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1.
C M James T K Ferguson J F Leykam J A Krzycki 《The Journal of biological chemistry》2001,276(36):34252-34258
Each of the genes encoding the methyltransferases initiating methanogenesis from trimethylamine, dimethylamine, or monomethylamine by various Methanosarcina species possesses one naturally occurring in-frame amber codon that does not appear to act as a translation stop during synthesis of the biochemically characterized methyltransferase. To investigate the means by which suppression of the amber codon within these genes occurs, MtmB, a methyltransferase initiating metabolism of monomethylamine, was examined. The C-terminal sequence of MtmB indicated that synthesis of this mtmB1 gene product did not cease at the internal amber codon, but at the following ochre codon. Antibody raised against MtmB revealed that Escherichia coli transformed with mtmB1 produced the amber termination product. The same antibody detected primarily a 50-kDa protein in Methanosarcina barkeri, which is the mass predicted for the amber readthrough product of the mtmB1 gene. Sequencing of peptide fragments from MtmB by Edman degradation and mass spectrometry revealed no change in the reading frame during mtmB1 expression. The amber codon position corresponded to a lysyl residue using either sequencing technique. The amber codon is thus read through during translation at apparently high efficiency and corresponds to lysine in tryptic fragments of MtmB even though canonical lysine codon usage is encountered in other Methanosarcina genes. 相似文献
2.
3.
Methylthiol:coenzyme M methyltransferase from Methanosarcina barkeri, an enzyme of methanogenesis from dimethylsulfide and methylmercaptopropionate. 总被引:1,自引:0,他引:1
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During growth on acetate, Methanosarcina barkeri expresses catabolic enzymes for other methanogenic substrates such as monomethylamine. The range of substrates used by cells grown on acetate was further explored, and it was found that cells grown on acetate also converted dimethylsulfide (DMS) and methylmercaptopropionate (MMPA) to methane. Cells or extracts of cells grown on trimethylamine or methanol did not utilize either DMS or MMPA. During growth on acetate, cultures demethylated MMPA, producing methane and mercaptopropionate. Extracts of acetate-grown cells possessed DMS- and MMPA-dependent coenzyme M (CoM) methylation activities. The activity peaks of CoM methylation with either DMS or MMPA coeluted upon gel permeation chromatography of extracts of acetate-grown cells consistent with an apparent molecular mass of 470 kDa. A 480-kDa corrinoid protein, previously demonstrated to be a CoM methylase but otherwise of unknown physiological function, was found to methylate CoM with either DMS or MMPA. MMPA was demethylated by the purified 480-kDa CoM methylase, consuming 1 mol of CoM and producing 1 mol of mercaptopropionate. DMS was demethylated by the purified protein, consuming 1 mol of CoM and producing 1 mol of methanethiol. The methylthiol:CoM methyltransferase reaction could be initiated only with the enzyme-bound corrinoid in the methylated state. CoM could demethylate, and DMS and MMPA could remethylate, the corrinoid cofactor. The monomethylamine corrinoid protein and the A isozyme of methylcobamide:CoM methyltransferase (proteins homologous to the two subunits comprising the 480-kDa CoM methylase) did not catalyze CoM methylation with methylated thiols. These results indicate that the 480-kDa corrinoid protein functions as a CoM methylase during methanogenesis from DMS or MMPA. 相似文献
4.
Gundllapalli S Ambrogelly A Umehara T Li D Polycarpo C Söll D 《FEBS letters》2008,582(23-24):3353-3358
Methanosarcina barkeri inserts pyrrolysine (Pyl) at an in-frame UAG codon in its monomethylamine methyltransferase gene. Pyrrolysyl-tRNA synthetase acylates Pyl onto tRNAPyl, the amber suppressor pyrrolysine Pyl tRNA. Here we show that M. barkeri Fusaro tRNAPyl can be misacylated with serine by the M. barkeri bacterial-type seryl-tRNA synthetase in vitro and in vivo in Escherichia coli. Compared to the M. barkeri Fusaro tRNA, the M. barkeri MS tRNAPyl contains two base changes; a G3:U70 pair, the known identity element for E. coli alanyl-tRNA synthetase (AlaRS). While M. barkeri MS tRNAPyl cannot be alanylated by E. coli AlaRS, mutation of the MS tRNAPyl A4:U69 pair into C4:G69 allows aminoacylation by E. coli AlaRS both in vitro and in vivo. 相似文献
5.
