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Originally published In Press as doi:10.1074/jbc.M506402200 on August 11, 2005

J. Biol. Chem., Vol. 280, Issue 44, 36962-36969, November 4, 2005
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The Residue Mass of L-Pyrrolysine in Three Distinct Methylamine Methyltransferases*{boxs}

Jitesh A. Soares{ddagger}, Liwen Zhang§, Rhonda L. Pitsch§, Nanette M. Kleinholz§, R. Benjamin Jones§, Jeremy J. Wolff¶, Jon Amster¶1, Kari B. Green-Church§2, and Joseph A. Krzycki{ddagger}||3

From the {ddagger}Department of Microbiology, §Campus Chemical Instrument Center Mass Spectrometry and Proteomics Facility, and the ||Ohio State University Biochemistry Program, Ohio State University, Columbus, Ohio 43210 and the Department of Chemistry, University of Georgia, Athens, Georgia 30602

Received for publication, June 13, 2005 , and in revised form, July 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Single in-frame amber (UAG) codons are found in the genes encoding MtmB, MtbB, or MttB, the methyltransferases initiating methane formation from monomethylamine, dimethylamine, or trimethylamine, respectively, in certain Archaea. The crystal structure of MtmB demonstrated that the amber codon codes for pyrrolysine, the 22nd genetically encoded amino acid found in nature. Previous attempts to visualize the amber-encoded residue by mass spectrometry identified only lysine, leaving information on the existence and structure of pyrrolysine resting entirely on crystallography of a single protein. Here we report successful mass spectral characterization of naturally occurring pyrrolysine and the first demonstration of the amber-encoded residue in proteins other than MtmB. The sequencing of chymotryptic fragments from acetonitrile-denatured proteins by tandem mass spectrometry revealed the mass of the amber-encoded residue in MtmB, MtbB, and MttB as 237.2 ± 0.2 Da. Fourier transform ion cyclotron resonance mass spectrometry produced an accurate measurement for the pyrrolysyl-residue as 237.1456 Da, within error limits of the predicted mass based on the empirical formula C12H19N3O2. These measurements support the structure of pyrrolysine in MtmB as 4-methylpyrroline-5-carboxylate in amide linkage with the {epsilon}N of lysine but not the alternative structure in which the 4-substituent of the pyrroline ring is an amine group. The presence of pyrrolysine with statistically identical mass in all three methyltransferases is in keeping with the proposed direct incorporation of pyrrolysine into protein during translation of the UAG codon and suggests that MtbB and MttB may exploit the unusual electrophilicity of pyrrolysine during catalysis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pyrrolysine is an unusual amino acid found in MtmB, the monomethylamine (MMA)4 methyltransferase that initiates methane formation in certain methanogenic Archaea of the family Methanosarcinacea (1). Unlike the many other atypical residues, pyrrolysine is genetically encoded, the 22nd such amino acid to be identified in nature (2-4). The mtmB1 gene encoding MtmB possesses an in-frame amber (TAG = UAG) codon that does not terminate translation during production of the isolated MtmB protein (5-7). A dedicated tRNAPyl has been identified that possesses the requisite anticodon to decode UAG as a sense codon (2). Chemically synthesized pyrrolysine (8) can be ligated to tRNAPyl by PylS, a homolog of class 2 aminoacyl-tRNA synthetases in in vitro charging assays (3, 9). Escherichia coli transformed with pylT (encoding tRNAPyl), pylS, and mtmB1 will suppress the amber codon in mtmB1 and produce full-length MtmB, but only if supplemented with exogenous pyrrolysine that is subsequently incorporated into the UAG position (3). This is consistent with the in vitro binding of Escherichia coli EF-Tu with charged tRNAPyl (10). UAG translation as pyrrolysine thus appears to involve ligation of cytoplasmic pyrrolysine to tRNAPyl by a dedicated pyrrolysyl-tRNA synthetase, much like the original set of 20 canonical amino acids. In contrast, the 21st amino acid, selenocysteine, follows a more indirect mechanism of genetic encoding. The selenocysteinyl-tRNASec required for UGA translation as selenocysteine is formed with seryl-tRNASec as an intermediate, not with free selenocysteine (11, 12).

The deduced structure of pyrrolysine rests to date entirely on crystallographic structures of MtmB at 1.55-2.0 Å resolution (1, 8). These data indicated that pyrrolysine is lysine with the {epsilon}-nitrogen in amide linkage to (4R,5R)-4-substituted pyrroline-5-carboxylate (Fig. 1). The identity of the 4-substituent as either a methyl, hydroxy, or amine group could not be initially established with certainty; however, further analysis of hydroxylamine-derivatized pyrrolysine at 2.0 Å resolution has best supported a methyl group, which fits the electron density with an occupancy of 0.89. An amine group remained an admitted possibility, having an occupancy of 0.69 (8). Attempts to further examine the pyrrolysyl residue by physical methods other than crystallography have had mixed success. Electrospray ionization mass spectrometry (MS) of intact MtmB revealed a mass 107 ± 2 Da larger than predicted if lysine were at the UAG-encoded position. However, the mass accuracy was insufficient to differentiate between the probable C-4 substituents (Fig. 1) or indeed to confirm if the increased mass of MtmB is due to the UAG-encoded residue (1). Edman degradation and tandem MS of HPLC-purified tryptic fragments previously indicated lysine was present at the UAG-encoded position of MtmB (7), a result possibly due to hydrolysis of pyrrolysine.

