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. 2011 Dec 14;133(49):19586-9.
doi: 10.1021/ja207327v. Epub 2011 Nov 18.

Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: methyl transfer by SN2 displacement and radical generation

Tyler L Grove  1 , Matthew I Radle, Carsten Krebs, Squire J Booker
Affiliations

Cfr and RlmN contain a single [4Fe-4S] cluster, which directs two distinct reactivities for S-adenosylmethionine: methyl transfer by SN2 displacement and radical generation

Tyler L Grove et al. J Am Chem Soc. .

Abstract

The radical SAM (RS) proteins RlmN and Cfr catalyze methylation of carbons 2 and 8, respectively, of adenosine 2503 in 23S rRNA. Both reactions are similar in scope, entailing the synthesis of a methyl group partially derived from S-adenosylmethionine (SAM) onto electrophilic sp(2)-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe-4S]-cluster cofactor; the remaining two are at opposite ends of the polypeptide. Here we show that each protein contains only the one "radical SAM" [4Fe-4S] cluster and the two remaining conserved cysteines do not coordinate additional iron-containing species. In addition, we show that, while wild-type RlmN bears the C355 mCys residue in its as-isolated state, RlmN that is either engineered to lack the [4Fe-4S] cluster by substitution of the coordinating cysteines or isolated from Escherichia coli cultured under iron-limiting conditions does not bear a C355 mCys residue. Reconstitution of the [4Fe-4S] cluster on wild-type apo RlmN followed by addition of SAM results in rapid production of S-adenosylhomocysteine (SAH) and the mCys residue, while treatment of apo RlmN with SAM affords no observable reaction. These results indicate that in Cfr and RlmN, SAM bound to the unique iron of the [4Fe-4S] cluster displays two reactivities. It serves to methylate C355 of RlmN (C338 of Cfr), or to generate the 5'-deoxyadenosyl 5'-radical, required for substrate-dependent methyl synthase activity.

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Figures

Figure 1
Figure 1
4.2-K/53-mT Mössbauer spectra of AI RlmNwt (A), RCN RlmNwt (B), AI Cfrwt (C), and RCN Cfrwt (D). Experimental spectra are shown as vertical bars. The solid lines are quadrupole doublet simulations with parameters quoted in the text, accounting for 93% (A), 95% (B), 86% (C), and 98% (D) of the total intensity.
Figure 2
Figure 2
Extracted ion chromatograms (EIC) for the C355-containing peptide from trypsin digests of RlmN. Black traces (A, B, C, D, E, F) are EIC of m/z 931.0 [M+2H], indicative of carbamidomethyl modification to C355. Red traces (A, B, C, D, E, F) are EIC of m/z 909.6 [M+2H], indicative of a methyl modification of C355. Traces correspond to A) AI RlmNwt; B) AI RlmNC125A-C129A-C132A; C) AI RlmNC118A; D) apo RlmNwt, inclusion bodies; E) apo RlmNwt, soluble fraction; F) apo RlmNwt→RCN after addition of 1.5 mM SAM. Spectral pair intensities are normalized to the most abundant EIC.
Figure 3
Figure 3
A) UV–vis traces of apo RlmNwt (5 µM, solid black line) and apo RlmNwt→RCN (12 µM, solid red line). B) Methyl transfer catalyzed by apo RlmNwt (150 µM) or apo RlmNwt→RCN (150 µM). Production of SAH by apo RlmNwt (purple diamonds) or apo RlmNwt→RCN (red circles) in the presence of 1.5 mM SAM; or apo RlmNwt→RCN (150 µM) in the presence of 1.5 mM d3-SAM (black triangles). Error bars indicate one standard deviation from the average of three assays. C) Q-Tof MS analysis of tryptic peptides from apo RlmNwt→RCN after incubation in the presence of SAM (black trace) or d3- SAM (red trace). Indicated m/z values correspond to the +2 charge state.
Scheme 1
Scheme 1
Reactions catalyzed by RlmN and Cfr in vivo.
Scheme 2
Scheme 2
SAM bound to the [4Fe–4S] cluster of RlmN or Cfr is activated toward two distinct reactivites.
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