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Substrate specificity and inhibitor resistance in OXA-class beta-lactamases

Reference	BB/C505916/1
Principal Investigator / Supervisor	Professor James Spencer
Co-Investigators / Co-Supervisors	Professor Anthony Clarke, Dr Timothy Walsh
Institution	University of Bristol
Department	Cellular and Molecular Medicine
Funding type	Research
Value (£)	230,536
Status	Completed
Type	Research Grant
Start date	23/08/2004
End date	22/09/2007
Duration	37 months

Abstract

Gram-negative pathogens such as Acinetobacter baumanii and Pseudomonas aeruginosa are responsible for an increasing number of opportunist nosocomial infections in severely immunocompromised patients. Beta-lactam antibiotics such as later generation cephalosporins and carbapenems are among the drugs of choice in such cases, where high levels of intrinsic resistance mean that treatment options are already limited. In this context the emergence and dissemination on mobile genetic elements of the class D (OXA) beta lactamases represents a considerable threat to the continued efficacy of these treatments. The class D enzymes are a diverse family of broad-spectrum serine beta lactamases that are generally not inhibited by clavulanic acid and whose mechanism of action remains poorly understood. An increasing number of class D enzymes display significant carbapenemase activity. We will study two uncharacterised class D enzymes with unusual properties. OXA-23 from A. baumanii is the prototypical class D carbapenemase whilst OXA-45 from P. aeruginosa is one of only two known class D enzymes that retain sensitivity to clavulanic acid. The comprehensive structural and functional dissection of these two enzymes that we propose will therefore elucidate the basis for clavulanate resistance and carbapenemase activity within the wider enzyme family. For both enzymes we will perform a comprehensive steady-state kinetic characterisation and use selected beta lactam substrates in stopped-flow and quenched-flow experiments to establish a minimal kinetic mechanism. We will use X-ray crystallography to determine structures of both enzymes either by molecular replacement or by anomalous scattering techniques. Crystals of OXA-45 will be soaked with substrates such as carbapenems and clavulanate to obtain structures of acylenzyme complexes. These data will illuminate the means by which class D enzymes achieve catalysis and enable us to identify variations in the enzyme active site likely toaccount for the observed differences in specificity. These conclusions will be tested and validated by a programme of mutagenic experiments. Upon the basis of available sequence and structural information we propose that a number of positions contribute to catalytic efficiency and specificity switching within the OXA enzyme family. We will study the effects on acylation and deacylation of substitutions within the omega and Ser-115-Val-117 loops; on clavulanate sensitivity of the presence of Met at 66 (adjacent to the Ser-67 nucleophile) and on carbapenemase activity of the Tyr-141-Phe switch and of alterations within the omega loop. These will be complemented by additional mutations identified from our structural work. Deacylation-deficient mutations of OXA-23 will be used in experiments to obtain structures of acylenzyme complexes. The understanding of the molecular recognition processes operating within the class D beta lactamase family that these data will provide will enable the development of pharmaceutical lead compounds, either new generations of beta-lactams with reduced susceptibility to hydrolysis by class D enzymes of specific class D inhibitors for use in combination therapies. Such agents will be of value in the treatment of nosocomial infections by Gram negative opportunists.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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