In addition to MMA, Methanosarcina barkeri and its close relatives can also form methane from trimethylamine (TMA) or dimethylamine (DMA), in reactions initiated by one of two methylamine methyltransferases that are not homologous to each other or to MtmB. MttB is specific for TMA (13), whereas MtbB is selective for DMA (14). The three types of methylamine methyltransferases each methylate a distinct corrinoid-binding protein (Fig. 2A) dedicated to the utilization of one type of methyltransferase. For MttB, MtbB, and MtmC, the cognate corrinoid proteins are MttC, MtbC, and MtmC, respectively. The corrinoid proteins are homologous to each other, as well as the cobalamin-binding domain of methionine synthase (15-17). The methylated cognate corrinoid protein of each methyltransferase can serve as substrate for a single methylcobamide:coenzyme M methyltransferase called MtbA (6, 13, 14, 18). The methyl-coenzyme M produced from this second methyltransferase reaction can then be converted to methane in the primary energy-yielding reaction of methanogenesis (19, 20).



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  		FIGURE 1.
The structure of pyrrolysine deduced from the electron density of MtmB and residue mass of the UAG-encoded residue in MtmB, MtbB, and MttB.

 



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  		FIGURE 2.
A, schemes for the initiation of methanogenesis from TMA, DMA, or MMA in Methanosarcina spp. MtmB can demethylate MMA (R represents NH2) and methylate the corrinoid cofactor bound to its cognate corrinoid protein MtmC. MtbB similarly demethylates DMA (R represents NHCH3) and methylates MtbC, whereas MttB demethylates trimethylamine (R represents N(CH3)2) and methylates the corrinoid cofactor of MttC. The three corrinoid proteins are homologous, although the methylamine methyltransferases share no readily identifiable homology. Each corrinoid protein can be demethylated by the same CoM methylase, MtbA. B, the genes in M. barkeri MS encoding the methylamine methyltransferases and corrinoid proteins. Transcriptional units are indicated by the arrows under the distinct gene clusters. The mtmB1 (GenBankTM accession number AF013713 [GenBank] ) and mtmB2 (GenBankTM accession number AF230870 [GenBank] ) genes are transcribed with nearly identical copies of mtmC. The single mttB gene (GenBankTM accession number AF102623 [GenBank] ) is co-transcribed with mtbB1 (GenBankTM accession number AF102623 [GenBank] ). Two genes with ~90% identity to mtbB1 have also been identified in M. barkeri MS that are designated mtbB2 (GenBankTM accession number AF153453 [GenBank] ) and mtbB3 (GenBankTM accession number AF153454 [GenBank] ). The first 10% of the 5' portions of mtbB2 and mtbB3 have not been sequenced in the strain used for these studies, and this would correspond to the first 48 residues encoded by the mtbB1 gene (shown in gray).

 
A deviation from the standard genetic code is required not only for expression of the gene encoding MtmB, but also during expression of the genes encoding either MtbB or MttB (Fig. 2B). In M. barkeri MS, the DMA and TMA methyltransferase genes feature a single in-frame amber codon (16). Two to three nearly identical copies of genes encoding each type of methyltransferase are present in the genomes of Methanosarcina spp., and all contain a single UAG codon (16, 21, 22). In M. barkeri MS, the 95% identical genes encoding MMA methyltransferase are termed mtmB1 and mtmB2, whereas the genes that could encode the DMA methyltransferase are termed mtbB1, mtbB2, and mtbB3. Only a single TMA methyltransferase gene, mttB, has been identified in M. barkeri MS (16). In all known examples of methanogen methylamine methyltransferases, the positioning of the UAG codon is conserved for homologous genes encoding a particular type of methylamine methyltransferase. Similar to MtmB, the UAG codons in the genes for MttB and MtbB do not appear to cease translation during production of the characterized 50-kDa methyltransferases. This was first suggested from the size of isolated TMA and DMA methyltransferases, which are much larger than would be predicted if the gene ended at the in-frame amber codon. Extension of both these genes beyond the amber codon to the following TAA or TGA codon would produce products of the measured size of the isolated methyltransferases (16). Sequencing of the N- and C-terminal sequences of the isolated DMA methyltransferase confirmed that amber codon read-through must occur during expression of the mtbB genes (16). The sequenced methylamine methyltransferase genes of Methanococcoides burtonii also possess in-frame amber codons, and sequencing of MttB peptides during a proteomic study also indicated that conservation of the reading frame occurs during expression of the TMA methyltransferase gene (23). However, although it has been largely speculated that the in-frame amber codon of the TMA and DMA methyltransferase genes would also be translated as pyrrolysine, this has never been demonstrated.

We have now successfully determined the mass of the UAG-encoded residue in the methylamine methyltransferases, thereby providing the first documentation of pyrrolysine as a naturally occurring protein residue by a method other than crystallography and further that UAG is translated as pyrrolysine in three nonhomologous methylamine methyltransferases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Cultivation and Extracts—M. barkeri MS (DSM800) cells were grown on 80 mM TMA or MMA in a phosphate-buffered medium (24) in 15-40-liter glass carboys with a nitrogen gas phase and harvested 7 days after inoculation. Cell extracts were anaerobically prepared with lysis using a French pressure cell and frozen at -70 °C prior to use.

