Enzymes
UniProtKB help_outline | 187 proteins |
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Namehelp_outline
adenosine2503 in 23S rRNA
Identifier
RHEA-COMP:10152
Reactive part
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- Name help_outline AMP residue Identifier CHEBI:74411 Charge -1 Formula C10H11N5O6P Positionhelp_outline 2503 SMILEShelp_outline NC1=NC=NC2=C1N=CN2[C@@H]3O[C@H](COP(=O)(*)[O-])[C@@H](O*)[C@H]3O 2D coordinates Mol file for the small molecule Search links Involved in 40 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
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Namehelp_outline
reduced [2Fe-2S]-[ferredoxin]
Identifier
RHEA-COMP:10001
Reactive part
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- Name help_outline [2Fe-2S]1+ Identifier CHEBI:33738 Charge 1 Formula Fe2S2 InChIKeyhelp_outline MAGIRAZQQVQNKP-UHFFFAOYSA-N SMILEShelp_outline S1[Fe]S[Fe+]1 2D coordinates Mol file for the small molecule Search links Involved in 238 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline S-adenosyl-L-methionine Identifier CHEBI:59789 Charge 1 Formula C15H23N6O5S InChIKeyhelp_outline MEFKEPWMEQBLKI-AIRLBKTGSA-O SMILEShelp_outline C[S+](CC[C@H]([NH3+])C([O-])=O)C[C@H]1O[C@H]([C@H](O)[C@@H]1O)n1cnc2c(N)ncnc12 2D coordinates Mol file for the small molecule Search links Involved in 868 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline 5'-deoxyadenosine Identifier CHEBI:17319 (CAS: 4754-39-6) help_outline Charge 0 Formula C10H13N5O3 InChIKeyhelp_outline XGYIMTFOTBMPFP-KQYNXXCUSA-N SMILEShelp_outline C[C@H]1O[C@H]([C@H](O)[C@@H]1O)n1cnc2c(N)ncnc12 2D coordinates Mol file for the small molecule Search links Involved in 69 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
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Namehelp_outline
8-methyladenosine2503 in 23S rRNA
Identifier
RHEA-COMP:10153
Reactive part
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- Name help_outline 8-methyladenosine 5'-phosphate residue Identifier CHEBI:74543 Charge -1 Formula C11H13N5O6P Positionhelp_outline 2503 SMILEShelp_outline C1(=NC=NC2=C1N=C(N2[C@@H]3O[C@H](COP(=O)(*)[O-])[C@@H](O*)[C@H]3O)C)N 2D coordinates Mol file for the small molecule Search links Involved in 1 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline L-methionine Identifier CHEBI:57844 Charge 0 Formula C5H11NO2S InChIKeyhelp_outline FFEARJCKVFRZRR-BYPYZUCNSA-N SMILEShelp_outline CSCC[C@H]([NH3+])C([O-])=O 2D coordinates Mol file for the small molecule Search links Involved in 121 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
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Namehelp_outline
oxidized [2Fe-2S]-[ferredoxin]
Identifier
RHEA-COMP:10000
Reactive part
help_outline
- Name help_outline [2Fe-2S]2+ Identifier CHEBI:33737 Charge 2 Formula Fe2S2 InChIKeyhelp_outline XSOVBBGAMBLACL-UHFFFAOYSA-N SMILEShelp_outline S1[Fe+]S[Fe+]1 2D coordinates Mol file for the small molecule Search links Involved in 238 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline S-adenosyl-L-homocysteine Identifier CHEBI:57856 Charge 0 Formula C14H20N6O5S InChIKeyhelp_outline ZJUKTBDSGOFHSH-WFMPWKQPSA-N SMILEShelp_outline Nc1ncnc2n(cnc12)[C@@H]1O[C@H](CSCC[C@H]([NH3+])C([O-])=O)[C@@H](O)[C@H]1O 2D coordinates Mol file for the small molecule Search links Involved in 792 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Cross-references
RHEA:42632 | RHEA:42633 | RHEA:42634 | RHEA:42635 | |
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Publications
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RNA methylation by radical SAM enzymes RlmN and Cfr proceeds via methylene transfer and hydride shift.
Yan F., Fujimori D.G.
