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Title: Crystallographic studies of the metallo-β-lactamase from Bacillus cereus
Author: Davies, Anna Marie
Awarding Body: University of London
Current Institution: King's College London (University of London)
Date of Award: 2005
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An emerging threat to the efficacy of commonly used β-lactam antibiotics is the bacterial production of metallo-β-lactamases. This family of enzymes, which require Zn²⁺ for activity, is unrelated to the serine β-lactamases, for which inhibitors have been developed. The metallo-β-lactamases are characterized by a broad substrate specificity and are unaffected by the serine β-lactamase inhibitors. Their mechanism is not well understood and there are no clinical inhibitors available. A better understanding of how these enzymes function is crucial if effective inhibitors are to be designed. Whilst all members of the metallo-β-lactamase family require Zn²⁺ ions for activity, they differ in their affinity for Zn²⁺ and the number of Zn²⁺ ions required for catalytic activity. The metallo-β-lactamase from Bacillus cereus is characterized by one higher affinity, and one lower affinity Zn²⁺ binding site (termed Zn1 and Zn2 respectively). Although only one Zn²⁺ ion is necessary for activity, maximum activity is achieved when both are present. Activity is also pH-dependent, decreasing at lower pH values. It has been proposed that a positively charged arginine residue (Arg121) located beneath the Zn2 binding site is responsible for the lower affinity of this enzyme for Zn²⁺ at this site, since other members of the family lacking this arginine residue bind two Zn²⁺ ions with equal, and higher, affinity. This thesis describes crystallographic studies of three B. cereus metallo-β-lactamase mutants, Arg121Cys, Arg121Glu and Arg121His, to investigate the binding of Zn2 in particular, and the mechanism in general. Each mutant has been studied in both mono- and bizinc forms at different pH values. Crystallographic evidence is presented in support of a proposed enzyme mechanism, in which Zn2 binding is flexible. A tri-zinc form of the enzyme is also described. This thesis also presents work relating to the crystallisation. and preliminary X-ray diffraction analysis of a putative phosphinothricin acetyltransferase from Pseudomonas aeruginosa.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available