Isolation of Methylamine Methyltransferases—MtmB was purified as the MtmBC complex from cell extracts of M. barkeri MS grown on MMA as previously described for the co-purification of MtbA and MtmC (25) with the exception that all steps were performed aerobically. In order to separate MtmB from the MtmBC complex, MtmBC was flush-evacuated under hydrogen and then reduced with 5 mM titanium (III)-citrate. The reduced MtmBC complex was then loaded onto an anoxic Sephacryl-S100 (Amersham Biosciences) gel filtration column (80 x 2.5 cm) operated in an anoxic chamber (Coy Laboratories, Grass Lake, MI). MtmB with trace amounts of MtmC eluted near the void volume under these conditions, whereas most of MtmC eluted later in the profile. The procedure was repeated to remove traces of MtmC from MtmB. The MtmB used in these experiments yielded only a single 50-kDa band when subjected to denaturing 12.5% polyacrylamide gel electrophoresis with detection by Coomassie staining.

The DMA methyltransferase, MtbB, was isolated entirely in the anoxic chamber. The dimethylamine:CoM methyltransferase assay was performed as described previously (14). The buffers and column matrices were made anaerobic before use by repeated cycles of flush/evacuation using N2. The purification was initiated by absorbing 1.5 liters (20 mg of protein/ml) of soluble extract prepared from cells grown on TMA onto a 40 x 5-cm DE-52 (Whatman Inc., Fairfield, NJ) column equilibrated with 50 mM NaCl in 50 mM Tris, pH 8.0. A gradient (2.4 liters) of 50-500 mM NaCl in the same buffer was applied to the column at 2 ml/min. MtbB eluted in 250 mM NaCl, and the pooled fractions (120 ml) of MtbB1 were concentrated 10-fold by ultrafiltration with a YM-10 membrane (Amicon, Inc., Beverly, MA). The sample was then diluted with 50 mM MOPS, pH 6.5. An aliquot (35 ml) was then chromatographed with a Mono-Q HR 10/10 column (Amersham Biosciences) equilibrated with 50 mM NaCl in 50 mM MOPS, pH 6.5. A 160-ml gradient of 50-500 mM NaCl in the same buffer was applied to the column at a flow rate of 2 ml/min. The MtbB activity eluted with ~300 mM NaCl in a total volume of 12 ml. The procedure was repeated with the remaining DE-52 aliquots, and MtbB fractions were pooled and concentrated 10-fold using a YM-10 membrane. The concentrated sample was rediluted 10-fold with 50 mM Tris-HCl, pH 8.0, and the pooled active fractions were chromatographed on two UNO-Q1 columns (Bio-Rad) that were connected in series and pre-equilibrated with 50 mM Tris, pH 8.0. A 160-ml gradient of 150-350 mM NaCl in 50 mM Tris, pH 8.0, was applied to the column at a flow rate of 0.5 ml/min. The MtbB activity eluted at 220 mM NaCl in a volume of 24 ml. The pooled active MtbB fractions from the UNO-Q column were concentrated with a YM-10 membrane and adjusted to 700 mM (NH4)2SO4 with a saturated solution. The sample was then loaded onto a phenyl-Sepharose HP cartridge (Amersham Biosciences) equilibrated with 500 mM (NH4)2SO4 in 50 mM MOPS, pH 7.0. A gradient (80 ml) of 500 to 0 mM (NH4)2SO4 in 50 mM MOPS, pH 7.0, was applied to the column at 0.5 ml/min. The active MtbB eluted at ~420 mM (NH4)2SO4 in 6 ml. The purified MtbB was concentrated in three Amicon Centricon 10 concentrators to a volume of 1 ml and adjusted to a volume of 4 ml with 50 mM MOPS, pH 7.0. The sample was homogeneous when 3 µg of protein was analyzed by polyacrylamide gel electrophoresis and stained with Coomassie.

The TMA methyltransferase, MttB, was isolated as described previously as the MttB-MttC complex (13). Repeated gel permeation chromatography was used to separate MttB from MttC, as described above for MtmB, but using 10 mM dithiothreitol as the reducing agent rather than titanium citrate.

Proteolysis—Intact protein was first reduced with dithiothreitol and then carbamidomethylated with iodoacetamide prior to proteolytic digestion using chymotrypsin (Roche Applied Science). The final buffer conditions for digestion of desalted samples were 25 mM ammonium bicarbonate and 5% acetonitrile. The final ratio of methyltransferase to chymotrypsin was 25:1 (w/w) in a total volume of 80 µl. The digestion was carried out at 37 °C for 4 h and stopped by acidification with 1 µl of trifluoroacetic acid.

Matrix-assisted Laser Desorption/Ionization (MALDI) Mass Spectrometry—MALDI-MS of the chymotryptic peptides was performed on a Bruker Reflex III (Bruker, Breman, Germany) mass spectrometer operated in reflectron positive ion mode with an N2 laser using {alpha}-cyano-4-hydroxycinnamic acid as the matrix prepared as a saturated solution in 50% acetonitrile, 0.1% trifluoroacetic acid (in water). Allotments of 5 µl of matrix and 1 µl of sample were thoroughly mixed together, and then 0.5 µl of this mixture was spotted on the target plate and allowed to dry. Surfactant-assisted MALDI (26) was performed on the digestion products to further increase the number of peptides detected as previously described (26).

