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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 language | English |
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Article number | 67979 |
Number of pages | 10 |
Journal | PLoS ONE |
Volume | 8 |
Issue number | 7 |
DOIs | |
Publication status | Published - 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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Dive into the research topics of 'Cysteine Methylation Controls Radical Generation in the Cfr Radical AdoMet rRNA Methyltransferase'. Together they form a unique fingerprint.Projects
- 1 Finished
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Unravelling a Novel Mode of Multiple Antibiotic Resistance: Mechanism and Inhibition of Radical-SAM RNA Methyltransferases
Spencer, J. (Principal Investigator)
1/07/12 → 30/09/15
Project: Research