Covalent intermediate in the catalytic mechanism of the radical S-adenosyl-L-methionine methyl synthase RlmN trapped by mutagenesis.

摘要: The posttranscriptional modification of ribosomal RNA (rRNA) modulates function and confers resistance to antibiotics targeted the ribosome. radical S-adenosyl-L-methionine (SAM) methyl synthases, RlmN Cfr, both methylate A2503 within peptidyl transferase center prokaryotic ribosomes, yielding 2-methyl- 8-methyl-adenosine, respectively. C2 C8 positions adenosine are unusual methylation substrates due their electrophilicity. To accomplish this reaction, Cfr use a shared radical-mediated mechanism. In addition SAM CX(3)CX(2)C motif, contain two conserved cysteine residues required for in vivo function, putatively form (cysteine 355 RlmN) resolve 118 covalent intermediate needed achieve challenging transformation. Currently, there is no direct evidence proposed intermediate. We have further investigated roles these cysteines mechanism RlmN. Cysteine mutants unable intermediate, either or vitro, enabling us isolate characterize Additionally, tandem mass spectrometric analyses mutant reveal methylene-linked at 355. Employing deuterium-labeled vitro has allowed clarify formation Together, experiments provide compelling species between its rRNA substrate well as catalysis.