Liquid Chromatography-Tandem Mass Spectrometry—In order to obtain sequence of individual peptide ions, a Micromass Q-TOF II (Micromass, Wythenshawe, UK) equipped with an orthogonal nanospray source (New Objective, Inc., Woburn, MA) was operated in positive ion mode in conjunction with a Dionex Capillary LC-System (LC Packings-A Dionex Co., Sunnyvale, CA). Samples (2.5 µl) were first injected onto a trapping column (Michrom BioResources, Auburn, CA) and then washed with 50 mM acetic acid and injected onto a 5-cm-long, 75-mm internal diameter ProteoPep II C18 column (New Objective, Inc.) packed directly in the nanospray tip. The column was then eluted with mobile phase A as 50 mM acetic acid and mobile phase B as acetonitrile. Peptides were eluted directly off the column into the Q-TOF system using a gradient of 2-80% B over 45 min with a flow rate of 0.3 µl/min. The total run time was 58 min. The nanospray capillary voltage was set at 3.0 kV, and the cone voltage was set at 55 V. The source temperature was maintained at 100 °C. Mass spectra were recorded using MassLynx 4.0 with automatic switching functions. Mass spectra were acquired from mass 400-2000 daltons every 1 swith a resolution of 8000 (full-width, half-maximum). When the desired peak (using include tables) was detected at a minimum of 15 ion counts, the mass spectrometer automatically switched to acquire a collision-induced dissociation (CID) MS/MS spectrum of the individual peptide. Collision energy was set dependent on charge state recognition properties. The PEAKS program from Bioinformatics Solutions was used for MS/MS data processing. Sequence information from the MS/MS data were processed using Mascot Distiller to form a peaklist file. Data were minimally processed with application of a three-point smoothing function and with the centroid calculated from the top 80% of the peak height. The charge state of each ion selected for MS/MS was calculated; however, the peaks were not deisotoped. Assigned peaks were judged valid only if they had a minimum of 5 counts (S/N of 3) and displayed the corresponding C13 ion. The mass accuracy of the precursor ions was set to 1.2 Da to accommodate accidental selection of the C13 ion, and the fragment mass accuracy was set to 0.3 Da. Considered modifications were methionine oxidation and carbamidomethyl cysteine. Pyrrolysine was also programmed into PEAKS as a modification. The data were acquired several times to ensure reproducibility.

MALDI-Fourier Transform Ion Cyclotron Resonance (MALDI-FTICR)—The chymotryptic digestion products of MtmB were mixed 1:1 with 1.3 M 2,5-dihydroxybenzoic acid and air-dried on a MALDI target plate. Ions from 15 laser shots from a Scout intermediate pressure MALDI source were accumulated into an external hexapole ion trap and then transferred into the analyzer cell of a Bruker BioApex 7 Tesla FTICR mass spectrometer. Twelve repetitions of this cycle were co-added for each mass spectrum. The mass spectra were externally calibrated using a bovine serum albumin tryptic digest and then internally calibrated on seven different MtmB chymotryptic peptides, achieving a final mass accuracy of 1.5 ppm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Initial solution phase proteolytic digestions of MtmB were attempted in aqueous buffers containing urea as protein denaturant and employing either chymotrypsin or a combination of GluC and ArgC. These approaches yielded adequate sequence coverage of the N-terminal and C-terminal portions of MtmB but did not yield peptides containing the UAG-encoded residue at position 202 of MtmB. Alternative digestion protocols were therefore developed using acetonitrile, which is highly compatible with mass spectrometry, to denature the protein (27). Whereas GluC/ArgC digestion of MtmB was inhibited by acetonitrile, organic solvent denaturation enhanced sequence coverage of MtmB with chymotrypsin. The observed m/z of intact peptide assignments identified in MALDI-time of flight MS (see supplemental Table S1), MALDI-FTICR (TABLE ONE), and sequence tags generated from LC-MS/MS (see supplemental Table S2) resulted in a total coverage of 83.4% of the protein (see supplemental Fig. S1). Digestion of MtmB with chymotrypsin in 25 mM ammonium bicarbonate and 5% acetonitrile for 18 h at room temperature allowed reproducible detection by all three methods of a MtmB peptide possessing the pyrrolysyl residue.


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  		TABLE ONE
Predicted and observed m/z from MALDI-FTICR data acquired on a Bruker MALDI-FTICR mass spectrometer from MtmB digested with chymotrypsin

 
From the crystallographic data, the mass of the pyrrolysyl residue can be calculated as 237.1477 Da (C12H19N3O2), 238.1429 Da (C11H18N4O2), or 239.1269 (C11H17N3O3), depending on the respective identity of the 4-substituent as a methyl, amine, or hydroxy group. The crystal structure obtained for MtmB also revealed that the primary structure of MtmB was that predicted for the gene product of the mtmB1 gene (1). Using these potential masses and the deduced mtmB1 reading frame, a predicted chymotryptic pyrrolysyl-containing peptide (residues 194-208) was observed as either the singly charged ion (M + H) by MALDI or a doubly charged ion (M + 2H) using electrospray ionization mass spectrometry.



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  		FIGURE 3.
CID spectrum of m/z 791.62+ reveals the mass of the pyrrolysyl residue. The b- and y-ions obtained by fragmentation of the peptide bonds of the peptide are indicated as are their m/z value. The peaks allowing calculation of masses of individual residues are indicated by the horizontal double-headed arrows.

 
MALDI-FTICR identified an M + H ion with an m/z value of 1581.8036 (n = 6, S.D. = 0.0009) in chymotryptic digests of MtmB. This is within 0.7 ppm of the theoretical mass (1581.8059 Da) of the MtmB peptide 194AGRPGMOXGVXGPETSL208, but only if residue X is pyrrolysyl with the 4-substituent a methyl group. The alternative possibilities would yield masses of 1582.8012 (hydroxyl) and 1583.7852 (amine) for the singly charged ions. The identification of this peptide further allows an estimate of the mass of the pyrrolysyl residue. The theoretical mass of the canonical residues within the peptide (i.e. excluding the UAG-encoded residue) is 1344.6582 Da. This would give the mass of the pyrrolysyl residue as 237.1456 with a mass accuracy of 10 ppm. This mass fits to an empirical formula for pyrrolysine of C12H19N3O2 with an error of 9 ppm.

