Cfr and RlmN Contain a Single [4Fe-4S] Cluster, which Directs Two Distinct Reactivities for S-Adenosylmethionine: Methyl Transfer by S_N2 Displacement and Radical Generation

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 sp2-hybridized carbon atoms via the intermediacy of a protein S-methylcysteinyl (mCys) residue. Both proteins contain five conserved Cys residues, each of which is required for turnover. Three cysteines lie in a canonical RS CxxxCxxC motif and coordinate a [4Fe–4S]-cluster cofactor. The remaining two cysteines are at opposite ends of the polypeptide. Herein we show that each protein contains only the one “radical SAM” [4Fe–4S] cluster, and that 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 it serves to generate the 5’-deoxyadenosyl 5’-radical, required for substrate-dependent methyl synthase activity.

Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

“…Initial work on these enzymes showed that SAM acts both as a source of Ado-CH 2 · and as a methyl group donor in the methylation reactions (36). Later, extensive mechanistic and crystallographic studies revealed that RlmN catalyzes an initial S N 2 nucleophilic substitution reaction to methylate a cysteine residue on the enzyme and uses subsequent radical SAM-dependent chemistry to generate a methyl group on the rRNA substrate (37)(38)(39). In the latter reaction, the methyl group that is appended to the adenosine residue retains only two hydrogens from the original SAM-derived methyl group.…”

Consecutive radical S -adenosylmethionine methylations form the ethyl side chain in thienamycin biosynthesis

“…Method 1 (compounds 2, 8, and 9): The methodology was similar to that reported previously (31). Mobile phase: The initial conditions included 95% solvent A (40 mM aqueous ammonium acetate, pH 6.0, with glacial acetic acid and 5% methanol) and 5% solvent B (methanol), 0.5-5 min up to 13% B, 5-6.5 min up to 27% B, 6.5-7.0 min up to 53% B, 7.0-7.5 min isocratic 53% B, 7.5-8 min down to 5% B, and 8.0-10.0 min reequilibration at 5% B.…”

Mechanistic Diversity of Radical S-Adenosylmethionine (SAM)-dependent Methylation

“…Studies by Booker and co-workers (33,38,39) showed that during the first turnover of RlmN and Cfr, when overproduced and purified with the [4Fe-4S] cluster intact, exogenous SAM is not the source of the appended methyl group. When RlmN and Cfr reactions were run in the presence of [methyl-2 H 3 ]SAM and a 7-nucleotide RNA substrate, the methylated products were not enriched with deuterium.…”