Covalent Intermediate in the Catalytic Mechanism of the Radical S

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 CX(3)CX(2)C 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

Elucidation of the biosynthesis of the methane catalyst coenzyme F430

Methane biogenesis in methanogens is mediated by methyl-coenzyme M reductase, an enzyme that is also responsible for the utilization of methane through anaerobic methane oxidation. The enzyme uses an ancillary factor called coenzyme F430, a nickel-containing modified tetrapyrrole that promotes catalysis through a methyl radical/Ni(II)-thiolate intermediate. However, it is unclear how coenzyme F430 is synthesized from the common primogenitor uroporphyrinogen III, incorporating 11 steric centres into the macrocycle, although the pathway must involve chelation, amidation, macrocyclic ring reduction, lactamization and carbocyclic ring formation. Here we identify the proteins that catalyse the biosynthesis of coenzyme F430 from sirohydrochlorin, termed CfbA–CfbE, and demonstrate their activity. The research completes our understanding of how the repertoire of tetrapyrrole-based pigments are constructed, permitting the development of recombinant systems to use these metalloprosthetic groups more widely