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

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.

Figure 1

SDS-PAGE analysis of RlmN C118 mutant proteins. Protein markers are in lane 1 of all gels. a) C118A RlmN purified by immobilized metal ion affinity chromatography (lane 2). b) C118G RlmN (lane 2) and C118S RlmN (lane 3) purified by immobilized metal ion affinity chromatography. c) Untreated C118A RlmN-RNA adduct purified by anion exchange (lane 2). Sample from lane 2, after treatment with RNases A&T₁ (lane 3). d) C118S RlmN grown under metal deficient conditions and purified by immobilized metal ion affinity chromatography (lane 2). In this sample, no RNA adduct could be detected.

Figure 2

Ion trap CID data of ³⁴⁸GDDIDAAC(CH₂adenine)GQLAGDVIDR³⁷⁵ formed from RlmN C118A protein in vivo and subsequently RNase treated. The precursor mass was m/z 650.9631(3+), within ~1 ppm of the calculated mass of 650.9623 (3+ ion of RlmN 348–375 peptide +147). The table at right lists all main sequence ions detected within 20 ppm of the calculated value. In comparison to the methyl derivative (Figure S3), the higher charge state of the precursor ion yielding the best CID data as well as the presence of doubly charged N- and C-terminal fragments clearly indicate the incorporation of a basic modification. The site of modification within the peptide and the deduced structure of the modification are highlighted in yellow.

Figure 3

In vitro gel-shift assay of RlmN C118 mutants. RlmN mutant proteins were incubated with RNA fragments as indicated, and RNA was separated by denaturing PAGE and visualized with ethidium bromide. Lane 1 contains RNA markers of the indicated size, in nt. Lane 2 contains only the 87 nt RNA fragment used in assays. Lane 3 is a negative control lacking NADPH. Lane 4 is the reaction with C118A RlmN, SAM and RNA. Lane 5 is the reaction with C118S RlmN, SAM and RNA. Lane 6 is the reaction with C118S RlmN, SAM and D-RNA. Lane 7 is the reaction with C118S RlmN, [methyl-²H₃]-SAM and RNA.

Figure 4

Ion trap CID data of ³⁴⁸GDDIDAAC(CD₂adenine)GQLAGDVIDR³⁷⁵ formed from RlmN C118S protein in vitro in the presence of [methyl-²H₃]-SAM. The precursor mass was m/z 651.6353(3+), within 3 ppm of the calculated mass of 651.6332 (3+ ion of RlmN 348–375 peptide +149). Fragments that showed the 2 Da shift due to deuterium incorporation are labeled with asterisks. Main sequence ions detected are shown in red in the table. The site of modification within the peptide and the deduced structure of the modification are highlighted in yellow.

Scheme 1

Proposed mechanism of RlmN-mediated methylation of RNA.

Scheme 2

Deuterium incorporation from substrates into covalent adduct between C118 mutants of RlmN and RNA.