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J. Biol. Chem., Vol. 275, Issue 37, 29053-29060, September 15, 2000
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§,
From the Department of Microbiology, The
Ohio State University, Columbus, Ohio 43210 and the ¶ Department
of Biochemistry and Molecular Biology, Uniformed Services University of
the Health Sciences, Bethesda, Maryland 20814
Received for publication, December 23, 1999, and in revised form, June 5, 2000
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Methyl transfer from dimethylamine to coenzyme M
was reconstituted in vitro for the first time using only
highly purified proteins. These proteins isolated from
Methanosarcina barkeri included the previously unidentified
corrinoid protein MtbC, which copurified with MtbA, the
methylcorrinoid:Coenzyme M methyltransferase specific for
methanogenesis from methylamines. MtbC binds 1.0 mol of corrinoid
cofactor/mol of 24-kDa polypeptide and stimulated dimethylamine:coenzyme M methyl transfer 3.4-fold in a cell
extract. Purified MtbC and MtbA were used to assay and purify a
dimethylamine:corrinoid methyltransferase, MtbB1. MtbB1 is a
230-kDa protein composed of 51-kDa subunits that do not possess a
corrinoid prosthetic group. Purified MtbB1, MtbC, and MtbA were the
sole protein requirements for in vitro
dimethylamine:coenzyme M methyl transfer. An MtbB1:MtbC ratio of 1 was
optimal for coenzyme M methylation with dimethylamine. MtbB1 methylated
either corrinoid bound to MtbC or free cob(I)alamin with dimethylamine,
indicating MtbB1 carries an active site for dimethylamine demethylation
and corrinoid methylation. Experiments in which different proteins of
the resolved monomethylamine:coenzyme M methyl transfer reaction
replaced proteins involved in dimethylamine:coenzyme M methyl transfer
indicated high specificity of MtbB1 and MtbC in
dimethylamine:coenzyme M methyl transfer activity. These results indicate MtbB1 demethylates dimethylamine and specifically methylates the corrinoid prosthetic group of MtbC, which is subsequently demethylated by MtbA to methylate coenzyme M during
methanogenesis from dimethylamine.
Methanosarcina barkeri is a methanogenic archaeon
capable of methanogenesis from a wide range of substrates. Aside from
H2:CO2 and acetate, the majority are
methylotrophic substrates such as methylated thiols, methanol, and
methylamines. Methanogenesis from a methylotrophic substrate requires
methyl group abstraction and subsequent methylation of the thiol of
2-mercaptoethanesulfonic acid (coenzyme M or
CoM).1 Methyl-CoM is then
converted to methane with reducing equivalents obtained from the
concomitant oxidation of the methylotrophic substrate to carbon dioxide
(1, 2).
The methylamines are important methylotrophic substrates in marine
environments where they arise from the anaerobic breakdown of choline
derivatives, betaine, and trimethylamine oxide (3). During the
breakdown of trimethylamine (TMA) methyl groups are sequentially
removed from the substrate with the intermediate production of
dimethylamine (DMA) and monomethylamine (MMA), which are subsequently
consumed for the production of methane. DMA or MMA can also serve as
sole carbon and energy source (4, 5).
The final step of CoM methylation with each methylamine is catalyzed by
the same polypeptide. This is the 37-kDa methylcorrinoid:CoM methyltransferase, termed MtbA, that was originally identified in cells
grown on acetate (6) but was found in highest abundance in cells grown
on TMA (7). MtbA was shown to be involved in the MMA:CoM methyl
transfer reaction due to its stimulation of this activity in cell
extracts (8). Removal of MtbA from cell extracts with immobilized
antibodies greatly diminished CoM methylation with TMA, DMA, or MMA,
but each activity could be fully restored by supplementing the
MtbA-depleted extract with purified MtbA (9). MtbA does not contain a
corrinoid prosthetic group, and since little corrinoid exists in
M. barkeri that is not protein-bound (10), these results
indicated that corrinoid-binding proteins must be involved in CoM
methylation from each methylamine.
This prediction was confirmed with the reconstitution of
MMA-dependent CoM methylation reaction (11) with highly
purified proteins (Fig. 1A;
also see Fig. 9). A 50-kDa polypeptide, MtmB, which lacks a detectable
prosthetic group, was required for the methylation with MMA of MtmC, a
29-kDa corrinoid-binding protein. The two independently isolated
proteins can form a complex and are most active in CoM methylation at a
MtmB:MtmC ratio of 2. Methyl-MtmC serves as substrate for CoM
methylation as catalyzed by MtbA. These three protein components are
sufficient for MMA:CoM methyl transfer in vitro in the
presence of Ti(III) citrate and methyl viologen. The latter two
components are required for reductive activation of MtmC to the active
Co(I) form, since MtmC is isolated in a mixture of the Co(II) and
Co(III) redox states. Use of this reductant and redox mediator
circumvents a requirement for a physiological ATP-dependent
reductive activation system that is not yet completely resolved (12,
13), allowing in vitro MMA:CoM methyl transfer with the
aforementioned three polypeptides.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
Proposed reaction schemes for in
vitro CoM methylation from two different methylamines.
A, CoM methylation from MMA requires a small corrinoid
protein, MtmC, and two methyltransferases (MtmB and MtbA). MtmB does
not bind a corrinoid prosthetic group but is required for the
methylation of the corrinoid cofactor of MtmC. MtmC is active in the
Co(I) state and inactive in the Co(II) state. Activity is maintained by
the presence of Ti(III) citrate and methyl viologen in the in
vitro assay. Methyl-MtmC is then used as a substrate by MtbA to
methylate CoM. B, DMA-dependent CoM methylation
as proposed by Wassenaar et al. (21, 22). A dimeric
methyltransferase, DMAMT, binds one corrinoid group/dimer and
automethylates the prosthetic group which is then demethylated by MtbA
to methylate CoM. The corrinoid bound to DMAMT is kept in the active
Co(I) state by components of a cellular ATP-dependent
reductive activation added to the in vitro assay as purified
MAP as well as hydrogenase added as a primary DEAE fraction of cell
extract.
