Description:
<jats:p>Cfr is a radical SAM methylase that catalyzes the radical‐mediated appendage of a methyl group at C8 of adenosine 2503 in 23S ribosomal RNA (rRNA). Methylation of C8 by Cfr confers resistance to several classes of antibiotics that bind to the peptidyl transferase center of the bacterial ribosome. RlmN, a homologous RS methyltransferase, exclusively methylates C2 of A2503, which tunes the ribosome's interaction with nascent peptide chains. Cfr can also catalyze methylation at C2 in vitro, but it is much slower than methylation at C8. The Cfr and RlmN reactions take place via a cross‐linked intermediate between the terminal carbon of a methylcysteine residue on Cfr or RlmN and the carbon atom undergoing methylation on the RNA. This intermediate is resolved by abstraction of the C8 proton during C8 methylation or the C2 proton during C2 methylation. However, the identity of the active site base that mediates this abstraction at C8 has not been determined. EPR and crystallographic studies on the C118A variant of RlmN provided evidence that C118 is the operative base in the RlmN reaction. Previous studies using deuterium‐labeled substrates showed that the C2 proton is transferred to the nascent methyl group without isotope dilution during C2 methylation, while partial solvent hydron exchange takes place during C8 methylation. These observations led to the proposal that a polyprotic base is responsible for abstracting the C8 proton to initiate resolution of the cross‐linked intermediate. Herein, using deuterated substrates in conjunction with mass spectrometry and EPR we provide evidence that C105 of Cfr, the cognate residue to C118 of RlmN, deprotonates C8 of A2503.</jats:p><jats:p><jats:bold>Support or Funding Information</jats:bold></jats:p><jats:p>This work was supported by NIH Grant GM 101957 (S.J.B.)</jats:p>