Ullah, Jakir Hussain; (1999) The determination of the crystal structure of the metallo β-lactamase from Stenotrophomonas maltophilia and mechanistic studies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The discovery of penicillin and its subsequent development for clinical use against bacterial infections was initially hailed as a magic bullet. However, it was soon apparent that this was not the case because of the appearance of penicillin resistant bacteria. β-lactam antibiotics, such as penicillin, function by inhibiting cell wall synthesis in growing bacterial cells. The major mechanism of resistance in bacteria to the β-lactams is mediated by β-lactamase production. These enzymes bind β-lactams with high affinity and hydrolyse the lactam ring, thus causing inactivation. Metallo β-lactamases are a potential problem to the efficiency of current β-lactam antibiotics. This is because they have a broader spectrum of activity than the more common serine β-lactamases and there are, as yet, no clinically useful inhibitors of the metallo β-lactamases. Currently, metallo β-lactamases have been found in only a few species that are pathogenic. However, recent reports of the plasmid mediated transfer of metallo β-lactamase genes between bacteria suggest that these genes may spread to pathogens that are more serious and thereby, severely compromise current antibiotic therapy. The work presented here describes the crystal structure of the metallo β-lactamase L1 from Stenotrophomonas maltophilia, which was determined to 1.7A resolution. The structure was solved using multiwavelength anomalous dispersion from data collected at the absorption edge of the intrinsic zincs and substituted seleno-methionine residues. Phasing was carried out by treating the data at different wavelengths as a special case of multiple isomorphous replacement with anomalous scattering (MIRas). The crystal structure showed that L1 was a tetramer, which was also confirmed by sedimentation equilibrium studies. The overall fold is composed of two roughly equivalent domains consisting of a β-sheet surrounded by α-helices. The active site is formed in a grove above the interface of the core β sheets and binds two zinc ions. Molecular modelling of substrates suggests that binding maybe facilitated by both the active site zincs. We propose that the substrate is bound to the enzymes via the zinc ions; hydrophobic residues near the active site then contribute to the binding of the various side chains of the β-lactams. The reaction is likely to proceed by nucleophilic attack by a zinc bound hydroxide ion on the β-lactam carbonyl to form the transition state. This complex is subsequently degraded by the donation of a proton, probably from a well positioned water molecule, to the β-lactam nitrogen.

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