Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase.
In: PLoS ONE, Jg. 8 (2013), Heft 7, S. e67979
Online
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Zugriff:
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.
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Cysteine methylation controls radical generation in the Cfr radical AdoMet rRNA methyltransferase.
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Autor/in / Beteiligte Person: | Martin R Challand ; Salvadori, Enrico ; Rebecca C Driesener ; Christopher W M Kay ; Peter L Roach ; Spencer, James |
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Zeitschrift: | PLoS ONE, Jg. 8 (2013), Heft 7, S. e67979 |
Veröffentlichung: | Public Library of Science (PLoS), 2013 |
Medientyp: | academicJournal |
ISSN: | 1932-6203 (print) |
DOI: | 10.1371/journal.pone.0067979 |
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