TMA:CoM methylation has also been resolved to three purified polypeptides (14). In this case, the 52-kDa MttB and the 26-kDa MttC polypeptides are required to catalyze TMA-specific CoM methylation in the presence of MtbA. MttB and MttC do not separate during purification. Dissociation of MttB and MttC using gel permeation in the presence of SDS demonstrated that each mole of MttC bound 1 mol of corrinoid, whereas no corrinoid was bound by MttB (14).
Methanol-dependent methylation of CoM also requires a minimum of three polypeptides. Methanol was the first methylotrophic substrate of M. barkeri for which the pathway of CoM methylation was elucidated. Methyltransferase I is a corrinoid-containing enzyme composed of two tightly bound polypeptides, MtaB and MtaC (15, 16). The 51-kDa MtaB subunit uses methanol to methylate the corrinoid prosthetic group of the 28-kDa MtaC subunit, as shown by the capability of recombinant MtaB to methylate free cob(I)alamin (17). Methyl-MtaC is then demethylated to generate methyl-CoM by MtaA, a methylcorrinoid:CoM methyltransferase (16, 18) which is homologous to MtbA (19, 20). Reductive activation of MtaC can be accomplished with either Ti(III) citrate (16) or the partially resolved ATP-dependent corrinoid activation system (12). MtbA does not function in the methylation of CoM with MMA or DMA but will function to a limited extent in the TMA-dependent CoM methylation pathway (14).
Thus, methanol and TMA-dependent CoM methylation systems are analogous to the MMA:CoM methylation scheme shown in Fig. 1A. Each system has in common a requirement for a small corrinoid-binding polypeptide, yet these are distinct corrinoid-binding proteins specific for each pathway. As demonstrated for the methanol and MMA systems, methylation of the corrinoid protein with a methylotrophic substrate requires a separate protein such as MtmB or MtaB. The methylated corrinoid is then demethylated by a methylcorrinoid:CoM methyltransferase such as MtaA or MtbA.
The DMA:CoM methyl transfer pathway has not previously been reconstituted with highly purified proteins. However, in addition to the CoM methylase MtbA, another protein was recently implicated in DMA:CoM methyl transfer by Wassenaar et al. (21, 22) and designated DMAMT (see Fig. 1B). Purified preparations of homodimeric DMAMT contained 0.45 mol of corrinoid/mol of 50-kDa DMAMT polypeptide. DMAMT and MtbA alone were insufficient for DMA:CoM methyl transfer and required addition of methyltransferase activation protein (MAP), a protein involved in ATP-dependent reductive activation of the corrinoid, as well as a fraction from a primary DEAE separation of cell extract that contained hydrogenase activity (22). A mechanism was proposed in which DMAMT methylated with DMA its own bound corrinoid prosthetic group, which was then demethylated by MtbA and CoM (Fig. 1B). This scheme stands in contrast with the resolved pathway for MMA:CoM methyl transfer (Fig. 1A) and the analogous pathways for TMA and methanol-dependent CoM methylation in that a small discrete corrinoid-binding protein is not required.
In this paper, we describe the isolation of a small corrinoid protein,
MtbC, from M. barkeri which stimulated DMA:CoM methyl transfer when added to cell extract. MtbC was used in an assay to
isolate MtbB1, a DMA:corrinoid methyltransferase that does not have a
corrinoid prosthetic group. Highly purified MtbB1, MtbC, and MtbA are
sufficient to catalyze in vitro CoM methylation by DMA in a
manner most analogous to the resolved MMA:CoM methylation reaction.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Cell Cultures and Preparation of Extracts--
M.
barkeri MS was cultured on 80 mM TMA (8, 23) as
described previously. Cell extracts were also prepared as described previously (24), except where indicated below for MtbC purification. Briefly, cell suspensions were lysed anaerobically at 20,000 pounds/square inch with a French pressure cell prior to
ultracentrifugation at 150,000 × g. The supernatant
was stored at 
70 °C in hydrogen-flushed stoppered serum vials
until used.
Materials-- Gases were purchased from Linde Specialty Gases (Columbus, OH) and passed through catalyst R3-11 (Chemical Dynamics Corp., South Plainfield, NJ) to remove O2. Column chromatography media were manufactured by Amersham Pharmacia Biotech unless otherwise indicated. MOPS, CoM, TMA, DMA, MMA, methyl viologen, hydroxocobalamin, methylcobalamin, ATP, and 5,5'-dithio(2-nitrobenzoic acid), or DTNB, were purchased from Sigma. Titanium (III) chloride (10% in aqueous solution) was purchased from Aldrich. Reagents for electrophoresis were purchased from Bio-Rad.