RlmN and Cfr are Radical SAM enzymes that modify a single adenosine nucleotide--A2503--in 23S ribosomal RNA. This nucleotide is positioned within the peptidyl transferase center of the ribosome, which is a target of numerous antibiotics. An unusual feature of these enzymes is their ability to carr ... >> More
RlmN and Cfr are Radical SAM enzymes that modify a single adenosine nucleotide--A2503--in 23S ribosomal RNA. This nucleotide is positioned within the peptidyl transferase center of the ribosome, which is a target of numerous antibiotics. An unusual feature of these enzymes is their ability to carry out methylation of amidine carbons of the adenosine substrate. To gain insight into the mechanism of methylation catalyzed by RlmN and Cfr, deuterium labeling experiments were carried out. These experiments demonstrate that the newly introduced methyl group is assembled from an S-adenosyl-L-methionine (SAM)-derived methylene fragment and a hydrogen atom that had migrated from the substrate amidine carbon. Rather than activating the adenosine nucleotide of the substrate by hydrogen atom abstraction from an amidine carbon, the 5'-deoxyadenosyl radical abstracts hydrogen from the second equivalent of SAM to form the SAM-derived radical cation. This species, or its corresponding sulfur ylide, subsequently adds into the substrate, initiating hydride shift and S-adenosylhomocysteine elimination to complete the formation of the methyl group. These findings indicate that rather than acting as methyltransferases, RlmN and Cfr are methyl synthases. Together with the previously described 5'-deoxyadenosyl and 3-amino-3-carboxypropyl radicals, these findings demonstrate that all three carbon atoms attached to the sulfonium center in SAM can serve as precursors to carbon-derived radicals in enzymatic reactions. << Less
Proc Natl Acad Sci U S A 108:3930-3934(2011) [PubMed] [EuropePMC]
This publication is cited by 2 other entries.
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A substrate radical intermediate in catalysis by the antibiotic resistance protein Cfr.
Grove T.L., Livada J., Schwalm E.L., Green M.T., Booker S.J., Silakov A.
Cfr-dependent methylation of C8 of A2503 in 23S ribosomal RNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a ... >> More
Cfr-dependent methylation of C8 of A2503 in 23S ribosomal RNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine enzyme, is the addition of a methylene radical, generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine, onto C8 of A2503 to form a protein-nucleic acid crosslinked species containing an unpaired electron. Herein we use continuous-wave and pulsed EPR techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid freeze-quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product. << Less
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RlmN and Cfr are radical SAM enzymes involved in methylation of ribosomal RNA.
Yan F., LaMarre J.M., Rohrich R., Wiesner J., Jomaa H., Mankin A.S., Fujimori D.G.
Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome's function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase ... >> More
Posttranscriptional modifications of ribosomal RNA (rRNA) nucleotides are a common mechanism of modulating the ribosome's function and conferring bacterial resistance to ribosome-targeting antibiotics. One such modification is methylation of an adenosine nucleotide within the peptidyl transferase center of the ribosome mediated by the endogenous methyltransferase RlmN and its evolutionarily related resistance enzyme Cfr. These methyltransferases catalyze methyl transfer to aromatic carbon atoms of the adenosine within a complex 23S rRNA substrate to form the 2,8-dimethylated product. RlmN and Cfr are members of the Radical SAM superfamily and contain the characteristic cysteine-rich CX(3)CX(2)C motif. We demonstrate that both enzymes are capable of accommodating the requisite [4Fe-4S] cluster. S-Adenosylmethionine (SAM) is both the methyl donor and the source of a 5'-deoxyadenosyl radical, which activates the substrate for methylation. Detailed analyses of the rRNA requirements show that the enzymes can utilize protein-free 23S rRNA as a substrate, but not the fully assembled large ribosomal subunit, suggesting that the methylations take place during the assembly of the ribosome. The key recognition elements in the 23S rRNA are helices 90-92 and the adjacent single stranded RNA that encompasses A2503. To our knowledge, this study represents the first in vitro description of a methyl transfer catalyzed by a member of the Radical SAM superfamily, and it expands the catalytic repertoire of this diverse enzyme class. Furthermore, by providing information on both the timing of methylation and its substrate requirements, our findings have important implications for the functional consequences of Cfr-mediated modification of rRNA in the acquisition of antibiotic resistance. << Less
J Am Chem Soc 132:3953-3964(2010) [PubMed] [EuropePMC]
This publication is cited by 2 other entries.
