2.1.1.192: 23S rRNA (adenine2503-C2)-methyltransferase
This is an abbreviated version!
For detailed information about 23S rRNA (adenine2503-C2)-methyltransferase, go to the full flat file.
Word Map on EC 2.1.1.192
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2.1.1.192
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5\'-deoxyadenosyl
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5'-deoxyadenosine
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radical-mediated
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ripps
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desii
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venezuelae
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maturase
- 2.1.1.192
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5\'-deoxyadenosyl
- 5'-deoxyadenosine
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radical-mediated
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ripps
- desii
- venezuelae
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maturase
Reaction
2 S-adenosyl-L-methionine + + 2 reduced [2Fe-2S] ferredoxin = + + + + 2 oxidized [2Fe-2S] ferredoxin
Synonyms
Cfr, EC 2.1.1.194, methyltransferase YfgB/RlmN, NlmA, radical AdoMet rRNA methyltransferase, radical S-adenosyl-L-methionine enzyme, radical SAM enzyme, RlmN, RlmN methyltransferase, YfgB, YfgB/RlmN
ECTree
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General Information
General Information on EC 2.1.1.192 - 23S rRNA (adenine2503-C2)-methyltransferase
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evolution
malfunction
physiological function
additional information
comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr and RlmN-like sequences. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. The differentiation between the two classes is supported by experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. The Cfr- and RlmN-specific conserved sites provide a very good indication of whether a gene is Cfr-like or RlmN-like. Most bacteria have an rlmN-like gene and all those that have a cfr-like gene also have an rlmN-like gene, evolutionary aspects of the bacterial distribution of Cfr and RlmN-like enzymes, overview
evolution
comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr and RlmN-like sequences. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. The differentiation between the two classes is supported by experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. The Cfr- and RlmN-specific conserved sites provide a very good indication of whether a gene is Cfr-like or RlmN-like. Most bacteria have an rlmN-like gene and all those that have a cfr-like gene also have an rlmN-like gene, evolutionary aspects of the bacterial distribution of Cfr and RlmN-like enzymes, overview
evolution
evolutionary relationship between the Cfr (EC 2.1.1.224) and RlmN enzymes, phylogenetic analysis, overview
evolution
RlmN and Cfr belong to the radical SAM (RS) superfamily of enzymes. RlmN is proposed to be an evolutionary precursor to Cfr. The catalytic residues in theactive site are strictly conserved as are most of the surrounding residues within the core of the barrel
evolution
unlike methylation of nitrogen or oxygen atoms, methylation of cytosine or uridine at the C5 position requires a different mechanism, because the target position is not nucleophilic. Covalent catalysis via a Michael addition activates the C5 carbon and accounts for methylation at these sites. In contrast, methylation at the unreactive C2 and C8 positions of adenosines requires a different enzymatic mechanism and is catalyzed by members of the radical S-adenosyl-L-methionine (SAM) superfamily
evolution
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comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr and RlmN-like sequences. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. The differentiation between the two classes is supported by experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. The Cfr- and RlmN-specific conserved sites provide a very good indication of whether a gene is Cfr-like or RlmN-like. Most bacteria have an rlmN-like gene and all those that have a cfr-like gene also have an rlmN-like gene, evolutionary aspects of the bacterial distribution of Cfr and RlmN-like enzymes, overview
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evolution
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
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comparative sequence analysis identifies differentially conserved residues that indicate functional sequence divergence between the two classes of Cfr and RlmN-like sequences. The enzymes are homologous and use the same mechanism involving radical S-adenosyl methionine to methylate RNA via an intermediate involving a methylated cysteine in the enzyme and a transient cross-linking to the RNA, but they differ in which carbon atom in the adenine they methylate. The differentiation between the two classes is supported by experimental evidence from antibiotic resistance, primer extensions, and mass spectrometry. The Cfr- and RlmN-specific conserved sites provide a very good indication of whether a gene is Cfr-like or RlmN-like. Most bacteria have an rlmN-like gene and all those that have a cfr-like gene also have an rlmN-like gene, evolutionary aspects of the bacterial distribution of Cfr and RlmN-like enzymes, overview
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an rlmN null mutant shows slightly increased susceptibility to sparsomycin and hygromycin A
malfunction