Krzycki JA 《Current opinion in chemical biology》2004,8(5):484-491
Methanogenesis from trimethylamine, dimethylamine or monomethylamine is initiated by a series of corrinoid-dependent methyltransferases. The non-homologous genes encoding the full-length methyltransferases each possess an in-frame UAG (amber) codon that does not terminate translation. The amber codon is decoded by a dedicated tRNA, and corresponds to the novel amino acid pyrrolysine in one of the methyltransferases, indicating pyrrolysine to be the 22nd genetically encoded amino acid. Pyrrolysine has the structure of lysine with the (epsilon)N in amide linkage with a pyrroline ring. The reactivity of the electrophilic imine bond is the basis for the proposed function of pyrrolysine in activating and optimally orienting methylamine for methyl transfer to the cobalt ion of a cognate corrinoid protein. This reaction is essential for methane formation from methylamines, and may underlie the retention of pyrrolysine in the genetic code of methanogens. 相似文献
6.
7.
In the methanogenic archae Methanosarcina barkeri, insertion of pyrrolysine, the 22nd amino acid, results from the decoding of an amber UAG codon in the mRNA of monomethylamine methyltransferases (MtmB). Sequence comparisons combined with structural enzymatic and chemical probing on M. barkeri MtmB1 mRNA demonstrate the presence of a hairpin motif located immediately after the redefined UAG codon. This structure of 86 nucleotides differs slightly from a proposal given in the literature and comprises four successive stems separated by three internal loops and closed by a large apical loop. Sequence alignments of MtmB mRNAs of different Methanosarcinacae reveal a conservation of the motif in both sequence and folding levels. The functional role of this motif as a signal leading to pyrrolysine insertion is discussed. 相似文献
8.
9.
10.
Polycarpo C Ambrogelly A Ruan B Tumbula-Hansen D Ataide SF Ishitani R Yokoyama S Nureki O Ibba M Söll D 《Molecular cell》2003,12(2):287-294
Monomethylamine methyltransferase of the archaeon Methanosarcina barkeri contains a rare amino acid, pyrrolysine, encoded by the termination codon UAG. Translation of this UAG requires the aminoacylation of the corresponding amber suppressor tRNAPyl. Previous studies reported that tRNAPyl could be aminoacylated by the synthetase-like protein PylS. We now show that tRNAPyl is efficiently aminoacylated in the presence of both the class I LysRS and class II LysRS of M. barkeri, but not by either enzyme acting alone or by PylS. In vitro studies show that both the class I and II LysRS enzymes must bind tRNAPyl in order for the aminoacylation reaction to proceed. Structural modeling and selective inhibition experiments indicate that the class I and II LysRSs form a ternary complex with tRNAPyl, with the aminoacylation activity residing in the class II enzyme. 相似文献
11.
Coenzyme M methylase activity of the 480-kilodalton corrinoid protein from Methanosarcina barkeri. 总被引:1,自引:0,他引:1
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Activity staining of extracts of Methanosarcina barkeri electrophoresed in polyacrylamide gels revealed an additional methylcobalamin:coenzyme M (methylcobalamin:CoM) methyltransferase present in cells grown on acetate but not in those grown on trimethylamine. This methyltransferase is the 480-kDa corrinoid protein previously identified by its methylation following inhibition of methyl-CoM reductase in otherwise methanogenic cell extracts. The methylcobalamin:CoM methyltransferase activity of the purified 480-kDa protein increased from 0.4 to 3.8 micromol/min/mg after incubation with sodium dodecyl sulfate (SDS). Following SDS-polyacrylamide gel electrophoresis analysis of unheated protein samples, a polypeptide with an apparent molecular mass of 48 kDa which possessed methylcobalamin:CoM methyltransferase activity was detected. This polypeptide migrated with an apparent mass of 41 kDa when the 480-kDa protein was heated before electrophoresis, indicating that the alpha subunit is responsible for the activity. The N-terminal sequence of this subunit was 47% similar to the N termini of the A and M isozymes of methylcobalamin:CoM methyltransferase (methyltransferase II). The endogenous methylated corrinoid bound to the beta subunit of the 480-kDa protein could be demethylated by CoM, but not by homocysteine or dithiothreitol, resulting in a Co(I) corrinoid. The Co(I) corrinoid could be remethylated by methyl iodide, and the protein catalyzed a methyl iodide:CoM transmethylation reaction at a rate of 2.3 micromol/min/mg. Methyl-CoM was stoichiometrically produced from CoM, as demonstrated by high-pressure liquid chromatography with indirect photometric detection. Two thiols, 2-mercaptoethanol and mercapto-2-propanol, were poorer substrates than CoM, while several others tested (including 3-mercaptopropanesulfonate) did not serve as methyl acceptors. These data indicate that the 480-kDa corrinoid protein is composed of a novel isozyme of methyltransferase II which remains firmly bound to a corrinoid cofactor binding subunit during isolation. 相似文献
12.
