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

• Published in: Chembiochem : a European journal of chemical biology Vol. 10; no. 18; pp. 2904 - 2912
• Main Authors: Raber, Mary L, Castillo, Alvaro, Greer, Alexander, Townsend, Craig A
• Format: Journal Article
• Language: English
• Published: Weinheim Wiley-VCH Verlag 14.12.2009; WILEY-VCH Verlag; WILEY‐VCH Verlag
• Subjects: Amidohydrolases - chemistry; Amidohydrolases - metabolism; Amino Acid Sequence; Amino Acid Substitution; Anti-Bacterial Agents - biosynthesis; beta-lactam; biosynthesis; Carbapenems - biosynthesis; clavulanic acid; Clavulanic Acids - biosynthesis; Crystallography, X-Ray; Cyclization; density functional theory; Ethylenes - chemistry; Hydrogen-Ion Concentration; Ketones - chemistry; Kinetics; Lysine - metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; pH-rate profiles; Sequence Alignment; Thermodynamics
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.200900389

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.