| Clavulanic acid (CA) is a widely used beta-lactamase inhibitor whose biosynthesis is initiated by D-glyceraldehyde-3-phosphate ( D-G3P) and L-arginine. The thiamine diphosphate/Mg2+-dependent enzyme N2-(2-carboxyethyl)arginine synthase (CEAS) condenses these substrates to yield N2-(2-carboxyethyl)-arginine (CEA). The key beta-lactam-forming step is then catalyzed by beta-lactam synthetase, which synthesizes deoxyguanidinoproclavaminate (DGPC) from CEA in an ATP/Mg2+-dependent manner. Overall, this thesis illuminated key residues and mechanistic features involved in the early steps of CA biosynthesis in an effort to guide future engineering efforts on this and other evolutionarily-related pathways.;The steady-state kinetics of beta-LS were studied using site-directed mutagenesis, pH-rate profiles, solvent isotope effects, proton inventory, radioactivity assays, viscosity variation, and Erying plots. The high level of protein and substrate pre-organization previously noted from crystallographic snapshots of beta-LS is presumably achieved from an active-site loop (residues 444-453) that squeezes down on the substrates after CEA binding and subsequently relaxes upon completion of the catalytic cycle. From SIEs and viscosity variation, loop relaxation to the open form of beta-LS was determined to be partially rate-determining. The overall activation energy of the beta-LS reaction was determined to be ca. 20 kcal/mol, which supported a conformational change as rate-determining.;The first chemical step of acyl-adenylation was determined to be effectively irreversible at optimal pH (pH 8-9) from a large forward commitment to beta-lactam formation. Ring cyclization is then initiated by the phenoxide oxygen of Y348, which deprotonates the alpha-amino group of CEA to allow intramolecular, nucleophilic acyl substitution. beta-LS utilizes a reverse-protonation mechanism, which results in a functional catalytic dyad: Y348/E382. The protonation state of a conserved, active-site lysine present in many known beta-lactam synthetases (K443 in beta-LS) also proved important to beta-lactam formation and possibly relaxation of the catalytic loop. Its proton assistance during formation of a tetrahedral intermediate is proposed since studies on a nearby histidine ruled out the intermediacy of a ketene intermediate in DGPC formation.;In vitro and in vivo assays demonstrated that beta-LS possessed the proper catalytic machinery to function as a carbapenam synthetase. The road to engineering CA biosynthesis through native and altered substrate tolerance was also probed with biochemical studies on the first enzyme in the pathway, CEAS. |