Purification of Methyltransferases and Corrinoid Proteins Involved in DMA:CoM and MMA:CoM Methyl Transfer-- The 24-kDa corrinoid protein MtbC was purified from cultures in exponential growth phase. The purification was performed under aerobic conditions at room temperature. Cell extract was prepared by French press disruption of 60 g of cell paste resuspended in 150 ml of 0.1 M potassium phosphate buffer, pH 7.5, containing 10% glycerol, 4 mM EDTA, 0.02% sodium azide, and 23 µg/ml DNase I and centrifuged to remove insoluble components. MtbA was assayed by the methylcobalamin:CoM reaction described previously (6). MtbC itself was followed by SDS-PAGE analyses of the fractions and in later steps also by absorbance at 360 and 546 nm (absorption maxima for the bound corrinoid cofactor). The corrinoid protein copurified with MtbA through the first two columns employed for purification. Cell extract was first applied at 5 ml/min to a 200-ml column of Q-Sepharose (5.0 × 10.0 cm) that had been equilibrated with a solution of 0.10 M KCl in Buffer A (20 mM potassium phosphate, 1 mM EDTA, and 0.02% sodium azide, pH 7.5). The column was then washed with two bed volumes of Buffer A, and elution was then carried out with a linear gradient of 0.10 to 0.25 M KCl in Buffer A. The bulk of both proteins, MtbA and the 24-kDa MtbC, was recovered in the fractions eluting between 0.10 and 0.15 M KCl. A separate 25-kDa corrinoid protein eluted in earlier fractions and was largely removed at this step. The pooled fractions containing MtbA and MtbC were then concentrated by ultrafiltration in a stirred cell apparatus using an YM-30 membrane (Amicon, Inc., Beverly, MA) and then diluted to approximately 0.07 M KCl. The protein solution was then applied to a 140-ml (1.5 × 20 cm) column of Q-Sepharose equilibrated and then eluted as described above. The resulting fraction of MtbA/MtbC was then concentrated and adjusted to 1.58 M (NH4)2SO4 in a final volume of 3 ml. The fraction was then applied at 0.5 ml/min to a Amersham Pharmacia Biotech HR 16/10 phenyl-Superose column equilibrated with 1.58 M (NH4)SO4 in Buffer A. Elution was carried out with a 30-ml gradient decreasing linearly in (NH4)2SO4 from 1.58 to 0 M and simultaneously increasing in 1,3-propanediol from 0% to a final concentration of 10%. After completion of the gradient, 10% 1,3-propanediol in Buffer A was applied to the column at the same flow rate. Immediately after application of this buffer, a peak containing mostly MtbC eluted from the column, which was then followed immediately by a protein peak comprised primarily of MtbA. The pooled MtbC fraction was dialyzed against Buffer A that was adjusted to pH 6.5 then concentrated to 2 ml and applied to a Amersham Pharmacia Biotech Mono Q HR 16/10 column equilibrated with the same buffer. During application of a linear gradient of 0-0.25 M KCl in Buffer A at pH 6.5, a peak of residual MtbA emerged, which was followed by a peak of MtbC at 150 mM KCl. The now homogeneous MtbC preparation was dialyzed against 20 mM HEPES, pH 7.5, concentrated by a Centricon 30 unit (Amicon) and stored as aliquots frozen in liquid nitrogen. The N terminus was determined by automated Edman degradation.
Purification of MtbB1 was performed in an anaerobic chamber containing 97% N2 and 3% H2 (Coy Laboratories, Grass Lake, MI) with buffers and column materials made anaerobic by repeated cycles of evacuation and flushing with N2. Protein purification was initiated by applying 35 ml of cell-free extract to a 25 × 2.5-cm DEAE-Sepharose (Sigma) equilibrated in 50 mM Tris, pH 8.0. A 500-ml gradient of 100-500 mM NaCl in 50 mM Tris-HCl, pH 8.0, was applied to the column at 2.2 ml/min. MtbB1 activity was assayed as described below and eluted in a volume of 85 ml after approximately 400 ml of the gradient had been applied. The MtbB1 active fraction was adjusted to pH 6.5 with 75 mM MOPS, pH 6.5, and 50 ml was loaded onto a Amersham Pharmacia Biotech Mono-Q HR 10/10 column. A 120-ml gradient of 50-500 mM NaCl in 50 mM MOPS, pH 6.5, was applied to the column at 2 ml/min. The remaining 35 ml of DEAE-Sepharose fraction was then subjected to the Mono-Q step. The MtbB1 activity from both Mono-Q runs eluted at approximately 320 mM NaCl in a total volume of 12 ml. The pooled active fractions were then loaded onto two UNO Q1 columns connected in series (Bio-Rad) equilibrated with 50 mM Tris, pH 8.0. A 100-ml gradient of 150-350 mM NaCl in 50 mM Tris, pH 8.0, was then applied to the column at 1 ml/min. The MtbB1 activity eluted at approximately 245 mM NaCl in a volume of 13 ml. The pooled active UNO Q1 fractions were adjusted to 700 mM (NH4)2SO4, and 6.5 ml were loaded onto a phenyl-Sepharose HP cartridge (Amersham Pharmacia Biotech) in 50 mM MOPS, pH 7.0, and a 39-ml gradient of 500 to 0 mM (NH4)2SO4 was applied to the column. The phenyl-Sepharose column was repeated with the remaining 6.5 ml of the MtbB1 UNO Q1 fraction. The active MtbB1 fractions from both runs eluted at a concentration of approximately 180 mM (NH4)2SO4 in a total volume of 20 ml. The pooled active fractions were then adjusted to pH 8.0 in 50 mM Tris and loaded onto a single UNO Q1 column. A gradient of 150-350 mM in 50 mM Tris, pH 8.0, was applied to the column at 2 ml/min over a volume of 80 ml. The active MtbB1 fractions eluted at a concentration of approximately 245 mM NaCl in a volume of 11 ml. The purified MtbB1 was then concentrated using four Amicon Centricon 10 concentrators to a volume of 1.5 ml and adjusted to a volume of 3.2 ml with 50 mM MOPS adjusted to pH 7.0.
The monomethylamine methyltransferase, MtmB, and its cognate corrinoid protein, MtmC, were purified as described previously (11). The methylcorrinoid:CoM methyltransferase, MtbA, was also purified as described previously (11), essentially following the protocol of Yeliseev et al. (7). It was found that both purifications could be performed aerobically at room temperature with no loss of activity.
Aerobically purified proteins were made anaerobic by repeated cycles of evacuation and flushing with H2 prior to use in the enzyme assays described below.
