The proposed biosynthetic pathway to the carbapenem antibiotics proceeds via epimerization/desaturation of a carbapenam in an unusual process catalyzed by an iron-and 2-oxoglutarate-dependent oxygenase, CarC. Crystal structures of CarC complexed with Fe(II) and 2-oxoglutarate reveal it to be hexameric (space group C222 1 ), consistent with solution studies. CarC monomers contain a double-stranded ␤-helix core that supports ligands binding a single Fe(II) to which 2-oxoglutarate complexes in a bi-dentate manner. A structure was obtained with L-N-acetylproline acting as a substrate analogue. Quantum mechanical/molecular mechanical modeling studies with stereoisomers of carbapenams and carbapenems were used to investigate substrate binding. The combined work will stimulate further mechanistic studies and aid in the engineering of carbapenem biosynthesis.

Carboxymethylproline synthase (CarB) catalyzes the committed step in the biosynthesis of (R)-1-carbapen-2-em-3-carboxylate, the simplest member of the carbapenem family of ␤-lactam antibiotics, some of which are used clinically. CarB displays sequence homology with members of the crotonase family including enoyl-CoA hydratase (crotonase) and methylmalonyl-CoA decarboxylase. The CarB reaction has been proposed to comprise condensation of acetyl coenzyme A (AcCoA) and glutamate semi-aldehyde to give (2S,5S)-carboxymethylproline ((2S,5S)-CMP). (2S,5S)-CMP is then cyclized in an ATP-driven reaction catalyzed by CarA to give a carbapenam, which is subsequently epimerized and desaturated to give a carbapenem in a CarC-mediated reaction. Here we report the purification of recombinant CarB and that it exists predominantly in a trimeric form as do other members of the crotonase family. AcCoA was not found to be a substrate for CarB. Instead malonyl-CoA was found to be a substrate, efficiently producing (2S,5S)-CMP in the presence of glutamate semi-aldehyde. In the absence of glutamate semi-aldehyde, mass spectrometric analysis indicated that CarB catalyzed the decarboxylation of malonyl-CoA to AcCoA. The reactions of CarB, CarA, and CarC were coupled in vitro demonstrating the viability of malonyl-CoA as a carbapenem precursor. CarB was also shown to accept methylmalonyl CoA as a substrate to form 6-methyl-(2S,5S)-CMP, which in turn is a substrate for CarA. The implications of the results for the biosynthesis of both carbapenem-3-carboxylate and C-2/C-6-substituted carbapenems, such as thienamycin, are discussed.

The enzyme-catalysed reactions involved in formation of the bicyclic clavam and carbapenem nuclei, including beta-amino acid and beta-lactam formation, are discussed and compared with those involved in penicillin and cephalosporin biosynthesis. The common role of unusual oxidation reactions in the biosynthetic pathways and the lack of synthetic reagents available to effect them are highlighted.

There are four main subfamilies of clinically used bicyclic ␤-lactam antibiotics comprised of the penicillins, the cephalosporins, the carbapenems, and the oxacephems. Three inhibitors of the serine ␤-lactamases have also found extensive clinical use and although these compounds are themselves bicyclic ␤-lactams, they possess limited antibiotic activity; hence are used in conjunction with a penicillin. With

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