Epsilon Open Archive

Abstract

The discovery of penicillin, a beta-lactam antibiotic, changed the way humans thought about infectious disease. Unfortunately, the widespread use of antibiotics has lead to a concomitant increase in bacterial resistance to these drugs. One of the most common mechanisms of bacterial resistance to beta-lactam antibiotics is mediated through the hydrolysis of the beta-lactam ring by beta-lactamases. In order to combat resistance two strategies have been employed: identification of antibiotics resistant to hydrolysis by beta-lactamases, and the development of beta-lactamase inhibitors. This thesis describes studies on enzymes/proteins in the biosynthetic pathways of beta-lactam antibiotics and beta-lactamase inhibitors. DAOCS is a non-heme Fe(II) dioxygenase that catalyses the oxidative ring expansion of the penicillins to cephalosporins. The expansion of the five-membered penicillin ring to a six-membered cephem ring provides increased resistance to beta-lactamases. The work described here led to the production of crystals with an alternate packing of molecules (belonging to a new space group), which did not show twinning, an anomaly hampering previous structural work. Clavulanic acid is a potent inhibitor of class A bacterial beta-lactamases. CAD is a short chain reductase responsible for the catalysis of the penultimate step in clavulanic acid biosynthesis: the NADPH-dependent reduction of the unstable intermediate clavulanate-9-aldehyde to clavulanic acid. Structures of CAD in complex with the NADPH co-factor, and clavulanic acid, are described here, leading to a proposed reaction mechanism, and an increased understanding of how the enzyme is able to catalyse a reaction involving such a labile intermediate. Approximately half of the genes in the clavulanic acid biosynthesis gene cluster are open reading frames, without a known function. The gene product of Orf15 has been shown to be essential for clavulanic acid production. The structure reported here reveals that it shares similarity with substrate-binding proteins. The complex of ORF15 with L-arginine, a precursor of clavulanic acid, suggests multiple roles for the protein.