Covalent Intermediate in the Catalytic Mechanism of the Radical S-Adenosyl-l-methionine Methyl Synthase RlmN Trapped by Mutagenesis

Abstract

The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical SAM (S-adenosyl-L-methionine) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center (PTC) of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr proceed by a shared radical-mediated mechanism. However, in addition to the radical SAM CX3CX2C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function. These conserved cysteine residues are putatively involved in a covalent intermediate employed by RlmN and Cfr in order to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further elucidate the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species formed between RlmN and its rRNA substrate and the roles of the conserved cysteine residues in catalysis.

Identification of an Intermediate Methyl Carrier in the Radical S-Adenosylmethionine Methylthiotransferases RimO and MiaB

“…In the best studied class, represented by the RS methylases RlmN and Cfr, which catalyze the synthesis of methyl groups at C2 and C8, respectively, of adenosine 2503 of 23 S rRNA, catalysis takes place via a ping-pong like mechanism, involving an initial S N 2-based transfer of a methyl group to a target Cys residue before it is transferred to the nucleotide substrate via radical-dependent chemistry 34,38 . Studies detailed herein provide strong evidence for an analogous ping-pong-like mechanism for MTTases.…”

“…In the second half-reaction a second molecule of SAM binds to the same site, but is reductively cleaved to a 5’-dA•, which initiates turnover by abstracting a hydrogen atom (H•) from the methylCys residue. After radical addition to C2 or C8 of the adenine ring and loss of an electron to an undetermined acceptor, a methylene-bridged protein-substrate crosslink is resolved by disulfide-bond formation with concomitant release of an enamine, which tautomerizes to the methyladenosine product upon acquiring a proton from a general acid in the active site 3438 .…”

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

“…In addition to the three cysteines that ligate the iron-sulfur (Fe/S) cluster cofactor, RlmN and Cfr contain two additional strictly conserved cysteines that are essential for catalysis (33)(34)(35). These additional cysteines distinguish Class A enzymes from other RS methylases and contribute to their unique reaction mechanism (11,33,35).…”

Determinants of tRNA Recognition by the Radical SAM Enzyme RlmN

“…A unique feature of these enzymes is their ability to utilize both homolytic and heterolytic reactivity of SAM to carry out methylation of the C2 and C8 amidine carbons of adenosine [19–21]. The first equivalent of SAM is used to methylate a conserved cysteine residue (C355), unassociated with the four iron-four sulfur ([4Fe-4S]) cluster, to form a protein-bound methyl thioether [19,22–24]. A second equivalent of SAM, coordinated by the [4Fe-4S] cluster in these proteins, is then cleaved homolytically to generate a 5′-deoxyadenosyl radical (5′-dA•), a canonical feature of radical SAM catalysis [25].…”

“…This highly reactive radical species then abstracts a hydrogen atom from the premethylated cysteine 355 to form a thiomethylene radical. The methylene radical then adds into the substrate carbon to form a covalent RNA-protein adduct, which has been trapped by mutagenesis [22] and characterized spectroscopically [23,24]. A second conserved cysteine residue resolves the covalent RNA-protein intermediate, forming the methylated product.…”