A conserved lysine in beta-lactam synthetase assists ring cyclization: Implications for clavam and carbapenem biosynthesis.

beta-Lactam synthetase (beta-LS) is the paradigm of a growing class of enzymes that form the critical beta-lactam ring in the clavam and carbapenem antibiotics. beta-LS catalyzes a two-stage reaction in which N(2)-(2-carboxyethyl)-L-arginine is first adenylated, and then undergoes intramolecular ring closure. It was previously shown that the forward kinetic commitment to beta-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. beta-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.