Dimethylamine-dependent Coenzyme M Methylation Assays-- All reactions were performed in anaerobic-sealed 2-ml glass vials under an atmosphere of H2 at 37 °C in a shaking water bath. An extract stimulation assay that identified fractions promoting DMA:CoM methyl transfer in crude cell extract was used to locate activity peaks eluting from the primary DEAE-Sepharose fractionation of total soluble proteins. These reactions were carried out in hydrogen-flushed vials containing 50 µl (850 µg) of MMA-grown cell extract, 1.5 µl (39 µg) of TMA-grown cell extract, 61.5 µl of column fraction, 1.5 mM Ti(III) citrate, 10 mM ATP, 20 mM MgCl2, 2 mM CoM, 100 mM DMA, and 1 mM bromoethanesulfonate in a total volume of 125 µl. The latter compound inhibits methane formation from the methyl-CoM product. The remaining free thiol of unmethylated CoM was quantitated by removing 10 µl of reaction mixture and added to 90 µl of 0.5 mM DTNB reagent at fixed time points as described previously (8). This method has previously been found to be an accurate measure of the DMA-dependent methylation of CoM in both whole and fractionated extracts of M. barkeri (21).
All other CoM methylation assays in subsequent experiments employed reactions supplemented with purified proteins. These reactions relied on activation of the methyltransferase reaction by Ti(III) citrate with methyl viologen as a redox mediator. Screening of columns following the initial DEAE column for purification of MtbB1 was done with hydrogen-flushed reaction vials containing 4 mM CoM, 100 mM DMA, 15 µg of MtbA, 15 µg of MtbC, 4.5 mM Ti(III) citrate, 0.75 mM methyl viologen, and appropriate amounts of column fractions in a total volume of 125 µl. For other experiments, 30 µg of purified MtbB1 were added in place of the column fractions in reactions containing purified MtbA and MtbC as the only other protein components. Protein concentrations in the assay were occasionally varied for some experiments, and this is indicated in the text. At intervals during the reaction time course, 5 µl of the reaction mixture were removed and reacted with 95 µl of 0.5 mM DTNB to determine the amount of unmethylated CoM. A low level of CoM loss (approximately 0.5-1 nmol/min) was observed in reactions lacking enzymes or DMA. This was not observed at 1.5 mM concentrations of Ti(III) citrate and was ascribed to interference of higher concentrations of Ti(III) citrate with DTNB detection of free thiols. This low background rate was subtracted from all of the CoM methylation reactions reported here in which the Ti(III) citrate/methyl viologen reducing system was employed.
Corrinoid Methylation Reactions-- The reactions contained 50 µg (10 µM) of either MtmB or MtbB1 and 35 µg of either MtmC or MtbC (12 or 15 µM, respectively) as indicated, as well as 4 mM Ti(III) citrate, 0.75 mM methyl viologen, and 100 mM MMA or DMA in a total volume of 100 µl. The vials were incubated at 37 °C for 20 min in a shaking water bath under dim red light. The corrinoid was then extracted as described by Kremer et al. (25) and methylcobalamin quantitated by C-18 reverse phase HPLC (11). The amount of methylated corrinoid extracted for each reaction was calculated by comparison to a standard curve of methylcobalamin analyzed by the same HPLC method.
DMA-dependent free cobalamin methylation by MtbB1 was
measured spectrally as follows. The reactions were performed in an
H2-flushed anaerobic cuvette with a 2-mm path length.
H2 rather than N2 was used to minimize Ti(III)
citrate oxidation by protons. The reactions contained 250 µg of
MtbB1, 15 mM Ti(III) citrate, 0.5 M DMA, and 2.5 mM hydroxocobalamin in 50 mM MOPS, pH 6.5, in a total volume of 0.6 ml. The spectrophotometer was blanked against
the cuvette containing only the DMA, purified MtbB1, and buffer. The
reactions were started by the addition of hydroxocobalamin and were
performed at 23 °C under dim red light. The conversion of
cob(I)alamin to methylcobalamin was quantified at 540 nm using an
extinction coefficient of 4.4 mM
1
cm1 (26). DMA did not methylate cob(I)alamin in the
absence of MtbB1.
Other Analytical Techniques--
Protein-bound cobamide
concentrations were determined using a dicyano derivatization method
(27). Protein concentrations were determined by using the bicinchoninic
acid protein assay (28) with reagent purchased from Pierce using bovine
serum albumin as standard. Plasma emission spectroscopy was performed
at the University of Georgia Chemical Analysis Laboratory using a
Jarrell-Ash 965 plasma emission spectrometer. Ultraviolet-visible
spectra were obtained using a Hewlett-Packard model 8453 photodiode
array spectrophotometer. SDS-gel electrophoresis was performed
following the procedure of Laemmli (29) using a Mini-slab
electrophoresis system (Idea Scientific Co., Minneapolis, MN).
Molecular size markers (Bio-Rad) were rabbit skeletal muscle myosin
(200,000), Escherichia coli 
-galactosidase (116,250),
phosphorylase b (97,400), bovine serum albumin (66, 200),
hen egg white ovalbumin (45,000), bovine carbonic anhydrase (31,000),
soybean trypsin inhibitor (21,500), hen egg white lysozyme (14,400),
and bovine pancreas aprotinin (6,500). HPLC size exclusion columns
G4000SWXL and G3000SWXL (Supelco, Bellefonte,
PA) equilibrated with 100 mM NaCl in 50 mM
MOPS, pH 7.0, connected in series and adapted to a Bio-Rad medium
pressure liquid chromatography system inside a Coy anaerobic chamber
were used to determine native protein masses. MtbB1 or MtbC (100 µg
each) was injected onto the columns in a volume of 250 µl and eluted
at a rate of 0.75 ml/min. The molecular mass standards used to generate
the standard curve were blue dextran (2,000,000), thyroglobulin
(669,000), apoferritin (443,000), -amylase (200,000), alcohol
dehydrogenase (150,000), bovine serum albumin (66,000), carbonic
anhydrase (29,000), and ferricyanide (324) purchased from Sigma.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Purification of MtbC, a Small Corrinoid Protein Involved in DMA:CoM
Methyl Transfer--
During the isolation of the amine-specific
methylcorrinoid:CoM methyltransferase, MtbA, from cell extracts of TMA
grown cells from M. barkeri, a previously uncharacterized
protein was identified and termed MtbC. The two proteins copurified
through several columns until a hydrophobic interaction column
separated MtbC from MtbA. Purified MtbC migrated as a single 24-kDa
protein band during SDS-gel electrophoresis (Fig.
