A Conserved Lysine in β‐Lactam Synthetase Assists Ring Cyclization: Implications for Clavam and Carbapenem Biosynthesis

β‐Lactam synthetase (β‐LS) is the paradigm of a growing class of enzymes that form the critical β‐lactam ring in the clavam and carbapenem antibiotics. β‐LS catalyzes a two‐stage reaction in which N²‐(2‐carboxyethyl)‐L ‐arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to β‐lactam formation is high, and that the overall rate of reaction is partially limited to a protein conformational change rather than to the chemical step alone of closing the strained ring. β‐Lactam formation was evaluated on the basis of X‐ray crystal structures, site‐specific mutation, and kinetic and computational studies. The combined evidence clearly points to a reaction coordinate involving the formation of a tetrahedral transition state/intermediate stabilized by a conserved Lys. The combination of substrate preorganization, a well‐stabilized transition state and an excellent leaving group facilitates this acyl substitution to account for the strong forward commitment to catalysis and to lower the barrier of four‐membered ring formation to the magnitude of a protein conformational change.