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.
Raber
Mary L
ML
Department of Chemistry, The Johns Hopkins University, Baltimore, MD 21218, USA.
Castillo
Alvaro
A
Greer
Alexander
A
Townsend
Craig A
CA
D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D015780:Carbapenems / Q000096:biosynthesis*; D002969:Clavulanic Acids / Q000096:biosynthesis*; D018360:Crystallography, X-Ray; D003500:Cyclization; D005030:Ethylenes / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000737:chemistry; D007700:Kinetics; D008239:Lysine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016415:Sequence Alignment; D013816:Thermodynamics
D000900:Anti-Bacterial Agents; D015780:Carbapenems; D002969:Clavulanic Acids; D005030:Ethylenes; D007659:Ketones; C483963:clavam; D000581:Amidohydrolases; C114020:beta-lactam synthetase; D008239:Lysine; C008223:ketene