2). The purified protein eluted with an
apparent molecular mass of 34 kDa from an HPLC silica gel permeation
column. The UV-visible spectra indicated MtbC is a corrinoid-binding
protein (Fig. 3A). The as
isolated aerobic protein had an absorbance maximum at 360 nm, typical
of protein-bound corrinoid in the Co(III) state with water or a
hydroxyl group as the -axial ligand. The stoichiometry of cofactor
bound to the protein was determined spectrophotometrically following
conversion of the cofactor to the dicyano derivative by SDS and heat
denaturation of the protein in the presence of KCN. Each mol of 24-kDa
polypeptide bound 1.0 mol of corrinoid. The N-terminal amino acid
residues were determined by Edman degradation to be
SXEELLQELADAIIS, where X was a residue that could
not be unambiguously assigned.
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Extracts of cells grown on TMA possess appreciable DMA:CoM methyl transfer activity (550 nmol/min/mg protein), but this activity is much less in MMA-grown cells (<2.5 nmol/min/mg protein). There is a highly abundant 24-kDa protein in TMA-grown cell extract, which comigrates with purified MtbC during SDS-gel electrophoresis. No such prominent 24-kDa band is detectable in electrophoresed extract from MMA-grown cells (Fig. 2). This was consistent with a possible role for MtbC in CoM methylation by DMA. CoM methylation with TMA, DMA, or MMA was therefore measured in an extract of TMA-grown cells before and after addition of purified MtbC to the extract. CoM methylation from TMA, DMA, or MMA was measured at rates (in nmol/min/mg total protein) of 30.4, 38.5, or 24.2, respectively. Addition of 20 µg of purified MtbC to the same extract resulted in rates of CoM methylation from TMA, DMA, or MMA of 35.9, 130.3, or 31.3, respectively. The marked stimulation of DMA:CoM methyl transfer by the addition of MtbC was consistent with a role for MtbC in the initiation of methanogenesis from DMA.
Isolation of MtbB1 Using Purified MtbC and MtbA--
A role for
MtbC in DMA:CoM methyl transfer implies the existence of a DMA:MtbC
methyltransferase. Extracts of TMA-grown cells were anaerobically
fractionated in order to identify such a methyltransferase and study
its possible interaction with MtbC. The ability of protein fractions to
stimulate the low rate of DMA:CoM methyl transfer in an extract of
MMA-grown cells was used to screen the initial DEAE-Sepharose column
(Fig. 4) for potential components of the DMA:CoM methyl transfer reaction. Two peaks of activity were detected. The first peak to elute (peak I) is most likely to be due to MtbC itself (see below). A protein that acts as a DMA:corrinoid
methyltransferase was isolated from the second activity peak to elute
(peak II) which was designated as MtbB1.
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Peak II fractions combined with purified MtbC and MtbA carried out DMA:CoM methyl transfer in the presence of Ti(III) citrate and methyl viologen. This assay, rather than the extract stimulation assay, was used to purify MtbB1 to a single homogeneous polypeptide that could carry out DMA:CoM methyl transfer in the presence of purified MtbA, MtbC, and no other proteins. A single peak of activity was detected in each of the subsequent columns on which MtbB1 was purified (Table I). The final preparation of MtbB1 revealed a single 51-kDa polypeptide upon SDS-gel electrophoresis (Fig. 2). The determined N terminus of the polypeptide (MATEYALRMGDGKRVYLTKE) matched 14 of 16 residues from the DMAMT polypeptide isolated by Wassenaar et al. (21, 22) from M. barkeri Fusaro. In marked contrast to the report for DMAMT preparations, our preparations of homogeneous MtbB1 polypeptide did not contain any detectable corrinoid prosthetic group when assayed by the dicyano method. The UV-visible spectrum of MtbB1 did not have any of the characteristic peaks of a corrinoid protein (Fig. 3B). Analysis of the metal content of MtbB1 by plasma emission spectroscopy revealed no detectable cobalt in two different preparations of the homogeneous enzyme. The lower limit of detection for cobalt using this procedure was 0.04 mol of cobalt/mol of polypeptide. No other metals were detected except 0.43 mol of zinc/mol of polypeptide. The protein eluted as a 230-kDa protein during size exclusion chromatography, indicating a homotetrameric or possibly homopentameric configuration.
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Minimum Requirements for in Vitro DMA:CoM Methyl Transfer-- Previous work with DMA:CoM methyl transfer in vitro had been performed primarily using purified proteins added to crude extracts or with extracts fractionated with only a single DEAE column (9, 21). Therefore, the minimal requirements for DMA:CoM methyl transfer could not be determined. However, as shown below, the isolation of MtbC and MtbB1 provided the final purified components required for a fully resolved in vitro DMA:CoM methyl transfer reaction.
To activate DMA:CoM methyl transfer, the presently unresolved cellular
ATP and hydrogen-dependent activation system was again replaced with Ti(III) citrate and methyl viologen. Purified MtbB1 and
MtbA were insufficient for DMA:CoM methyl transfer when incubated with
DMA and Ti(III) citrate/methyl viologen. However, DMA:CoM methyl
transfer occurred when MtbC was also added to these reactions. Activity
was completely dependent on each protein (Fig.