Chymotryptic digests were also analyzed by LC-MS/MS, and peptides identified with high confidence by their sequences are indicated. The doubly charged peptide of 194AGRPGMOXGVXGPETSL208 was detected as a m/z = 791.62+ ion. (The acidic conditions used to produce the electrospray tend to protonate all available basic sites in the peptide, and since this peptide contains an arginine and the N-terminal amine, a doubly charged ion is expected.) The theoretical m/z value is 791.42+ for the sequence 194AGRPGMOXGVXGPETSL208 again, only if X is pyrrolysine with the 4-substituent as a methyl group. The collision-induced dissociation spectrum (CID) of m/z 791.62+ confirmed this sequence assignment (Fig. 3). The y and b ions detected are listed in TABLE TWO. The mass differences between the b9/b8 or y7/y6 ion pairs indicate that the mass of the UAG-encoded pyrrolysyl residue is 237.3 Da. Within the 0.2-Da error of this measurement, this value is consistent with the theoretical mass of the pyrrolysyl residue if the C-4 substituent is a methyl group but not an amine group.


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  		TABLE TWO
The y- and b-ions observed in the CID mass spectrum of m/z 791.62+ obtained from proteolytic digestion of MtmB

The observed sequence matches that of the predicted gene product of the mtmB1 with the UAG codon translated as pyrrolysine.

 
In order to determine whether UAG encodes pyrrolysine in a methylamine methyltransferase that is not homologous to MtmB, the isolated DMA methyltransferase MtbB was subjected to denaturation with acetonitrile, treatment with iodoacetamide, and digestion with chymotrypsin. The peptides were then analyzed by LC-MS/MS. Since nearly identical copies of genes that could encode the DMA methyltransferase have been identified in M. barkeri MS, the identified peptides were examined for unique peptides that would arise from the predicted gene products of the mtbB1, mtbB2, and mtbB3 genes (16). This analysis revealed 10 peptides that could be encoded by either the mtbB1 or mtbB2 genes but not the mtbB3 gene (see supplemental Table S3). However, eight peptide ions were identified that could only arise from the mtbB1 gene that is cotranscribed with the TMA methyltransferase gene, mttB (16). No peptides were identified that are unique to the mtbB2 or mtbB3 gene. Since a single MtbB peak was identified in previous isolations of the DMA methyltransferase (14) as well as in the isolation for this study, this result indicates that the mtbB1 gene produces the major isolatable form of the DMA methyltransferase.

The identified peptides from the chymotryptic digest of MtbB covered 86% of the protein predicted from the gene sequence of mtbB1. Peptides from before and after the in-frame amber codon were identified (see Supplemental Fig. S2 and Table S3). Among the peptides identified that were uniquely predicted from the mtbB1 gene were two peptides that contained the UAG-encoded residue found at position 356 in the gene product of mtbB1. 341RASKAMVEIAGVDGIXIGVGDPL363 was identified as an m/z = 802.523+ ion, whereas 347VEIAGVDGIXIGVGDPLGMPIAHIM371 was identified as both an m/z = 871.213+ ion and an m/z = 1306.322+ species. For both peptides, CID spectrometry confirmed the sequence assignment of these ions, and the UAG-encoded residue was of a similar mass in both peptides. In TABLE THREE are listed the identified b- and y-ions obtained upon collision-induced dissociation of the m/z = 871.213+ ion. The mass difference between the y-16/y-15 ion pair yielded a predicted residue mass for the UAG-encoded residue of 237.22 Da. The difference between the b-10 and b-9 ion pair also allows calculation of the mass of the UAG-encoded residue as 237.22 Da. This is not significantly different from the mass as observed for the UAG-encoded residue in MtmB. This leads us to conclude that the DMA methyltransferase possesses pyrrolysine as the UAG-encoded residue.


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  		TABLE THREE
List of b- and y-ions identified in a CID spectrum of the m/z 871.213+ peptide fragment observed in chymotryptic digests of the DMA methyltransferase, MtbB

The sequence matches that of the predicted gene product of the mtbB1 gene.

 
The TMA methyltransferase was also examined for the presence of pyrrolysine, following chymotryptic digestion using the methods described for the other two methyltransferases. Multiple homologs of the TMA methyltransferase gene have been identified in the Methanosarcina acetivorans genome (21) as well as the Methanosarcina mazei genome (22). However, only a single TMA methyltransferase gene, mttB, has been identifiable in the unsequenced genome of M. barkeri MS. Southern analysis reveals only a single band in Southern blots of M. barkeri MS that hybridizes with mttB probes. LC-MS/MS of the chymotryptic MttB peptides resulted in identification of peptides covering 85% of the predicted sequence from the sole M. barkeri mttB gene, including sequence encoded before and after the in-frame amber codon (see supplemental Fig. S3). Several chymotryptic peptides were identified that contained the UAG-encoded residue from MttB (see supplemental Table S4). These included 330VAGSXSDAKVPDDQAGHEKTMox350, identified as m/z = 766.093+, and 325GLPSYVAGSXSDAKVPDDQAGHEKTM 350, identified as m/z = 932.78+ and as m/z = 1398.682+. The latter sequence was also identified as an independent m/z = 938.443+ ion in which Met350 was oxidized. For all of these ions, the sequence was confirmed by CID mass spectrometry, and the UAG-encoded residue was found to have no significant difference from the mass predicted for pyrrolysine. TABLE FOUR lists the b- and y-ions obtained following collision-induced dissociation of the m/z 932.783+ ion. The difference between the y-17/y-16 ion pair yields a measurement of 237.1 Da for the UAG-encoded residue of MttB, again consistent with the insertion of pyrrolysine into the protein under the direction of a UAG codon.