13.
Reconstitution of trimethylamine-dependent coenzyme M methylation with the trimethylamine corrinoid protein and the isozymes of methyltransferase II from Methanosarcina barkeri. 总被引:4,自引:0,他引:4
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Reconstitution of trimethylamine-dependent coenzyme M (CoM) methylation was achieved with three purified polypeptides. Two of these polypeptides copurified as a trimethylamine methyl transfer (TMA-MT) activity detected by stimulation of the TMA:CoM methyl transfer reaction in cell extracts. The purified TMA-MT fraction stimulated the rate of methyl-CoM formation sevenfold, up to 1.7 micromol/min/mg of TMA-MT protein. The TMA-MT polypeptides had molecular masses of 52 and 26 kDa. Gel permeation of the TMA-MT fraction demonstrated that the 52-kDa polypeptide eluted with an apparent molecular mass of 280 kDa. The 26-kDa protein eluted primarily as a monomer, but some 26-kDa polypeptides also eluted with the 280-kDa peak, indicating that the two proteins weakly associate. The two polypeptides could be completely separated using gel permeation in the presence of sodium dodecyl sulfate. The corrinoid remained associated with the 26-kDa polypeptide at a molar ratio of 1.1 corrin/26-kDa polypeptide. This polypeptide was therefore designated the TMA corrinoid protein, or TCP. The TMA-MT polypeptides, when supplemented with purified methylcorrinoid:CoM methyltransferase (MT2), could effect the demethylation of TMA with the subsequent methylation of CoM and the production of dimethylamine at specific activities of up to 600 nmol/min/mg of TMA-MT protein. Neither dimethylamine nor monomethylamine served as the substrate, and the activity required Ti(III) citrate and methyl viologen. TMA-MT could interact with either isozyme of MT2 but had the greatest affinity for the A isozyme. These results suggest that TCP is uniquely involved in TMA-dependent methanogenesis, that this corrinoid protein is methylated by the substrate and demethylated by either isozyme of MT2, and that the predominant isozyme of MT2 found in TMA-grown cells is the favored participant in the TMA:CoM methyl transfer reaction. 相似文献
14.
Monomethylamine methyltransferase of the archaebacterium Methanosarcina barkeri contains a novel amino acid, pyrrolysine, encoded by the termination codon UAG. Initial studies suggest that pyrrolysine may be co-translationally inserted during protein synthesis, probably by a mechanism analogous to that operating during selenocysteine incorporation. 相似文献
15.
16.
Tsuneo Ferguson Jitesh A. Soares Tanja Lienard Gerhard Gottschalk Joseph A. Krzycki 《The Journal of biological chemistry》2009,284(4):2285-2295
Archaeal methane formation from methylamines is initiated by distinct
methyltransferases with specificity for monomethylamine, dimethylamine, or
trimethylamine. Each methylamine methyltransferase methylates a cognate
corrinoid protein, which is subsequently demethylated by a second
methyltransferase to form methyl-coenzyme M, the direct methane precursor.