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inactivation of the yfgB gene in Escherichia coli leads to the loss of modification at nucleotide A2503 of 23S rRNA. The A2503 modification is restored when YfgB protein is expressed in the yfgB knockout strain. In a co-growth competition experiment, the rlmN knockout mutant shows reduced fitness compared to wild type. The absence of m2A2503 modification produces a subtle but significant effect on cell fitness apparently through its effect on protein synthesis or ribosome assembly. Escherichia coli rlmN knockout strain shows a small but reproducible twofold increased susceptibility to tiamulin, hygromycin A, and sparsomycin compared to the wild-type strain
malfunction
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a naturally occurring rlmN mutation, by codon insertion in the rlmN gene, in a clinical Staphylococcus aureus isolate, strain JKD6229, decreases susceptibility to oxazolidinone antibiotic linezolid and is thought to increase the extent of A2503 modification, but mutation in fact abolishes RlmN activity, resulting in a lack of A2503 modification. rlmN knockout mutant Staphylococcus aureus Newman strain, SAV1218, shows a slight decrease in linezolid resistance, overview
malfunction
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the DrlmN mutant lacks m2A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m2A37 from tRNA is expected to produce a hyperaccurate phenotype, the error-prone phenotype exhibited by the DrlmN mutant is due to loss of m2A from 23S rRNA
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RlmN introduces m2A at position 2503 in the peptidyl transferase center of 23S RNA. RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA, see for EC 2.1.1.66. The m2A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center
physiological function
the RlmN methyltransferase primarily methylates C-2 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. Enzyme RlmN does not confer resistance to antibiotics
physiological function
the RlmN methyltransferase primarily methylates C-2 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. Enzyme RlmN does not confer resistance to antibiotics
physiological function
antibiotic resistance effects of wild-type and mutant enzymes, overview
physiological function
methyl transfer is essential in the synthesis of cellular metabolites and clinically relevant natural products, and in the modification of RNA, DNA, lipids, and proteins. Enzymes RlmN and Cfr catalyze methylation of a 23S rRNA nucleotide (adenosine 2503, A2503) ultimately located within the peptidyltransferase center of the 50S subunit of the bacterial ribosome near the entrance to the nascent peptide exit tunnel. RlmN methylates the C2 position of A2503, a housekeeping modification important in translational fidelity and the nascent peptide response. The radical SAM (RS) 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. RlmN accomplishes its complex reaction with structural economy, harnessing the two most important reactivities of SAM within a single site
physiological function
RlmN is a bacterial radical SAM methylating enzyme, that has the unusual ability to modify two distinct types of RNA: 23S rRNA and tRNA. In rRNA, RlmN installs a methyl group at the C2 position of A2503 of 23S rRNA, while in tRNA the modification occurs at nucleotide A37, immediately adjacent to the anticodon triplet
physiological function
RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The RlmN mutant lacks 2-methyladenine in both tRNA set and in the peptidyl transferase center of 23S RNA
physiological function
RlmN is a radical S-adenosylmethionine (SAM) enzyme that is best known for catalyzing the methylation of C2 of adenosine 2503 (A2503) (1-3) in domain V of 23S rRNA. But RlmN is a dual-specificity RNA methylase that modifies C2 of adenosine 2503 (A2503) in 23S rRNA and C2 of adenosine 37 (A37) in several Escherichia coli tRNAs. RlmN thus joins a pseudouridine synthase, RluA, as a known dual-specificity RNA modification enzymes capable of acting both on ribosomal and on transfer RNA
physiological function
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the RlmN methyltransferase primarily methylates C-2 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. Enzyme RlmN does not confer resistance to antibiotics
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physiological function
Thermus thermophilus HB8 / ATCC 27634 / DSM 579
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the RlmN methyltransferase primarily methylates C-2 in A2503 of 23S rRNA in the peptidyl transferase region of bacterial ribosomes. Enzyme RlmN does not confer resistance to antibiotics
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although SAM is the source of the appended methyl carbon in the reactions catalyzed by RlmN and Cfr, these enzymes operate by a mechanism that is distinctly different from that of typical SAM-dependent methyltransferases. As radical SAM (RS) enzymes, RlmN and Cfr employ very similar radical-based mechanisms of catalysis, initiated by the abstraction of a hydrogen atom from a Cys-appended methyl group via a 5'-deoxyadenosyl 5'-radical. Subsequent attack of the resulting methylene radical upon the carbon atom undergoing methylation affords a protein/RNA cross-linked intermediate whose resolution requires prior proton abstraction from C2 (RlmN) or C8 (Cfr) of the substrate by an unidentified base. Conversion of the intermediate to the methylated product has also been demonstrated in the Cfr reaction. The proximity (5.0 A) of the Cys 355 side chain (the proposed site of thiyl radical formation) to the sulfur atom of Met176, a strictly conserved residue in RlmN and Cfr, might allow formation of a transient thiosulfuranyl radical. Structure analysis of the key intermediate in the RlmN reaction, in which a Cys118->Ala variant of the protein is cross-linked to a tRNAGlu substrate through the terminal methylene carbon of a formerly methylcysteinyl residue and C2 of A37. RlmN contacts the entire length of tRNAGlu, accessing A37 using an induced-fit strategy that completely unfolds the tRNA anticodon stem loop, which is likely critical for recognition of both tRNA and rRNA substrates. The most extensive RlmN-tRNA interactions involve the anti-codon stem loop (ACSL) of tRNAGlu near A37. The protein binds in the minor groove of the ACSL and interacts more intimately with the nucleobases. Binding structure, overview