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  		TABLE FOUR
List of b- and y-ions identified in a CID spectrum of the m/z 932.78 peptide fragment observed in chymotrypic digests of the TMA methyltransferase, MttB

The sequence matches that predicted by the sequence of mttB, where UAG encodes a residue with the mass of pyrrolysine.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This report is the first documentation of pyrrolysine in MtmB1 by a technique other than x-ray crystallographic analysis and the first demonstration of pyrrolysine in the mtbB1 and mttB gene products. Earlier attempts to visualize pyrrolysine in MtmB with mass spectroscopy resulted in identification of lysine as the UAG-encoded residue (7). Synthetic pyrrolysine has since been shown to tolerate short exposure to acidic or basic conditions but was unstable upon prolonged exposure to either condition (8). Such sensitivity may underlie the apparent hydrolysis of the amide bond of the pyrrolysyl residue in MtmB in earlier attempts to analyze the UAG-encoded residue, since MtmB was first alkylated with undiluted iodoacetic acid, and tryptic fragments were isolated by reverse chromatography in trifluoroacetic acid-containing buffers that were subsequently sequenced. Direct LC-MS coupled with solvent denaturation and chymotrypsin digestion of iodoacetamide-treated protein minimized hydrolysis of the pyrrolysyl residue. In the current study, we were unable to identify a peptide from any of the methylamine methyltransferases in which lysine, rather than pyrrolysine, was present at the UAG-encoded position. Indeed, we did not observe modifications of the pyrrolysyl residue in any methyltransferase.

In the most recent crystal structures of MtmB, the electron density of the 4-substituent of pyrrolysine was assigned to a methyl group; however, an amine group remained a distinct possibility (8). The mass of the pyrrolysyl residue observed in all three methylamine methyltransferases is consistent with the proposed structure of pyrrolysine as lysine in {epsilon}N-amide linkage with (4R,5R)-4-methyl-pyrroline-5-carboxylate, but not the alternative structure in which the methyl group is replaced by an amine. This has important implications for future investigations into the biosynthesis of pyrrolysine, since radically different precursors would have to be envisioned, depending on the nature of the 4-substituent. CID spectra of intact pyrrolysyl-containing peptides will greatly facilitate labeling studies needed to ascertain and test paths of pyrrolysine biosynthesis. The mass of pyrrolysine will also be useful in mass spectrometry-based proteomic studies that may result in identification of new proteins containing this novel residue.

The presence of pyrrolysine in corrinoid-dependent methyltransferases specific for either TMA, DMA, or MMA suggests a conserved function for the single pyrrolysyl residue in these proteins. This function may employ the demonstrated chemical reactivity of the pyrrolysyl residue, which has been proposed to play a role in MMA-corrinoid protein methyl transfer. Structures of MtmB have been obtained in which the C-2 position of the pyrroline ring is modified with hydroxylamine, sulfite (8), and ammonia (1). These observations supported the existence of the reactive imine bond of pyrrolysine, which gives the residue a potential for electrophilic chemistry that is found in few proteins (28). A model was proposed for pyrrolysine function in binding, orienting, and activating MMA as a methyl donor to the Co(I) corrinoid cofactor of its cognate corrinoid protein, MtmC (1). Pyrrolysine is positioned in the bottom of a putative active site cleft in the TIM barrel of MtmB (1). The methyl-tetrahydrofolate:corrinoid methyltransferase domain of methionine synthase (MetH) (29) is a structural homolog of MtmB, and the active site of the methionine synthase domain and the pyrrolysine cleft of MtmB are superimposable. A substituent at the C-2 position of pyrrolysine, and the methyl group of methyltetrahydrofolate would occupy similar relative positions within their respective clefts, presumably at positions optimal for methyl transfer to their homologous cognate corrinoid proteins (30).

Like MMA, unionized DMA and TMA have lone electron pairs, which could interact with the imine bond of pyrrolysine in MtbB and MttB. The model of pyrrolysine binding of MMA as postulated for MtmB can be extrapolated to the reactions carried out by the TMA and DMA methyltransferases, which are essentially analogous to the reaction catalyzed by MtmB. Each of these proteins methylate a corrinoid protein that is homologous to the cognate corrinoid protein of MtmB. Alternative models are also possible that would exploit the electrophilicity of pyrrolysine; e.g. the demethylation product of TMA, DMA, or MMA could bind pyrrolysine, which could act to facilitate methyl transfer to Co(I). Currently, we are testing whether compounds that interact with some specificity for imine bonds modify the pyrrolysyl residue and act as inhibitors of the methylamine methyltransferases.