Methylation of the corrinoid protein requires reduction of the central cobalt
to the highly reducing and nucleophilic Co(I) state. RamA, a 60-kDa monomeric
iron-sulfur protein, was isolated from Methanosarcina barkeri and is
required for in vitro ATP-dependent reductive activation of
methylamine:CoM methyl transfer from all three methylamines. In the absence of
the methyltransferases, highly purified RamA was shown to mediate the
ATP-dependent reductive activation of Co(II) corrinoid to the Co(I) state for
the monomethylamine corrinoid protein, MtmC. The ramA gene is located
near a cluster of genes required for monomethylamine methyltransferase
activity, including MtbA, the methylamine-specific CoM methylase and the
pyl operon required for co-translational insertion of pyrrolysine
into the active site of methylamine methyltransferases. RamA possesses a
C-terminal ferredoxin-like domain capable of binding two tetranuclear
iron-sulfur proteins. Mutliple ramA homologs were identified in
genomes of methanogenic Archaea, often encoded near methyltrophic
methyltransferase genes. RamA homologs are also encoded in a diverse selection
of bacterial genomes, often located near genes for corrinoid-dependent
methyltransferases. These results suggest that RamA mediates reductive
activation of corrinoid proteins and that it is the first functional archetype
of COG3894, a family of redox proteins of unknown function.Most methanogenic Archaea are capable of producing methane only from carbon
dioxide. The Methanosarcinaceae are a notable exception as representatives are
capable of methylotrophic methanogenesis from methylated amines, methylated
thiols, or methanol. Methanogenesis from these substrates requires methylation
of 2-mercaptoethanesulfonic acid (coenzyme M or CoM) that is subsequently used
by methylreductase to generate methane and a mixed disulfide whose reduction
leads to energy conservation
(1–4).Methylation of CoM with trimethylamine
(TMA),4 dimethylamine
(DMA), or monomethylamine (MMA) is initiated by three distinct
methyltransferases that methylate cognate corrinoid-binding proteins
(3). MtmB, the MMA
methyltransferase, specifically methylates cognate corrinoid protein, MtmC,
with MMA (see Fig. 1)
(5,
6). The DMA methyltransferase,
MtbB, and its cognate corrinoid protein, MtbC, interact specifically to
demethylate DMA (7,
8). TMA is demethylated by the
TMA methyltransferase (MttB) in conjunction with the TMA corrinoid protein
(MttC) (8,
9). Each of the methylated
corrinoid proteins is a substrate for a methylcobamide:CoM methyltransferase,
MtbA, which produces methyl-CoM
(10–12).Open in a separate windowFIGURE 1.MMA:CoM methyl transfer. A schematic of the reactions catalyzed by
MtmB, MtmC, and MtbA is shown that emphasizes the key role of MtmC in the
catalytic cycle of both methyltransferases. Oxidation to Co(II)-MtmC of the
supernucleophilic Co(I)-MtmC catalytic intermediate inactivates methyl
transfer from MMA to the thiolate of coenzyme M (HSCoM). In
vitro reduction of the Co(II)-MtmC with either methyl viologen reduced to
the neutral species or with RamA in an ATP-dependent reaction can regenerate
the Co(I) species. In either case in vitro Ti(III)-citrate is the
ultimate source of reducing power.CoM methylation with methanol requires the methyltransferase MtaB and the
corrinoid protein MtaC, which is then demethylated by another
methylcobamide:CoM methyltransferase, MtaA
(13–15).
The methylation of CoM with methylated thiols such as dimethyl sulfide in
Methanosarcina barkeri is catalyzed by a corrinoid protein that is
methylated by dimethyl sulfide and demethylated by CoM, but in this case an
associated CoM methylase carries out both methylation reactions
(16).In bacteria, analogous methyltransferase systems relying on small corrinoid
proteins are used to achieve methylation of tetrahydrofolate. In
Methylobacterium spp., CmuA, a single methyltransferase with a
corrinoid binding domain, along with a separate pterin methylase, effect the
methylation of tetrahydrofolate with chloromethane
(17,
18). In Acetobacterium
dehalogenans and Moorella thermoacetica various three-component
systems exist for specific demethylation of different phenylmethyl ethers,
such as vanillate (19) and
veratrol (20), again for the
methylation of tetrahydrofolate. Sequencing of the genes encoding the
corrinoid proteins central to the archaeal and bacterial methylotrophic
pathways revealed they are close homologs. Furthermore, genes predicted to
encode such corrinoid proteins and pterin methyltransferases are widespread in
bacterial genomes, often without demonstrated metabolic function. All of these
corrinoid proteins are similar to the well characterized cobalamin binding
domain of methionine synthase
(21,
22).In contrast, the TMA, DMA, MMA, and methanol methyltransferases are not
homologous proteins. The methylamine methyltransferases do share the common
distinction of having in-frame amber codons
(6,
8) within their encoding genes
that corresponds to the genetically encoded amino acid pyrrolysine
(23–25).
