eprintid: 1401337 rev_number: 31 eprint_status: archive userid: 608 dir: disk0/01/40/13/37 datestamp: 2013-08-09 20:56:39 lastmod: 2021-10-13 23:23:46 status_changed: 2013-08-09 20:56:39 type: article metadata_visibility: show item_issues_count: 0 creators_name: Challand, MR creators_name: Salvadori, E creators_name: Driesener, RC creators_name: Kay, CW creators_name: Roach, PL creators_name: Spencer, J title: Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase ispublished: pub divisions: UCL divisions: B04 divisions: C06 divisions: F64 note: © 2013 Challand et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. PMCID: PMC3702613 abstract: The 'radical S-adenosyl-L-methionine (AdoMet)' enzyme Cfr methylates adenosine 2503 of the 23S rRNA in the peptidyltransferase centre (P-site) of the bacterial ribosome. This modification protects host bacteria, notably methicillin-resistant Staphylococcus aureus (MRSA), from numerous antibiotics, including agents (e.g. linezolid, retapamulin) that were developed to treat such organisms. Cfr contains a single [4Fe-4S] cluster that binds two separate molecules of AdoMet during the reaction cycle. These are used sequentially to first methylate a cysteine residue, Cys338; and subsequently generate an oxidative radical intermediate that facilitates methyl transfer to the unreactive C8 (and/or C2) carbon centres of adenosine 2503. How the Cfr active site, with its single [4Fe-4S] cluster, catalyses these two distinct activities that each utilise AdoMet as a substrate remains to be established. Here, we use absorbance and electron paramagnetic resonance (EPR) spectroscopy to investigate the interactions of AdoMet with the [4Fe-4S] clusters of wild-type Cfr and a Cys338 Ala mutant, which is unable to accept a methyl group. Cfr binds AdoMet with high (∼ 10 µM) affinity notwithstanding the absence of the RNA cosubstrate. In wild-type Cfr, where Cys338 is methylated, AdoMet binding leads to rapid oxidation of the [4Fe-4S] cluster and production of 5'-deoxyadenosine (DOA). In contrast, while Cys338 Ala Cfr binds AdoMet with equivalent affinity, oxidation of the [4Fe-4S] cluster is not observed. Our results indicate that the presence of a methyl group on Cfr Cys338 is a key determinant of the activity of the enzyme towards AdoMet, thus enabling a single active site to support two distinct modes of AdoMet cleavage. date: 2013-07-05 official_url: http://dx.doi.org/10.1371/journal.pone.0067979 vfaculties: VFLS oa_status: green full_text_type: pub language: eng primo: open primo_central: open_green article_type_text: Journal Article verified: verified_manual elements_source: PubMed elements_id: 889543 doi: 10.1371/journal.pone.0067979 pii: PONE-D-13-08964 lyricists_name: Kay, Christopher lyricists_name: Salvadori, Enrico lyricists_id: CWKAY79 lyricists_id: ESALV65 full_text_status: public publication: PLoS One volume: 8 number: 7 article_number: e67979 pagerange: - event_location: United States issn: 1932-6203 citation: Challand, MR; Salvadori, E; Driesener, RC; Kay, CW; Roach, PL; Spencer, J; (2013) Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase. PLoS One , 8 (7) , Article e67979. 10.1371/journal.pone.0067979 <https://doi.org/10.1371/journal.pone.0067979>. Green open access document_url: https://discovery.ucl.ac.uk/id/eprint/1401337/1/journal.pone.0067979.pdf