Our current results indicate that the nearly universal function of UAG as a stop codon must be overcome during the synthesis of not only the MMA methyltransferases but of all major methylamine methyltransferases of M. barkeri. MtmB, MtbB, and MttB are abundant cellular proteins, each being estimated at several percent of total soluble protein during growth on trimethylamine (6, 13, 14). Such abundance underscores the necessity for efficient translation of UAG with minimal termination that would be required to provide these levels of full-length methyltransferases. The limited energy available from methanogenesis (6) would be a strong driving force for evolution of a mechanism for suppressing UAG-directed termination during translation of these abundant proteins. UAG translation as pyrrolysine in MtmB does appear to be highly efficient, since cells possess little of the MtmB truncation product (7). The mechanism by which UAG-directed termination is circumvented and with subsequent efficient translation as pyrrolysine is currently unknown. A possible structural element, termed PYLIS, has been noted 3' of the UAG codon in mtmB transcripts, and this may act to increase translation of UAG as pyrrolysine (31). Whereas a variation of this element can be seen in mtmB1 and mttB transcripts, it is apparently lacking in mtbB transcripts (32). A broad comparison of all known examples of methylamine methyltransferase homologs with inframe amber codons indicates that the PYLIS element is not conserved among them (33). Any downstream cis-acting element that promotes UAG translation may therefore be said to have limited sequence or structural similarity. Since our current results establish that the DMA and TMA methyltransferases have in-frame amber codons translated as pyrrolysine, the genes encoding these proteins will be of utility in further investigations of the mechanisms that underlie UAG translation as pyrrolysine.

Pyrrolysine is unique in that it is the only noncanonical amino acid that is translationally inserted into proteins in a manner similar to the common set of 20 amino acids. Pyrrolysine has its own cognate aminoacyl-tRNA synthetase in PylS, which can charge tRNAPyl with chemically synthesized L-pyrrolysine (3), having the stereochemistry shown in Fig. 1, and with R representing CH3 (8). E. coli expressing the genes encoding the pyrrolysyl-tRNA synthetase and tRNAPyl incorporated exogenously supplied pyrrolysine into recombinant MtmB under the direction of a UAG codon (3). The mass of the residue found at the UAG-encoded position of the recombinant protein was 237.2 Da, within error limits of the mass observed here for pyrrolysyl residue in native MtmB, MtbB, or MttB directly isolated from the methanogen. These results suggest that the chemically synthesized form of pyrrolysine used in the experiments with E. coli is identical to that recognized by PylS in vivo in the methanogen. This result further supports the contention that pyrrolysine is the native substrate of PylS and that pyrrolysine is a cellular metabolite. A PylS substrate is present in the low molecular mass pool of M. barkeri (3). The mass of the native pyrrolysyl residue allows prediction of the mass of pyrrolysine as a free cellular intermediate as 255.16 Da, with the proviso that the amino acid is unchanged during incorporation into MtmB during protein synthesis. This proviso is completely consistent with the statistically equivalent masses of the UAG-encoded residue found in the three nonhomologous methylamine methyltransferases of M. barkeri.


   FOOTNOTES
 
* Purchase of the mass spectrometry equipment was supported by the Hayes Academic Investment Funds of the Ohio Board of Regents. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S4. Back

1 Supported by National Science Foundation Grant CHE-0316002. Back

2 To whom correspondence may be addressed: Mass Spectrometry and Proteomics Facility, 243 Fontana Labs, 116 W. 19th Ave., Columbus OH 43210. Tel.: 614-688-0521; Fax: 614-292-5955; E-mail: Green-Church.1{at}osu.edu.

3 Supported by Department of Energy Grant DE-FG0202-91ER200042 and National Science Foundation Grant MCB-9808914. To whom correspondence may be addressed: Dept. of Microbiology, Ohio State University, 484 West 12th Ave., Columbus, OH 43210. Tel.: 614-292-1578; Fax: 614-292-8120; E-mail: Krzycki.1{at}osu.edu.