Pyrrolysine has been proposed to act in presenting a methylammonium adduct to
the central cobalt ion of the corrinoid protein for methyl transfer
(3,
23,
26). However, nucleophilic
attack on a methyl donor requires the central cobalt ion of a corrinoid
cofactor is in the nucleophilic Co(I) state rather than the inactive Co(II)
state (27). Subsequent
demethylation of the methyl-Co(III) corrinoid cofactor regenerates the
nucleophilic Co(I) cofactor. The Co(I)/Co(II) in the cobalamin binding domain
of methionine synthase has an Em value of -525 mV at pH 7.5
(28). It is likely to be
similarly low in the homologous methyltrophic corrinoid proteins. These low
redox potentials make the corrinoid cofactor subject to adventitious oxidation
to the inactive Co(II) state (Fig.
1).During isolation, these corrinoid proteins are usually recovered in a
mixture of Co(II) or hydroxy-Co(III) states. For in vitro studies,
chemical reduction can maintain the corrinoid protein in the active Co(I)
form. The methanol:CoM or the phenylmethyl ether:tetrahydrofolate
methyltransferase systems can be activated in vitro by the addition
of Ti(III) alone as an artificial reductant
(14,
19). In contrast, activation
of the methylamine corrinoid proteins further requires the addition of methyl
viologen as a redox mediator. Ti(III) reduces methyl viologen to the extremely
low potential neutral species. In vitro activation with these agents
does not require ATP (5,
7,
9).Cellular mechanisms also exist to achieve the reductive activation of
corrinoid cofactors in methyltransferase systems. Activation of human
methionine synthase involves reduction of the co(II)balamin by methionine
synthase reductase (29),
whereas the Escherichia coli enzyme requires flavodoxin
(30). The endergonic reduction
is coupled with the exergonic methylation of the corrinoid with
S-adenosylmethionine
(27). An activation system
exists in cellular extracts of A. dehalogenans that can activate the
veratrol:tetrahydrofolate three-component system and catalyze the direct
reduction of the veratrol-specific corrinoid protein to the Co(I) state;
however, the activating protein has not been purified
(31).For the methanogen methylamine and methanol methyltransferase systems, an
activation process is readily detectable in cell extracts that is ATP- and
hydrogen-dependent (32,
33). Daas et al.
(34,
35) examined the activation of
the methanol methyltransferase system in M. barkeri and purified in
low yield a methyltransferase activation protein (MAP) which in the presence
of a preparation of hydrogenase and uncharacterized proteins was required for
ATP-dependent reductive activation of methanol:CoM methyl transfer. MAP was
found to be a heterodimeric protein without a UV-visible detectable prosthetic
group. Unfortunately, no protein sequence has been reported for MAP, leaving
the identity of the gene in question. The same MAP protein was also suggested
to activate methylamine:CoM methyl transfer, but this suggestion was based on
results with crude protein fractions containing many cellular proteins other
than MAP (36).Here we report of the identification and purification to near-homogeneity
of RamA (reductive activation of
methyltransfer, amines), a protein mediating activation
of methylamine:CoM methyl transfer in a highly purified system
(Fig. 1). Quite unlike MAP,
which was reported to lack prosthetic groups, RamA is an iron-sulfur protein
that can catalyze reduction of a corrinoid protein such as MtmC to the Co(I)
state in an ATP-dependent reaction (Fig.
1). Peptide mapping of RamA allowed identification of the gene
encoding RamA and its homologs in the genomes of Methanosarcina spp.
RamA belongs to COG3894, a group of uncharacterized metal-binding proteins
found in a number of genomes. RamA, thus, provides a functional example for a
family of proteins widespread among bacteria and Archaea whose physiological
role had been largely unknown. 相似文献
17.
