin: H4folate
methyltransferase.
MtvA was found to catalyze the
H4folate-dependent demethylation of methyl-cob(III)alamin,
which was followed photometrically at 525 nm (Fig.
6). The 525-nm peak decreased as a peak
at 475 nm formed, which is characteristic of cob(II)alamin. Based on the published extinction coefficient for methylcobalamin
(
540 = 7.70 mM1 cm1
[19]), the rate of demethylation is 72 nmol
min1 (mg of MtvA)1. Under the conditions of
the assay, which lacked reductant, cob(I)alamin is rapidly oxidized to
cob(II)alamin. MtvA showed no activity in the vanillate- or
dicamba-dependent methylation of cob(I)alamin.
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FIG. 6.
Methylcobalamin-tetrahydrofolate methyl transfer
catalyzed by MtvA. The reaction was performed at 55°C under anaerobic
conditions as described in Materials and Methods. Spectral changes
occurring during the methyl group transfer from methylcobalamin (20 µM) to H4folate (140 µM) were recorded at 0, 5, 10, 20, 40, and 60 min after addition of H4folate.
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Functionally, MtvA is similar to the A. dehalogenans
component D (15, 17). The reaction of MtvA (equation 4) is
also analogous to that of AcsE (equation 5), the
CH3-H4folate- and corrinoid-dependent methyltransferase involved in transferring the methyl group of the
CH3-H4folate to the corrinoid iron-sulfur
protein in the Wood-Ljungdahl pathway (33, 34). However,
MtvA is unable to substitute for AcsE, and AcsE cannot substitute for
MtvA. Similarly, in methanogens, the cognate corrinoid proteins and the
methyltransferases involved in dimethylsulfide, trimethylamine,
dimethylamine, and monomethylamine metabolism (28)
are discrete gene products that cannot substitute for each other
(10, 26, 28).
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(4)
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(5)
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Based on SDS-PAGE analysis, MtvA is a 29.5-kDa protein.
Similarly, component D from A. dehalogenans and AcsE from
M. thermoacetica have monomeric molecular masses of 30 kDa
(15, 17) and 28.6 kDa (30), respectively. The
UV-visible absorption spectrum of MtvA does not reveal any chromophores
other than the amino acids. AcsE also lacks prosthetic groups
(30).
The N-terminal sequence of MtvA is highly similar to that of the
CH3-H4folate- and cobalamin-dependent
methyltransferase from M. thermoacetica (AcsE) (Fig.
7), which folds into a TIM barrel structure containing the pterin binding site within the barrel (7). Given the strong sequence homology among the N
termini of MtvA, AcsE, and component D, perhaps the
O-demethylases adopt a similar fold.
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FIG. 7.
Comparison of N-terminal sequences of MtvA and AcsE (the
corrinoid iron-sulfur protein methyltransferase) from M. thermoacetica and component D from A. dehalogenans.
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| 1.
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Andreesen, J. R.,
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Bache, R., and N. Pfennig.
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Berman, M. H., and A. C. Frazer.
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Blum, H.,
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Daniel, S. L.,
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52:25-28[CrossRef].
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Doukov, T.,
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Structure
8:817-830[Medline].
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El Kasmi, A.,
S. Rajasekharan, and S. W. Ragsdale.
1994.
Anaerobic pathway for conversion of the methyl group of aromatic methyl ethers to acetic acid by Clostridium thermoaceticum.
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33:11217-11224[CrossRef][Medline].
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Elliott, J. I., and J. M. Brewer.
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The inactivation of yeast enolase by 2,3-butanedione.
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Ferguson, D. J.,
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Abstract
Introduction