4 The abbreviations used are: MMA, monomethylamine; MS, mass spectrometry; MS/MS, tandem mass spectrometry; DMA, dimethylamine; TMA, trimethylamine; HPLC, high performance liquid chromatography; LC, liquid chromatography; MALDI, matrix-assisted laser desorption/ionization; FTICR, Fourier transform ion cyclotron resonance; CID, collision-induced dissociation; MOPS, 4-morpholinepropanesulfonic acid. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Hao, B., Gong, W., Ferguson, T. K., James, C. M., Krzycki, J. A., and Chan, M. K. (2002) Science 296, 1462-1466[Abstract/Free Full Text]
  2. Srinivasan, G., James, C. M., and Krzycki, J. A. (2002) Science 296, 1459-1462[Abstract/Free Full Text]
  3. Blight, S. K., Larue, R. C., Mahapatra, A., Longstaff, D. G., Chang, E., Zhao, G., Kang, P. T., Green-Church, K. B., Chan, M., K., and Krzycki, J. A. (2004) Nature 431, 333-335[CrossRef][Medline] [Order article via Infotrieve]
  4. Atkins, J. F., and Gesteland, R. (2002) Science 296, 1409-1411[Abstract/Free Full Text]
  5. Burke, S. A., Lo, S. L., and Krzycki, J. A. (1998) J. Bacteriol. 180, 3432-3440[Abstract/Free Full Text]
  6. Burke, S. A., and Krzycki, J. A. (1997) J. Biol. Chem. 272, 16570-16577[Abstract/Free Full Text]
  7. James, C. M., Ferguson, T. K., Leykam, J. F., and Krzycki, J. A. (2001) J. Biol. Chem. 276, 34252-34258[Abstract/Free Full Text]
  8. Hao, B., Zhao, G., Kang, P. T., Soares, J. A., Ferguson, T. K., Gallucci, J., Krzycki, J. A., and Chan, M. K. (2004) Chem. Biol. 11, 1317-1324[CrossRef][Medline] [Order article via Infotrieve]
  9. Polycarpo, C., Ambrogelly, A., Berube, A., Winbush, S. M., McCloskey, J. A., Crain, P. F., Wood, J. L., and Soll, D. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 12450-12454[Abstract/Free Full Text]
  10. Theobald-Dietrich, A., Frugier, M., Giege, R., and Rudinger-Thirion, J. (2004) Nucleic Acids Res. 32, 1091-1096[Abstract/Free Full Text]
  11. Driscoll, D. M., and Copeland, P. R. (2003) Annu. Rev. Nutr. 23, 17-40[Medline] [Order article via Infotrieve]
  12. Commans, S., and Bock, A. (1999) FEMS Microbiol. Rev. 23, 335-351[CrossRef][Medline] [Order article via Infotrieve]
  13. Ferguson, D. J., Jr., and Krzycki, J. A. (1997) J. Bacteriol. 179, 846-852[Abstract/Free Full Text]
  14. Ferguson, D. J., Jr., Gorlatova, N., Grahame, D. A., and Krzycki, J. A. (2000) J. Biol. Chem. 275, 29053-29060[Abstract/Free Full Text]
  15. Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science 266, 1669-1674[Abstract/Free Full Text]
  16. Paul, L., Ferguson, D. J., and Krzycki, J. A. (2000) J. Bacteriol. 182, 2520-2529[Abstract/Free Full Text]
  17. Paul, L., and Krzycki, J. A. (1996) J. Bacteriol. 178, 6599-6607[Abstract/Free Full Text]
  18. Ferguson, D. J., Jr., Krzycki, J. A., and Grahame, D. A. (1996) J. Biol. Chem. 271, 5189-5194[Abstract/Free Full Text]
  19. Thauer, R. K. (1998) Microbiology 144, 2377-2406[Abstract/Free Full Text]
  20. Deppenmeier, U. (2004) J. Bioenerg. Biomembr. 36, 55-64[CrossRef][Medline] [Order article via Infotrieve]
  21. Galagan, J. E., Nusbaum, C., Roy, A., Endrizzi, M. G., Macdonald, P., FitzHugh, W., Calvo, S., Engels, R., Smirnov, S., Atnoor, D., Brown, A., Allen, N., Naylor, J., Stange-Thomann, N., DeArellano, K., Johnson, R., Linton, L., McEwan, P., McKernan, K., Talamas, J., Tirrell, A., Ye, W., Zimmer, A., Barber, R. D., Cann, I., Graham, D. E., Grahame, D. A., Guss, A. M., Hedderich, R., Ingram-Smith, C., Kuettner, H. C., Krzycki, J. A., Leigh, J. A., Li, W., Liu, J., Mukhopadhyay, B., Reeve, J. N., Smith, K., Springer, T. A., Umayam, L. A., White, O., White, R. H., Conway de Macario, E., Ferry, J. G., Jarrell, K. F., Jing, H., Macario, A. J., Paulsen, I., Pritchett, M., Sowers, K. R., Swanson, R. V., Zinder, S. H., Lander, E., Metcalf, W. W., and Birren, B. (2002) Genome Res. 12, 532-542[Abstract/Free Full Text]
  22. Deppenmeier, U., Johann, A., Hartsch, T., Merkl, R., Schmitz, R. A., Martinez-Arias, R., Henne, A., Wiezer, A., Bäumer, S., Jacobi, C., Brüggemann, H., Lienard, T., Christmann, A., Bömeke, M., Steckel, S., Bhattacharyya, A., Lykidis, A., Overbeek, R., Klenk, H.-P., Robert P. Gunsalus, R. P. H.-J., Fritz, H.-J., and Gottschalk, G. (2002) J. Mol. Microbiol. Biotechnol. 4, 453-461[Medline] [Order article via Infotrieve]
  23. Amber Goodchild, A., Saunders, N. F., Ertan, H., Raftery, M., Guilhaus, M., Curmi, P. M. G., and Cavicchioli, R. (2004) Mol. Microbiol. 53, 309-321[CrossRef][Medline] [Order article via Infotrieve]
  24. Krzycki, J. A., Mortenson, L. E., and Prince, R. C. (1989) J. Biol. Chem. 264, 7217-7221[Abstract/Free Full Text]
  25. Burke, S. A., and Krzycki, J. A. (1995) J. Bacteriol. 177, 4410-4416[Abstract/Free Full Text]
  26. Breaux, G. A., Green-Church, K. B., France, A., and Limbach, P. A. (2000) Anal. Chem. 72, 1169-1174[Medline] [Order article via Infotrieve]
  27. Russell, W. K., Park, Z.-Y., and Russell, D. H. (2001) Anal. Chem. 73, 2682-2685[Medline] [Order article via Infotrieve]
  28. Retey, J. (2003) Biochim. Biophys. Acta 1647, 179-184[Medline] [Order article via Infotrieve]
  29. Evans, J. C., Huddler, D. P., Hilgers, M. T., Romanchuk, G., Matthews, R. G., and Ludwig, M. L. (2004) Proc. Natl. Acad. U. S. A. 101, 3729-3736[Abstract/Free Full Text]
  30. Krzycki, J. A. (2004) Curr. Opin. Chem. Biol. 8, 484-491[CrossRef][Medline] [Order article via Infotrieve]
  31. Namy, O., Rousset, J. P., Napthine, S., and Brierley, I. (2004) Mol. Cell 13, 157-168[CrossRef][Medline] [Order article via Infotrieve]
  32. Paul, L. (2000) Analysis of the Genes Encoding the Enzymes Initiating Methanogenesis from Methlthiols, Trimethylamine, and Dimethylamine in Methanosarcina barkeri MS. Ph.D. thesis, Ohio State University, Columbus, OH
  33. Zhang, Y., V., P., Baranov, P. V., Atkins, J. F., Gladyshev, V. N. (2005) J. Biol. Chem. 280, 20740-20751[Abstract/Free Full Text]

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