吡咯赖氨酸在产甲烷菌的甲胺甲基转移酶中发现,是目前已知的第 22 种参与蛋白质生物合成的氨基酸,与标准氨基酸不同的是,它由终止密码子 UAG 的有义编码形成 . 与之对应的在产甲烷菌中也含有特异的吡咯赖氨酰 -tRNA 合成酶 (PylRS) 和吡咯赖氨酸 tRNA (tRNAPyl). tRNAPyl具有不同于经典 tRNA 的特殊结构 . 产甲烷菌通过直接途径和间接途径这两种途径生成吡咯赖氨酰 -tRNAPyl(Pyl-tRNAPyl) ,它还可能通过 mRNA 上的特殊结构以及其他还未发现的机制,控制 UAG 编码成为终止密码子或者吡咯赖氨酸 . 比较了吡咯赖氨酸与另一种非标准氨基酸,第 21 种氨基酸———硒代半胱氨酸的相似点与不同点 . 相似文献
18.
Krzycki JA 《Current opinion in microbiology》2005,8(6):706-712
Pyrrolysine is an amino acid encoded by the amber codon in genes required for methylamine utilization by members of the Methanosarcinaceae. Pyrrolysine and selenocysteine share the distinction of being the only two non-canonical amino acids that have entered natural genetic codes. Recent experiments have shown that encoding of pyrrolysine, unlike that of selenocysteine, also shares an important trait of the original set of twenty amino acids. UAG is translated as pyrrolysine with the participation of a dedicated aminoacyl-tRNA synthetase. Expression of the genes encoding the pyrrolysyl-tRNA synthetase and its cognate tRNA is sufficient to add pyrrolysine to the genetic code of a recombinant organism. Thus, the recruitment of pyrrolysine into the genetic code involved evolution of the first non-canonical aminoacyl-tRNA synthetase and cognate tRNA to be described from nature. 相似文献
19.
Summary A spontaneous mutant was isolated that harbors a weak suppressing activity towards a UAG mutation, together with an inability to grow at 43° C in rich medium. The mutation is shown to be associated with an increased misreading of UAG at certain codon contexts and UAA. UGA, missense or frameshift mutations do not appear to be misread to a similar extent. The mutation gives an increased efficiency to several amber tRNA suppressors with-out increasing their ambiguity towards UAA. The ochre suppressors SuB and Su5 are stimulated in their reading of both UAG and UAA with preference for UAG. An opal suppressor is not affected. The effect of the mutation on the efficiency of amber and ochre suppressors is dependent on the codon context of the nonsense codon.The mutated gene (uar) has been mapped and found to be recessive both with respect to suppressor-enhancing ability as well as for temperature sensitivity. The phenotype is partly suppressed by the ochre suppressor SuC. It is suggested that uar codes for a protein, which is involved in translational termination at UAG and UAA stop codons. 相似文献
20.
Biochemical evidence suggests that methanol catabolism in Methanosarcina species requires the concerted effort of methanol:5-hydroxybenzimidazolylcobamide methyltransferase (MtaB), a corrinoid-containing methyl-accepting protein (MtaC) and Co-methyl-5-hydroxybenzimidazolylcobamide:2-mercapto-ethanesulphonic acid methyltransferase (MtaA). Here we show that Methanosarcina acetivorans possesses three operons encoding putative methanol-specific MtaB and corrinoid proteins: mtaCB1, mtaCB2 and mtaCB3. Deletion mutants lacking the three operons, in all possible combinations, were constructed and characterized. Strains deleted for any two of the operons grew on methanol, whereas strains lacking all three did not. Therefore, each operon encodes a bona fide methanol-utilizing MtaB/corrinoid protein pair. Most of the mutants were similar to the wild-type strain, with the exception of the DeltamtaCB1 DeltamtaCB2 double mutant, which grew more slowly and had reduced cell yields on methanol medium. However, all mutants displayed significantly longer lag times when switching from growth on trimethylamine to growth on methanol. This indicates that all three operons are required for wild-type growth on methanol and suggests that each operon has a distinct role in the metabolism of this substrate. The combined methanol:CoM methyltransferase activity of strains carrying only mtaCB1 was twofold higher than strains carrying only mtaCB2 and fourfold higher than strains carrying only mtaCB3. Interestingly, the presence of the mtaCB2 and mtaCB3 operons, in addition to the mtaCB1 operon, did not increase the overall methyltransferase activity, suggesting that these strains may be limited by MtaA availability. All deletion mutants were unaffected with respect to growth on trimethylamine and acetate corroborating biochemical evidence indicating that each methanogenic substrate has specific methyltransfer enzymes. 相似文献