ABSTRACT
A cunning and dangerous plan foiled! An enzyme-specific molecular construct exploits the overexpression of beta-lactamase in several drug-resistant bacteria. Specific photodynamic toxicity was detected towards beta-lactam-resistant methicillin-resistant Staphylococcus aureus (MRSA), whereby the usual mechanism for antibiotic resistance (cleavage of the beta-lactam ring) releases the phototoxic component from the prodrug (see picture; Q = quencher).
Subject(s)
Methicillin-Resistant Staphylococcus aureus/radiation effects , beta-Lactamases/radiation effects , Kinetics , Methicillin-Resistant Staphylococcus aureus/metabolism , Microbial Sensitivity Tests , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Prodrugs/chemistry , Prodrugs/pharmacology , beta-Lactamases/metabolism , beta-Lactamases/toxicityABSTRACT
Enzyme functions such as catalysis, binding and regulation are directly related to a variety of conformational changes. A sensitive and useful method for their investigation is circular dichroism (CD) and a rotational strength (R) is its fundamental characteristic. In this study, we show how the sensitivity of the mechanisms of rotational strengths to important conformational changes depends on the chromophore environment in two beta-lactamases from class A (from Escherichia coli and B. licheniformis). Rotational strengths have been calculated using the matrix method and including the effects of local environment (LE). X-ray structures (of protein components) of several enzyme-ligand complexes from the catalytic cycle of the TEM-1 enzyme and for both crystallographic monomers of the enzyme from B. licheniformis were used. An analysis of the relative degree of perturbation of the rotational strengths upon local interactions is performed.
Subject(s)
Computer Simulation , beta-Lactamases/chemistry , Bacillus/classification , Bacillus/enzymology , Catalysis , Computational Biology/methods , Crystallography, X-Ray , Escherichia coli/classification , Escherichia coli/enzymology , Ligands , Models, Molecular , Optical Rotation , Protein Conformation , Protein Structure, Tertiary , Sensitivity and Specificity , Structure-Activity Relationship , Ultraviolet Rays , beta-Lactamases/radiation effectsABSTRACT
Bacterial beta-lactamases hydrolyze beta-lactam antibiotics such as penicillins and cephalosporins. The TEM-type class A beta-lactamase SHV-2 is a natural variant that exhibits activity against third-generation cephalosporins normally resistant to hydrolysis by class A enzymes. SHV-2 contains a single Gly238Ser change relative to the wild-type enzyme SHV-1. Crystallographic refinement of a model including hydrogen atoms gave R and R(free) of 12.4% and 15.0% for data to 0.91 A resolution. The hydrogen atom on the O(gamma) atom of the reactive Ser70 is clearly seen for the first time, bridging to the water molecule activated by Glu166. Though hydrogen atoms on the nearby Lys73 are not seen, this observation of the Ser70 hydrogen atom and the hydrogen bonding pattern around Lys73 indicate that Lys73 is protonated. These findings support a role for the Glu166-water couple, rather than Lys73, as the general base in the deprotonation of Ser70 in the acylation process of class A beta-lactamases. Overlay of SHV-2 with SHV-1 shows a significant 1-3 A displacement in the 238-242 beta-strand-turn segment, making the beta-lactam binding site more open to newer cephalosporins with large C7 substituents and thereby expanding the substrate spectrum of the variant enzyme. The OH group of the buried Ser238 side-chain hydrogen bonds to the main-chain CO of Asn170 on the Omega loop, that is unaltered in position relative to SHV-1. This structural role for Ser238 in protein-protein binding makes less likely its hydrogen bonding to oximino cephalosporins such as cefotaxime or ceftazidime.