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A radically different mechanism for S-adenosylmethionine-dependent methyltransferases.
Grove T.L., Benner J.S., Radle M.I., Ahlum J.H., Landgraf B.J., Krebs C., Booker S.J.
Methylation of small molecules and macromolecules is crucial in metabolism, cell signaling, and epigenetic programming and is most often achieved by S-adenosylmethionine (SAM)-dependent methyltransferases. Most employ an S(N)2 mechanism to methylate nucleophilic sites on their substrates, but rece ... >> More
Methylation of small molecules and macromolecules is crucial in metabolism, cell signaling, and epigenetic programming and is most often achieved by S-adenosylmethionine (SAM)-dependent methyltransferases. Most employ an S(N)2 mechanism to methylate nucleophilic sites on their substrates, but recently, radical SAM enzymes have been identified that methylate carbon atoms that are not inherently nucleophilic via the intermediacy of a 5'-deoxyadenosyl 5'-radical. We have determined the mechanisms of two such reactions targeting the sp(2)-hybridized carbons at positions 2 and 8 of adenosine 2503 in 23S ribosomal RNA, catalyzed by RlmN and Cfr, respectively. In neither case is a methyl group transferred directly from SAM to the RNA; rather, both reactions proceed by a ping-pong mechanism involving intermediate methylation of a conserved cysteine residue. << Less
Science 332:604-607(2011) [PubMed] [EuropePMC]
This publication is cited by 2 other entries.
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Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.
McCusker K.P., Medzihradszky K.F., Shiver A.L., Nichols R.J., Yan F., Maltby D.A., Gross C.A., Fujimori D.G.
The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokary ... >> More
The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr use a shared radical-mediated mechanism. In addition to the radical SAM CX(3)CX(2)C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function, putatively to form (cysteine 355 in RlmN) and resolve (cysteine 118 in RlmN) a covalent intermediate needed to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further clarify the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species between RlmN and its rRNA substrate and well as the roles of the conserved cysteine residues in catalysis. << Less
J Am Chem Soc 134:18074-18081(2012) [PubMed] [EuropePMC]
This publication is cited by 2 other entries.
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Characterization of a cross-linked protein-nucleic acid substrate radical in the reaction catalyzed by RlmN.
Silakov A., Grove T.L., Radle M.I., Bauerle M.R., Green M.T., Rosenzweig A.C., Boal A.K., Booker S.J.
RlmN and Cfr are methyltransferases/methylsynthases that belong to the radical S-adenosylmethionine superfamily of enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of 23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide, and will subsequently catalyze C2 methyl ... >> More
RlmN and Cfr are methyltransferases/methylsynthases that belong to the radical S-adenosylmethionine superfamily of enzymes. RlmN catalyzes C2 methylation of adenosine 2503 (A2503) of 23S rRNA, while Cfr catalyzes C8 methylation of the exact same nucleotide, and will subsequently catalyze C2 methylation if the site is unmethylated. A key feature of the unusual mechanisms of catalysis proposed for these enzymes is the attack of a methylene radical, derived from a methylcysteine residue, onto the carbon center undergoing methylation to generate a paramagnetic protein-nucleic acid cross-linked species. This species has been thoroughly characterized during Cfr-dependent C8 methylation, but does not accumulate to detectible levels in RlmN-dependent C2 methylation. Herein, we show that inactive C118S/A variants of RlmN accumulate a substrate-derived paramagnetic species. Characterization of this species by electron paramagnetic resonance spectroscopy in concert with strategic isotopic labeling shows that the radical is delocalized throughout the adenine ring of A2503, although predominant spin density is on N1 and N3. Moreover, (13)C hyperfine interactions between the radical and the methylene carbon of the formerly [methyl-(13)C]Cys355 residue show that the radical species exists in a covalent cross-link between the protein and the nucleic acid substrate. X-ray structures of RlmN C118A show that, in the presence of SAM, the substitution does not alter the active site structure compared to that of the wild-type enzyme. Together, these findings have new mechanistic implications for the role(s) of C118 and its counterpart in Cfr (C105) in catalysis, and suggest involvement of the residue in resolution of the cross-linked species via a radical mediated process. << Less
J. Am. Chem. Soc. 136:8221-8228(2014) [PubMed] [EuropePMC]
This publication is cited by 2 other entries.