5). The preparations of all three
proteins used in this and the following experiments were nearly
homogeneous as illustrated in Fig. 2. Preincubation of the proteins
with 10 mM ATP and 20 mM MgCl2 had no effect on the rate of the reaction. A low molecular weight fraction,
obtained as the filtrate from Amicon Centricon 3 membrane filtration unit treatment of a TMA-grown cell extract, also did not
stimulate the rate of CoM methylation. No CoM methylation was
detectable if MtbB1, MtbC, or MtbA were incubated with TMA or MMA as
methyl donors. MtbC (typically present as 15 µg or 0.65 µM in the resolved reaction) could not be replaced by the
addition of 20 µM hydroxocobalamin.
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The resolved DMA:CoM methyl transfer reaction was used to verify that MtbC was the protein responsible for activity of peak I that eluted from the DEAE-Sepharose column depicted in Fig. 4. The pooled peak I fractions, purified MtbB1, and purified MtbA carried out DMA:CoM methyl transfer in the presence of Ti(III) citrate and methyl viologen (data not shown). However, peak I incubated with MtbC and MtbA could not carry out CoM methylation with DMA. These results indicated the first peak of DMA:CoM methyl transfer stimulating activity to elute from the DEAE column is likely due to the presence of MtbC in this fraction, since this fraction replaced purified MtbC but not MtbB1.
Specific Activities of Individual Protein Components in
DMA:CoM Methyl Transfer--
Purified MtbB1 was titrated against a
constant amount of purified MtbC (0.65 nmol) and purified MtbA (0.41 nmol), and the rate of DMA:CoM methyl transfer was measured (Fig.
6). Activity was seen to increase with
increasing MtbB1 with saturation observed at approximately 0.6 nmol of
MtbB1. This corresponds to an approximate molar ratio of 1:1 between
MtbB1 and MtbC. No increase in activity was observed in reactions
containing 1 nmol of MtbB1, 0.6 nmol of MtbC, and either 0.4, 0.8, or
1.2 nmol of MtbA, indicating that MtbA was also saturating under these
conditions. These data allow estimation of the specific activities of
both MtbB1 and MtbC in the DMA:CoM methyl transfer reaction when the
other components are in excess. When MtbC and MtbA were not limiting,
methylation of CoM occurred at an average specific activity of 2.2 µmol/min/mg MtbB1. In the presence of excess MtbB1 and MtbA, the
methylation of CoM occurred at an average specific activity of 2.5 µmol/min/mg purified MtbC.
|
The specific activity of MtbC found in extracts of TMA-grown cells was determined in the presence of saturating amounts of MtbB1 and MtbA (1 and 0.4 nmol, respectively) as 42 nmol of CoM methylated per min/mg of protein. This indicates that an approximately 60-fold purification of MtbC was achieved during the course of MtbC isolation.
The specific activity of methyl transfer by MtbA was measured during the DMA:CoM methyl transfer reaction in the presence of excess MtbC and MtbB1 (0.6 nmol of both MtbB1 and MtbC with a range of 0.05 to 0.25 nmol MtbA) and found to be an average of 2.1 µmol/min/mg purified MtbA.
MtbB1 Is a DMA:Cob(I)alamin Methyltransferase--
The requirement
for a methylcorrinoid:CoM methyltransferase, a corrinoid protein, and
MtbB1 for DMA:CoM methylation suggested that MtbB1 could function in
the demethylation of DMA and methylation of the corrinoid prosthetic
group of MtbC. To test this hypothesis, the ability of MtbB1 to
methylate free cob(I)alamin with DMA was examined. Hydroxocobalamin was
reduced to the Co(I) state with Ti(III) citrate in the presence of DMA,
and MtbB1-dependent formation of methylcobalamin was
monitored spectrophotometrically. Upon initiation of the reaction, a
shift in the cob(I)alamin 553 nm absorbance peak occurred toward the
methylcobalamin peak of 532 nm. An isosbestic point at 578 nm for time
points following T0 indicated that the
transformation of cob(I)alamin to methylcobalamin proceeded
without a detectable intermediate (Fig.
7A). The spectrum taken
at T0 may have contributions from
cob(II)alamin since the reaction was initiated by introduction of
hydroxocobalamin into the cuvette. The conversion of
cob(I)alamin to methylcobalamin can be followed at 540 nm,
the isosbestic point of Co(I)/Co(II) cobalamin (26). MtbB1 methylated
2.5 mM cob(I)alamin at a rate of 14.4 nmol/min/mg MtbB1
(Fig. 7B). No change in the cob(I)alamin spectrum was noted
in the absence of MtbB1. The formation of methylcobalamin from DMA and
cob(I)alamin as catalyzed by MtbB1 was confirmed by HPLC analysis (data
not shown).
|
Methylation of MtbC by MtbB1-- The methylation of free cobalamin with DMA by MtbB1 indicates that its role in the DMA:CoM reaction is in the methylation of the prosthetic group of MtbC. In order to test this directly, 35 µg of MtbC (1500 pmol of corrinoid) and 1 nmol of MtbB1 were incubated in the presence of Ti(III) citrate, methyl viologen, and DMA but in the absence of MtbA and CoM (Table II). The corrinoid prosthetic group of MtbC was then aerobically extracted with ethanol and analyzed by HPLC. No hydroxylated corrinoid peak was detected, and 1250 pmol of methylated corrinoid was recovered from MtbC. Methylation of MtbC was completely dependent on DMA.
|
Specificity of MtbB1 and MtbC for CoM Methylation by DMA-- The preceding results indicated that the resolved in vitro DMA:CoM methyl transfer system was analogous to the resolved three-component MMA:CoM methyl transfer system (11). In addition to the methylcorrinoid:CoM methyltransferase MtbA, each of these systems requires a methylamine-specific methyltransferase and a corrinoid protein. In the following experiments we examined the ability of these protein components to substitute for one another during CoM methylation from DMA or MMA.
