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Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase

Research output: Contribution to journalArticle

  • Martin R. Challand
  • Enrico Salvadori
  • Rebecca C. Driesener
  • Christopher W. M. Kay
  • Peter L. Roach
  • James Spencer
Original languageEnglish
Article number67979
Number of pages10
JournalPLoS ONE
Volume8
Issue number7
DOIs
DatePublished - 5 Jul 2013

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 (similar to 10 mu M) 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.

    Research areas

  • LYASE-ACTIVATING ENZYME, IRON-SULFUR CLUSTER, ADENOSYL-L-METHIONINE, S-ADENOSYLMETHIONINE, BIOTIN SYNTHASE, ESCHERICHIA-COLI, LYSINE 2,3-AMINOMUTASE, CRYSTAL-STRUCTURE, SAM ENZYMES, ENTEROCOCCUS-FAECALIS

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