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

Martin R. Challand, Enrico Salvadori, Rebecca C. Driesener, Christopher W. M. Kay*, Peter L. Roach, James Spencer

*Corresponding author for this work

Research output: Contribution to journalArticle (Academic Journal)peer-review

5 Citations (Scopus)

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.

Original languageEnglish
Article number67979
Number of pages10
JournalPLoS ONE
Volume8
Issue number7
DOIs
Publication statusPublished - 5 Jul 2013

Keywords

  • 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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