MtmB and MtbB1 were incubated with either MtmC or MtbC in the presence
of MtbA and CoM methylation from either DMA or MMA monitored (Fig.
8). Only the combination of MtbB1 and
MtbC supported CoM methylation with DMA, whereas only the combination
of MtmB and MtmC supported CoM methylation with MMA. MtmC and MtbB1
incubated with MtbA did not result in detectable methylation of CoM
with either DMA or MMA. Similarly, MtbC incubated with MtmB and MtbA resulted in no detectable CoM methylation with either MMA or DMA. These
experiments demonstrate that MtmB and MtmC determine the specificity of
CoM methylation for MMA, while MtbB1 and MtbC are essential for CoM
methylation by DMA. MtbB1/MtbC and MtmB/MtmC thus form specific cognate
methyltransferase/corrinoid protein pairs initiating methanogenesis
from DMA or MMA, respectively.
|
Specificity of the MtbB1 and MtmB in Methylation of Their Cognate Corrinoid Proteins-- Neither MtbB1 nor MtmB carried out significant methylation of the non-cognate corrinoid protein with their corresponding methylamine substrates. Methylation of MtbC or MtmC was analyzed following extended incubation with either MtbB1 or MtmC in the presence of either DMA or MMA and Ti(III) citrate and methyl viologen (Table II). Following incubation, the corrinoid prosthetic groups were aerobically extracted from the reaction mixture, and methylcorrinoid was quantitated by HPLC. In control reactions, MtmB methylated MtmC with MMA, as observed previously (11), and MtbB1 methylated MtbC with DMA as discussed above. No detectable unmethylated corrinoid remained in either of these reactions.
In contrast, when MtmB was incubated with reduced MtbC in the presence
of MMA only 1.8% of the initial corrinoid bound to MtbC was recovered
in the methylated form. No methylation of the corrinoid bound to MtmC
by MtbB1 with DMA was detectable. The remainder of the corrinoid
cofactor extracted from both reactions eluted as hydroxylated corrinoid
during HPLC analysis.
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DISCUSSION |
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|
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
|
|---|
This work describes the first complete resolution of a pathway for
the methylation of CoM with DMA using only highly purified proteins.
MtbB1, MtbC, and MtbA are sufficient, and each necessary, for in
vitro transfer of methyl groups from DMA to CoM. Our data support
a model for the interaction of these proteins in the methylation of CoM
with DMA that is analogous to the reactions for CoM methylation with
TMA, MMA, and methanol (Fig. 9). In each
of these pathways, a small corrinoid protein (26-29 kDa) specific for
that pathway interacts with two methyltransferases to initiate CoM
methylation from the methylotrophic substrate. The first
methyltransferase methylates the corrinoid protein with the
methanogenic precursor, whereas the second methyltransferase catalyzes
the demethylation of the corrinoid protein and methylation of CoM.
|
Several lines of evidence support this model of DMA:CoM methylation reaction in M. barkeri. MtbC is a 24-kDa corrinoid protein that specifically stimulated CoM methylation from DMA when added to cell extracts. Furthermore, purified MtbC was used as a component of the DMA:CoM methyl transfer assay employed to isolate MtbB1, a DMA:corrinoid methyltransferase. MtbB1 is the first protein reported to catalyze the direct methylation of non-protein bound cob(I)alamin with DMA. This indicates that MtbB1 possesses the active site used in DMA-dependent methylation of the corrinoid prosthetic group of MtbC. Purified MtbB1, MtbC, and MtbA can methylate CoM with DMA but not other methylamines. These results are consistent with a model in which MtbB1 specifically methylates the corrinoid prosthetic group of MtbC with DMA, and methyl-MtbC is subsequently demethylated by the methylcorrinoid:CoM methyltransferase, MtbA.
Wassenaar et al. (21, 22) have postulated the existence of a different pathway of DMA:CoM methylation in M. barkeri (Fig. 1B). In their work, they proposed that a 50-kDa DMA methyltransferase, DMAMT, binds corrinoid and automethylates its prosthetic group which is then demethylated by MtbA to methylate CoM. Their proposed pathway does not include a small corrinoid protein such as MtbC, rather, DMAMT was reported to bind 0.45 mol of corrinoid/mol of polypeptide and was suggested to serve both as a corrinoid-binding protein and as a DMA:corrinoid methyltransferase. The full relationship of DMAMT to the DMA methyltransferase isolated here (MtbB1) is unknown, but the N termini are 88% identical. The key difference between the two proteins is that our homogeneous preparations of MtbB1 do not bind detectable corrinoid and clearly methylate with DMA a discrete corrinoid protein, MtbC. The complete sequence of DMAMT has not yet been published. We have obtained the full sequence of MtbB1, and it has no detectable homology with any corrinoid-binding protein (30) (see also GenbankTM accession number AF102623). The specific activity of DMA demethylation measured for purified MtbB1 is comparable to that reported for DMAMT. The native structure of the two proteins may also be different. DMAMT is reported to be a dimer, whereas MtbB1 is a tetramer or pentamer.
If both DMAMT and MtbB1 represent gene products that are homologous across their entire length, then it seems clear that DMAMT-bound corrinoid is not essential for activity, and a small corrinoid protein, MtbC, supplies the corrinoid cofactor for the DMA:CoM methyltransferase reaction. However, it is possible that both pathways are present in methanogens. Recently, it was demonstrated that chloromethane utilization by an aerobic methylotroph requires a protein that is a fusion of a methylotrophic corrinoid protein and MtbA homolog (31). This observation indicates that the DMA methyltransferase-corrinoid protein fusion proposed by Wassenaar et al. (21, 22) might be feasible. However, since DMAMT required unresolved protein fractions to carry out in vitro DMA:CoM methyl transfer, it is difficult to determine if a small corrinoid protein is functioning in the DMAMT-dependent methylation of CoM. Wassenaar et al. (22) acknowledged that a small corrinoid protein could have been added to their assays as part of non-homogeneous MAP and MtbA preparations. Further resolution of the DMAMT-dependent DMA:CoM methyl transfer system, and the sequencing of the gene encoding DMAMT, should establish whether a pathway of DMA:CoM methyl transfer exists other than that illustrated in Fig. 9.