additional information
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determinants of tRNA recognition by the radical SAM enzyme RlmN, overview. Usage of in vitro transcribed tRNAs as model substrates to interrogate RNA recognition by RlmN. Structure and sequence of RNA influence methylation, identifying position 38 of tRNAs as a critical determinant of substrate recognition, tRNA methylation requirements are consistent with radical S-adenosyl-L-methionine (SAM) reactivity. Studies on RlmN and Cfr, two bacterial radical SAM methylating enzymes, have established key mechanistic features of radical SAM methylation of RNA. A unique feature of these enzymes is their ability to utilize both homolytic and heterolytic reactivity of SAM to carry out methylation of the C2 and C8 amidine carbons of adenosine. The first equivalent of SAM is used to methylate a conserved cysteine residue (C355), unassociated with the four iron-four sulfur ([4Fe-4S]) cluster, to form a protein-bound methyl thioether. A second equivalent of SAM, coordinated by the [4Fe-4S] cluster in these proteins, is then cleaved homolytically to generate a 5'-deoxyadenosyl radical, a canonical feature of radical SAM catalysis. This highly reactive radical species then abstracts a hydrogen atom from the premethylated cysteine 355 to form a thiomethylene radical. The methylene radical then adds into the substrate carbon to form a covalent RNA-protein adduct, which has been trapped by mutagenesis and characterized spectroscopically. A second conserved cysteine residue resolves the covalent RNA-protein intermediate, forming the methylated product
additional information
determinants of tRNA recognition by the radical SAM enzyme RlmN, overview. Usage of in vitro transcribed tRNAs as model substrates to interrogate RNA recognition by RlmN. Structure and sequence of RNA influence methylation, identifying position 38 of tRNAs as a critical determinant of substrate recognition, tRNA methylation requirements are consistent with radical S-adenosyl-L-methionine (SAM) reactivity. Studies on RlmN and Cfr, two bacterial radical SAM methylating enzymes, have established key mechanistic features of radical SAM methylation of RNA. A unique feature of these enzymes is their ability to utilize both homolytic and heterolytic reactivity of SAM to carry out methylation of the C2 and C8 amidine carbons of adenosine. The first equivalent of SAM is used to methylate a conserved cysteine residue (C355), unassociated with the four iron-four sulfur ([4Fe-4S]) cluster, to form a protein-bound methyl thioether. A second equivalent of SAM, coordinated by the [4Fe-4S] cluster in these proteins, is then cleaved homolytically to generate a 5'-deoxyadenosyl radical, a canonical feature of radical SAM catalysis. This highly reactive radical species then abstracts a hydrogen atom from the premethylated cysteine 355 to form a thiomethylene radical. The methylene radical then adds into the substrate carbon to form a covalent RNA-protein adduct, which has been trapped by mutagenesis and characterized spectroscopically. A second conserved cysteine residue resolves the covalent RNA-protein intermediate, forming the methylated product
additional information
mechanisms of catalytic action of Cfr (EC 2.1.1.224) and related RlmN, the methylation mechanism involves a transitory methylation of Cys338 for Cfr and Cys355 for RlmN, investigation of target binding to the active sites of the two enzymes, overview. Cfr and RlmN are methylated before transfer of the methyl group to the substrate. Molecular dynamics simulations, and calculation of the binding free energy, using the structure of Escherichia coli RlmN (PDB ID 3RFA). Defining regions of the active site to be interchanged to investigate C8/C2 specificity
additional information
structure modelling and structure-function analysis of RlmN compared to Cfr from Staphylococcus aureus (EC 2.1.1.224), Escherichia coli RlmN and Staphylococcus aureus Cfr are mapped onto the RlmN structure, detailed overview. The RlmN reactions proceed by a ping-pong mechanism. The methyl group from one SAM molecule is initially appended to a conserved Cys355 in RlmN by a typical SN2 displacement. This SAM-derived one-carbon unit is then attached to the RNA by radical addition initiated by a 5'-deoxyadenosyl 5'-radical formed from a second molecule of SAM. Finally, this covalent intermediate is resolved by formation of a disulfide bond between the methyl-carrying Cys (mCys) residue and a second conserved Cys118. Nature and location of the rRNA binding site. The electrostatic surface potential for RlmN suggests the substrate may approach the active site from the bottom of the barrel. This extensive surface involves the core barrel, its extensions, and the extra domain, implicating the accessory elements in substrate interaction