Mechanism of RadicalS-Adenosyl-L-methionine Adenosylation:Radical Intermediates and the Catalytic Competence of the 5′-Deoxyadenosyl Radical

RadicalS-adenosyl-L-methionine (SAM) enzymes employa [4Fe-4S] cluster and SAM to initiate diverse radical reactions via eitherH-atom abstraction or substrate adenosylation. Here we use freeze-quench techniques together with electron paramagnetic resonance (EPR)spectroscopy to provide snapshots of the reaction pathway in anadenosylation reaction catalyzed by the radical SAM enzyme pyruvateformate-lyase activating enzyme on a peptide substrate containing adehydroalanine residue in place of the target glycine. The reactionproceeds via the initial formation of the organometallic intermediate Omega,asevidenced by the characteristic EPR signal withg parallel to= 2.035 andg perpendicular to= 2.004observed when the reaction is freeze-quenched at 500 ms. Thermalannealing of frozen Omega converts it into a second paramagnetic species centered atgiso= 2.004; this second species was generateddirectly using freeze-quench at intermediate times (similar to 8 s) and unequivocally identified via isotopic labeling and EPR spectroscopy asthe tertiary peptide radical resulting from adenosylation of the peptide substrate. An additional paramagnetic species observed insamples quenched at intermediate times was revealed through thermal annealing while frozen and spectral subtraction as the SAM-derived 5 '-deoxyadenosyl radical (5 '-dAdo center dot). The time course of the 5 '-dAdo center dot and tertiary peptide radical EPR signals reveals thatthe former generates the latter. These results thus support a mechanism in which Omega liberates 5 '-dAdo center dot by Fe-C5 ' bond homolysis,and the 5 '-dAdo center dot attacks the dehydroalanine residue of the peptide substrate to form the adenosylated peptide radical species. Theresults thus provide a picture of a catalytically competent 5 '-dAdo center dot intermediate trapped just prior to reaction with the substrate.

Automated summary

Using freeze-quench techniques and electron paramagnetic resonance (EPR) spectroscopy, the reaction pathway of an adenosylation reaction catalyzed by the radical SAM enzyme is studied. The results provide snapshots of the process, revealing the formation of organic intermediate Omega and the generation of 5'-deoxyadenosyl radical and adenosylated peptide radical. These findings help to understand the mechanism of the reaction.