Recently, the genes encoding two polypeptides required to initiate TMA:CoM methylation, MttB and MttC (see Fig. 9), were cloned and sequenced (30) (see also GenBankTM accession number AF102623). Two open reading frames were identified near these genes, which initially could not be assigned to gene products. However, the N termini of MtbB1 and MtbC obtained in this study indicate that each of these open reading frames encode proteins involved in the DMA-dependent methylation of CoM. Interestingly, the genes mttB, mttC, mtbC, and mttB are encoded on a single transcript found in TMA-grown cells (30). The cotranscription of the genes encoding MtbC and MtbB1 is consistent with the function of these two independently isolated proteins in the DMA:CoM methyl transfer reaction. It is of further interest that both the mtbB1 and mtbC genes are cotranscribed with genes for initiating TMA catabolism. Since M. barkeri generates DMA as an intermediate of methanogenesis from TMA, cotranscription of both TMA and DMA methyltransferase genes would be an efficient means of coordinating expression of these methyltransferases.
The protein sequence predicted for MtbC from its gene, mtbC, is approximately 50% similar to the predicted protein sequences from the genes encoding the methylotrophic corrinoid proteins of TMA (MttC) (30), MMA metabolism (MtmC) (32), and methanol metabolism (MtaC) (16). Each of these corrinoid proteins are in turn homologous to the cobalamin-binding domain of corrinoid-dependent methionine synthase and mutases (16, 32-34). The homology of the corrinoid protein isolated here, MtbC, to these other small discrete corrinoid proteins involved in methanogenesis from methylotrophic substrates is consistent with our results demonstrating a role for MtbC in a CoM methylation pathway, that is DMA:CoM methyl transfer.
MtmB, MtbB1, and MttB are each implicated in analogous reactions, the methylation of corrinoid with methylamines. However, now that proteins and genes for all three methyltransferases have been identified, it is somewhat surprising to observe that the methylamine methyltransferases are not homologous (30, 32), see also GenBankTM accession number AF102623. This may explain the presence of the different, yet homologous, methylotrophic corrinoid proteins of methylamine-dependent methanogenesis. Although each corrinoid protein serves as a methyl donor for the same CoM methylase, MtbA, the differences in primary sequence must be necessary in order to optimize interaction of a particular corrinoid protein with one of the non-homologous methylamine:corrinoid methyltransferases. These differences appear to lead to highly specific interactions between a methyltransferase and its cognate corrinoid protein and the dedication of a particular corrinoid protein to a particular pathway. Presumably, this specificity occurs with all three of the small corrinoid proteins identified as involved in methylamine metabolism. Unfortunately, this cannot be easily tested with the proteins initiating CoM methylation with TMA, since MttB and MttC form a complex that is difficult to separate. However, our results with the resolved MMA:CoM and DMA:CoM methyl transfer systems do illustrate this specificity. There is little detectable interaction between the MMA methyltransferase and the DMA corrinoid protein or between the DMA methyltransferase and the DMA corrinoid protein. Neither corrinoid protein will detectably support the non-cognate methyltransferase in CoM methylation. This lack of activity in CoM methylation coincides with the inability of either methyltransferase to methylate rapidly the corrinoid prosthetic group of the non-cognate corrinoid protein.
MtbC greatly enhances the interaction of MtbB1 with corrinoid. During
the course of this study, the observed rates of
DMA-dependent methylation of CoM with the reconstituted
system varied from 0.8 to 2.2 µmol/min/mg MtbB1. MtbB1 methylation of
the corrinoid bound to MtbC is therefore much faster than observed for
the methylation of free cobalamin, since 20 µM cobalamin
did not replace MtbC in the resolved DMA:CoM reaction. Indeed, even
with 2.5 mM cobalamin, the rate of
MtbB1-dependent methylation of corrinoid with DMA was slow
relative to the rates observed for MtbB1-, MtbC-, and MtbA-mediated
methylation of CoM with DMA. These results further illustrate the
unique role of the corrinoid-binding protein MtbC in greatly, and
specifically, enhancing the interaction of corrinoid with the
DMA:corrinoid methyltransferase, MtbB1.
| |
ACKNOWLEDGEMENT |
|---|
We thank Tsuneo Ferguson for the assistance in isolation of some proteins used in these experiments.
| |
FOOTNOTES |
|---|
* This work was supported by United States Department of Energy Grant DE-FG-02-91ER20042 (to J. A. K.) and by National Science Foundation Grant MCB-9905068 (to D. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Dept. of Veterans Affairs Medical Center Research Service, Cincinnati, OH 45220.
To whom correspondence should be addressed. Tel.:
614-292-1578; Fax: 614-292-8120; E-mail: Krzycki.1@osu.edu.
Published, JBC Papers in Press, June 13, 2000, DOI 10.1074/jbc.M910218199
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CoM, coenzyme M; MMA, monomethylamine; DMA, dimethylamine; TMA, trimethylamine; PAGE, polyacrylamide gel electrophoresis; HPLC, high performance liquid chromatography; MOPS, 3-(N-morpholino)propanesulfonic acid; DTNB, 5,5'-dithio(2-nitrobenzoic acid); MAP, methyltransferase activation protein.
| |
REFERENCES |
|---|
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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), as well as the complete reaction minus MtbB1 (
), minus
MtbA (
), or minus MtbC (
).
); MtbB1, MtmC, and DMA (
);
MtbB1, MtmC, and MMA (
); MtmB, MtmC, and MMA (+); MtmB, MtbC, and
DMA (