Identification and characterization of beta-lactam synthetase of clavulanic acid and biosynthesis

The biosynthesis of the β-lactam ring in many natural products is thought to entail enzyme-catalyzed intramolecular acyl-addition of an amine to an activated β-carboxylate. In the case of clavulanic acid biosynthesis in Streptomyces clavuligerus, it was envisioned that the β-amino acid N2 (carboxyethyl)arginine (CEA) would cyclize to form the known clavulanate precursor deoxyguanidoproclavaminate (DGPC). Analysis of the clavulanic acid biosynthetic gene cluster identified a gene ( orf3), the translated product of which shows significant homology to Class B asparagine synthetases. These enzymes activate aspartate for intermolecular acyl-addition of glutamyl-derived ammonia, suggesting a possible role for Orf 3 in β-lactam formation. Targeted disruption of orf3 in S. clavuligerus led to the accumulation of CEA, and complete cessation of clavulanic acid production, which could be restored by chemical complementation with authentic DGPC. Partially purified recombinant Orf 3 was demonstrated to catalyze the in vitro conversion of CEA to DGPC in the presence of ATP/Mg2+. Therefore, the orf3 encoded protein was named β-lactam synthetase (β-LS). The recombinant β-LS was purified to homogeneity in sufficient quantity to allow initial X-ray crystallographic studies and detailed biochemical characterization. Extensive kinetic studies led to the proposal of a detailed kinetic mechanism for β-lactam formation, and allowed a rigorous chemical mechanism for the enzyme to be put forth. Furthermore, a series of substrate analogs were synthesized and evaluated as substrates of the enzyme. These results shed light on important substrate-enzyme interactions in the active site of β-LS, providing a preliminary map of the CEA binding site and partially exploring product lassitude. β-Lactam synthetase from the clavulanic acid pathway is the first of a new class of enzymes, and future studies will provide valuable insight into the biosynthesis of a large number of β-lactam containing natural products.