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Pathway elucidation by in vitro analyses and theoretical investigations. a) Mechanistic probes and their conversion by AsqJ to obtain first mechanistic insights. b) Proposed enzymatic pathways P1 and P2 to the newly discovered product class. c) Calculated AsqJ model for P1 versus P2 at the uB3LYP‐D3(BJ)/def2‐TZVP//uB3LYP‐D3(BJ)/def2‐SVP level of theory. Pathway P1 is strongly preferred with a low barrier for α‐lactam formation.
