"Covalent Intermediate in the Catalytic Mechanism of the Radical S-Adenosyl-l-methionine Methyl Synthase RlmN Trapped by Mutagenesis."

McCusker KP, Medzihradszky KF, Shiver AL, Nichols RJ, Yan F, Maltby DA, Gross CA, Galonic Fujimori D...



Published 2012-10-22 in J Am Chem Soc .

Pubmed ID: 23088750
DOI identifier: -

Abstract:
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.


This publication refers to following proteins:



Last modification of this entry: Oct. 29, 2012