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2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
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2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
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2 S-adenosyl-L-methionine + adenine37 in tRNA + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNA + 2 oxidized [2Fe-2S] ferredoxin
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2 S-adenosyl-L-methionine + adenine37 in tRNAArg(ACG) + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAArg(ACG) + 2 oxidized [2Fe-2S] ferredoxin
low activity with tRNAArg(ACG)
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2 S-adenosyl-L-methionine + adenine37 in tRNAAsp(GUC) + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAAsp(GUC) + 2 oxidized [2Fe-2S] ferredoxin
very low activity with tRNAAsp(GUC)
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2 S-adenosyl-L-methionine + adenine37 in tRNAGln(CUG) + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAGln(CUG) + 2 oxidized [2Fe-2S] ferredoxin
moderate activity with tRNAGln(CUG)
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2 S-adenosyl-L-methionine + adenine37 in tRNAGln(UUG) + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAGln(UUG) + 2 oxidized [2Fe-2S] ferredoxin
high activity with tRNAGln(UUG)
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2 S-adenosyl-L-methionine + adenine37 in tRNAGlu(UUC) + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAGlu(UUC) + 2 oxidized [2Fe-2S] ferredoxin
low activity with tRNAGlu(UUC)
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2 S-adenosyl-L-methionine + adenine37 in tRNAHis(GUG) + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNAHis(GUG) + 2 oxidized [2Fe-2S] ferredoxin
best tRNA substrate
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S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
additional information
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2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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the enzyme catalyzes the natural post-transcriptional modification of A2503 to m2A in Escherichia coli 23S rRNA
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S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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the enzyme only catalyzes C2 methylation of adenine2503 in 23S rRNA
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S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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the enzyme primarily methylates C-8 in A2503 of 23S rRNA
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additional information
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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, see for EC 2.1.1.66, substrate specificity analysis, overview
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additional information
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recombinant His-RlmN protein is able to catalyze the synthesis of m2A in a SAM-dependent manner on tRNAchimera UUG purified from DELTArlmN cells
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additional information
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enzyme additionally methylates adenine37 in tRNA such as EScherichia coli tRNAArgACG, tRNAAspGUC, tRNAGlnCUG, tRNAGlnUUG, tRNAGluUUC, and tRNAHisGUG. tRNA methylation requirements are consistent with radical SAM reactivity
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additional information
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enzyme additionally methylates adenine37 in tRNA such as EScherichia coli tRNAArgACG, tRNAAspGUC, tRNAGlnCUG, tRNAGlnUUG, tRNAGluUUC, and tRNAHisGUG. tRNA methylation requirements are consistent with radical SAM reactivity
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additional information
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only a subset of tRNAs that contain an adenosine at position 37 are substrates for RlmN, structural comparison of substrates and non-substrates, and generation of chimeric tRNA substrates, substrate specificity, detailed overview. No activity with tRNAGly(CCC), in vitro methylation of chimeric constructs
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additional information
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only a subset of tRNAs that contain an adenosine at position 37 are substrates for RlmN, structural comparison of substrates and non-substrates, and generation of chimeric tRNA substrates, substrate specificity, detailed overview. No activity with tRNAGly(CCC), in vitro methylation of chimeric constructs
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2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA + 2 reduced [2Fe-2S] ferredoxin
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 8-methyladenine2503 in 23S rRNA + 2 oxidized [2Fe-2S] ferredoxin
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2 S-adenosyl-L-methionine + adenine37 in tRNA + 2 reduced [2Fe-2S] ferredoxin
2 S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine37 in tRNA + 2 oxidized [2Fe-2S] ferredoxin
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S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
additional information
?
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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, see for EC 2.1.1.66, substrate specificity analysis, overview
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2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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2 S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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the enzyme catalyzes the natural post-transcriptional modification of A2503 to m2A in Escherichia coli 23S rRNA
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S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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the enzyme only catalyzes C2 methylation of adenine2503 in 23S rRNA
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S-adenosyl-L-methionine + adenine2503 in 23S rRNA
S-adenosyl-L-homocysteine + L-methionine + 5'-deoxyadenosine + 2-methyladenine2503 in 23S rRNA
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the enzyme primarily methylates C-8 in A2503 of 23S rRNA
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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
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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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
physiological function
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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
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
additional information
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
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
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C118A enzyme mutant complexed with tRNAGlu substrate, X-ray diffraction structure determination and analysis at 2.4 A resolution, structure modelling
homology modeling of structure and comparison with Cfr from Staphylococcus sciuri, EC 2.1.1.194
mutant C118A, hanging drop vapor diffusion method, using 7.5% (w/v) PEG 6000, 0.1 M HEPES, pH 7.5, and 5% (v/v) 2-methyl-2,4-pentanediol as the precipitant
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structure of a key intermediate in the RlmN reaction, in which a Cys118Ala variant of the protein is crosslinked 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
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Toh, S.M.; Xiong, L.; Bae, T.; Mankin, A.S.
The methyltransferase YfgB/RlmN is responsible for modification of adenosine 2503 in 23S rRNA
RNA
14
98-106
2008
Escherichia coli
brenda
Benitez-Paez, A.; Villarroya, M.; Armengod, M.E.
The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy
RNA
18
1783-1795
2012
Escherichia coli
brenda
Atkinson, G.C.; Hansen, L.H.; Tenson, T.; Rasmussen, A.; Kirpekar, F.; Vester, B.
Distinction between the Cfr methyltransferase conferring antibiotic resistance and the housekeeping RlmN methyltransferase
Antimicrob. Agents Chemother.
57
4019-4026
2013
Bacillus subtilis (O34617), Bacillus subtilis 168 (O34617), Escherichia coli, Mammaliicoccus sciuri (Q9FBG4), Thermus thermophilus (Q5SGZ3), Thermus thermophilus HB8 / ATCC 27634 / DSM 579 (Q5SGZ3)
brenda
Silakov, A.; Grove, T.L.; Radle, M.I.; Bauerle, M.R.; Green, M.T.; Rosenzweig, A.C.; Boal, A.K.; Booker, S.J.
Characterization of a cross-linked protein-nucleic acid substrate radical in the reaction catalyzed by RlmN
J. Am. Chem. Soc.
136
8221-8228
2014
Escherichia coli
brenda
Ntokou, E.; Hansen, L.; Kongsted, J.; Vester, B.
Biochemical and computational analysis of the substrate specificities of Cfr and RlmN Methyltransferases
PLoS ONE
10
e0145655
2015
Escherichia coli (P36979), Mammaliicoccus sciuri (A0A1X0U0N1)
brenda
Fitzsimmons, C.M.; Fujimori, D.G.
Determinants of tRNA recognition by the radical SAM enzyme RlmN
PLoS ONE
11
e0167298
2016
Escherichia coli, Escherichia coli (P36979)
brenda
Benitez-Paez, A.; Villarroya, M.; Armengod, M.-A.
The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy
RNA
18
1783-1795
2012
Escherichia coli (P36979), Escherichia coli
brenda
Boal, A.K.; Grove, T.L.; McLaughlin, M.I.; Yennawar, N.H.; Booker, S.J.; Rosenzweig, A.C.
Structural basis for methyl transfer by a radical SAM enzyme
Science
332
1089-1092
2011
Escherichia coli (P36979)
brenda
Schwalm, E.; Grove, T.; Booker, S.; Boal, A.
Crystallographic capture of a radical S-adenosylmethionine enzyme in the act of modifying tRNA
Science
352
309-312
2016
Escherichia coli (P36979)
brenda