| Carbapenem antibiotics are among the most recently discovered beta-lactam classes and they exhibit a broad spectrum of antibacterial activity as well as relative resistance to most clinically encountered beta-lactamases. Thienamycin was the first naturally occurring carbapenem isolated from Streptomycetes in 1976. Since then, more than 60 structurally related carbapenems have been identified. Two Gram negative bacteria, Serratia sp. ATCC 39006 and Pectobacterium carotovorum, were later shown to produce the simple carbapenem nucleus: (5R)-carbapen-2-em-3-carboxylic acid and two related beta-lactam containing compounds (3S,5 S)- and (3S,5R)-carbapenam-3-carboxylic acids. Unlike the carbapenem, the other two homologous saturated carbapenams both lack antibacterial activity and are resistant to beta-lactamases. Since the isolation of the carbapenem and the carbapenams, little information has been reported regarding the course of carbapenem and carbapenam assembly either in vivo or in vitro. Confusion also persisted in the literature about the signs of rotation and absolute configurations of the carbapenams that are finally resolved here.; In this work, we report the utilization of a series of Escherichia coli transformants harboring various genes of the carbapenem pathway to elucidate the assembly of the carbapenem antibiotic which takes place through the action of just three enzymes, CarA, B and C. This efficient biosynthetic process is accompanied by a remarkable stereoinversion at the bridgehead stereocenter mediated by CarC, which has modest homology to clavaminate synthase from the clavulanic acid biosynthetic pathway. L-Proline labeled with either deuterium or tritium at the C-5 diastereotopic methylene loci was shown to incorporate label into the (3S,5S)-carbapenem-3-carboxylic acid, but not into the "inverted" (3S,5 R)-carbapenem-3-carboxylic acid or the carbapenem antibiotic product. Synthetic methods were developed to synthesize both the (3S,5 S)- and (3S,5R)-carbapenem-3-carboxylates for in vitro assays with purified CarC. The results of these tests demonstrated that CarC is an alpha-ketoglutarate dependent oxygenase, in accord with the predicted homology with clavaminate synthase.; Purification and crystallization of CarA, which has good homology to beta-lactam synthetase from the clavulanic acid biosynthetic pathway, shows unambiguously the stereochemistry of the preferred substrate. The synthesis of the substrate for CarA and in vitro assay testing with alternate substrates is also described. |