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Structural basis for methyl transfer by a radical SAM enzyme.
Boal A.K., Grove T.L., McLaughlin M.I., Yennawar N.H., Booker S.J., Rosenzweig A.C.
The radical S-adenosyl-L-methionine (SAM) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys(355)) by SAM. Methyl transfer to ... >> More
The radical S-adenosyl-L-methionine (SAM) enzymes RlmN and Cfr methylate 23S ribosomal RNA, modifying the C2 or C8 position of adenosine 2503. The methyl groups are installed by a two-step sequence involving initial methylation of a conserved Cys residue (RlmN Cys(355)) by SAM. Methyl transfer to the substrate requires reductive cleavage of a second equivalent of SAM. Crystal structures of RlmN and RlmN with SAM show that a single molecule of SAM coordinates the [4Fe-4S] cluster. Residue Cys(355) is S-methylated and located proximal to the SAM methyl group, suggesting the SAM that is involved in the initial methyl transfer binds at the same site. Thus, RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site. << Less
Science 332:1089-1092(2011) [PubMed] [EuropePMC]
This publication is cited by 2 other entries.
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Identification of 8-methyladenosine as the modification catalyzed by the radical SAM methyltransferase Cfr that confers antibiotic resistance in bacteria.
Giessing A.M.B., Jensen S.S., Rasmussen A., Hansen L.H., Gondela A., Long K., Vester B., Kirpekar F.
The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an ad ... >> More
The Cfr methyltransferase confers combined resistance to five different classes of antibiotics that bind to the peptidyl transferase center of bacterial ribosomes. The Cfr-mediated modification has previously been shown to occur on nucleotide A2503 of 23S rRNA and has a mass corresponding to an additional methyl group, but its specific identity and position remained to be elucidated. A novel tandem mass spectrometry approach has been developed to further characterize the Cfr-catalyzed modification. Comparison of nucleoside fragmentation patterns of A2503 from Escherichia coli cfr+ and cfr-strains with those of a chemically synthesized nucleoside standard shows that Cfr catalyzes formation of 8-methyladenosine. In addition, analysis of RNA derived from E. coli strains lacking the m(2)A2503 methyltransferase reveals that Cfr also has the ability to catalyze methylation at position 2 to form 2,8-dimethyladenosine. The mutation of single conserved cysteine residues in the radical SAM motif CxxxCxxC of Cfr abolishes its activity, lending support to the notion that the Cfr modification reaction occurs via a radical-based mechanism. Antibiotic susceptibility data confirm that the antibiotic resistance conferred by Cfr is provided by methylation at the 8 position and is independent of methylation at the 2 position of A2503. This investigation is, to our knowledge, the first instance where the 8-methyladenosine modification has been described in natural RNA molecules. << Less
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Insights into the structure, function and evolution of the radical-SAM 23S rRNA methyltransferase Cfr that confers antibiotic resistance in bacteria.
Kaminska K.H., Purta E., Hansen L.H., Bujnicki J.M., Vester B., Long K.S.
The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase ... >> More
The Cfr methyltransferase confers combined resistance to five classes of antibiotics that bind to the peptidyl tranferase center of bacterial ribosomes by catalyzing methylation of the C-8 position of 23S rRNA nucleotide A2503. The same nucleotide is targeted by the housekeeping methyltransferase RlmN that methylates the C-2 position. Database searches with the Cfr sequence have revealed a large group of closely related sequences from all domains of life that contain the conserved CX(3)CX(2)C motif characteristic of radical S-adenosyl-l-methionine (SAM) enzymes. Phylogenetic analysis of the Cfr/RlmN family suggests that the RlmN subfamily is likely the ancestral form, whereas the Cfr subfamily arose via duplication and horizontal gene transfer. A structural model of Cfr has been calculated and used as a guide for alanine mutagenesis studies that corroborate the model-based predictions of a 4Fe-4S cluster, a SAM molecule coordinated to the iron-sulfur cluster (SAM1) and a SAM molecule that is the putative methyl group donor (SAM2). All mutations at predicted functional sites affect Cfr activity significantly as assayed by antibiotic susceptibility testing and primer extension analysis. The investigation has identified essential amino acids and Cfr variants with altered reaction mechanisms and represents a first step towards understanding the structural basis of Cfr activity. << Less