posted on 2022-03-08, 18:07authored byMaike
N. Lundahl, Raymond Sarksian, Hao Yang, Richard J. Jodts, Adrien Pagnier, Donald F. Smith, Martín A. Mosquera, Wilfred A. van der Donk, Brian M. Hoffman, William E. Broderick, Joan B. Broderick
Radical S-adenosyl-l-methionine (SAM)
enzymes employ a [4Fe–4S] cluster and SAM to initiate diverse
radical reactions via either H-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 an adenosylation reaction catalyzed by the radical SAM
enzyme pyruvate formate-lyase activating enzyme on a peptide substrate
containing a dehydroalanine residue in place of the target glycine.
The reaction proceeds via the initial formation of the organometallic
intermediate Ω, as evidenced by the characteristic EPR signal
with g∥ = 2.035 and g⊥ = 2.004 observed when the reaction is freeze-quenched
at 500 ms. Thermal annealing of frozen Ω converts it into a
second paramagnetic species centered at giso = 2.004; this second species was generated directly using freeze-quench
at intermediate times (∼8 s) and unequivocally identified via
isotopic labeling and EPR spectroscopy as the tertiary peptide radical
resulting from adenosylation of the peptide substrate. An additional
paramagnetic species observed in samples quenched at intermediate
times was revealed through thermal annealing while frozen and spectral
subtraction as the SAM-derived 5′-deoxyadenosyl radical (5′-dAdo•).
The time course of the 5′-dAdo• and tertiary peptide
radical EPR signals reveals that the former generates the latter.
These results thus support a mechanism in which Ω liberates
5′-dAdo• by Fe–C5′ bond homolysis, and
the 5′-dAdo• attacks the dehydroalanine residue of the
peptide substrate to form the adenosylated peptide radical species.
The results thus provide a picture of a catalytically competent 5′-dAdo•
intermediate trapped just prior to reaction with the substrate.