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Results: 6

1.
Scheme 1

Scheme 1. From: Covalent Intermediate in the Catalytic Mechanism of the Radical SAM Methyl Synthase RlmN Trapped by Mutagenesis.

Proposed mechanism of RlmN-mediated methylation of RNA.

Kevin P. McCusker, et al. J Am Chem Soc. 2012 October 31;134(43):18074-18081.
2.
Scheme 2

Scheme 2. From: Covalent Intermediate in the Catalytic Mechanism of the Radical SAM Methyl Synthase RlmN Trapped by Mutagenesis.

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

Kevin P. McCusker, et al. J Am Chem Soc. 2012 October 31;134(43):18074-18081.
3.
Figure 3

Figure 3. From: Covalent Intermediate in the Catalytic Mechanism of the Radical SAM Methyl Synthase RlmN Trapped by Mutagenesis.

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.

Kevin P. McCusker, et al. J Am Chem Soc. 2012 October 31;134(43):18074-18081.
4.
Figure 1

Figure 1. From: Covalent Intermediate in the Catalytic Mechanism of the Radical SAM Methyl Synthase RlmN Trapped by Mutagenesis.

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.

Kevin P. McCusker, et al. J Am Chem Soc. 2012 October 31;134(43):18074-18081.
5.
Figure 4

Figure 4. From: Covalent Intermediate in the Catalytic Mechanism of the Radical SAM Methyl Synthase RlmN Trapped by Mutagenesis.

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.

Kevin P. McCusker, et al. J Am Chem Soc. 2012 October 31;134(43):18074-18081.
6.
Figure 2

Figure 2. From: Covalent Intermediate in the Catalytic Mechanism of the Radical SAM Methyl Synthase RlmN Trapped by Mutagenesis.

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.

Kevin P. McCusker, et al. J Am Chem Soc. 2012 October 31;134(43):18074-18081.

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