EC Number   |
General Information |
Reference |
|---|
   2.1.1.192 | 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 |
-, 733111 |
| 2.1.1.192 | evolution |
evolutionary relationship between the Cfr (EC 2.1.1.224) and RlmN enzymes, phylogenetic analysis, overview |
758128 |
| 2.1.1.192 | 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 |
758461 |
| 2.1.1.192 | 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 |
758141 |
| 2.1.1.192 | malfunction |
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 |
718586 |
| 2.1.1.192 | malfunction |
an rlmN null mutant shows slightly increased susceptibility to sparsomycin and hygromycin A |
704203 |
| 2.1.1.192 | malfunction |
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 |
706722 |
| 2.1.1.192 | malfunction |
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 |
721038 |
| 2.1.1.192 | more |
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 |
758463 |
| 2.1.1.192 | more |
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 |
758141 |
