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Estructura cristalográfica de la B-lactamasa oxa-24: bases estructurales que determinan su actividad carbapenemasa

AuthorsSantillana, Elena
AdvisorRomero, Antonio
Carbapenem resistance
Enzyme mechanism
Protein crystallography
Acinetobacter baumannii
Issue DateJan-2014
PublisherCSIC - Centro de Investigaciones Biológicas Margarita Salas (CIB)
Universidad Autónoma de Madrid
AbstractOXA-24 is a class D carbapenem-hydrolyzing oxacillinase extracted from a clinically epidemic multidrug-resistant Acinetobacter baumannii strain. In contrast to other oxacillinases, most of these OXA-type carbapenemases do not present hydrolytic activity against oxacillin, cloxacillin, and methicillin, and they display activity against carbapenems. Moreover, these enzymes are widely dispersed in clinically relevant species, such as A. baumannii and Pseudomonas aeruginosa. Carbapenems are the remaining drug of choice to treat these multiresistant pathogens responsible for severe nosocomial infections. Thus, the emergence of OXA-type carbapenemases represents a particularly disturbing problem. The native form of OXA-24 is a monomer with 275 residues. OXA-24 folds into an α/β structure composed by two domains, one helical and one mixed α/β containing a six stranded antiparallel β-sheet covered by the N- and C-terminal α- helices. The active site of OXA-24 lies in a cleft at the interface of the β-sheet and the helical domain and is composed of three highly conserved motifs in all Class D β- lactamases, which are referred to as the active site elements 1 (S81-T82-F83-K84), 2 (S128-A129-V130) and 3 (K218-S219-G220). The first element is located at the N-terminal end of the 310 helix 3 and contains the catalytic serine (S81) and the carboxylated lysine (K84), which plays a regulatory role in the acylation- deacylation process. The second motif is located at the loop connecting helices 4 and 5 and the third active site element is made up by residues from the β4 strand. The interaction between Y112 and M223 is essential for the carbapenem specificity in OXA-24. The interaction between these residues creates a hydrophobic barrier that limits the access to the catalytic site, defining a tunnel- like entrance to the active site. The substrate is anchored into the tunnel-like cavity of the binding site through the Y112 that binds the 6-hydroxyethyl group of the carbapenemic antibiotics, playing an important role in the catalytic efficiency of OXA-24 for carbapenems. The key role of the Y112 and the M223 has been confirmed by mutagenesis studies. The substitution of the residues Y112 and M223 by alanine eliminates the hydrophobic barrier essential for the carbapenem specificity and decreased significantly the hydrolytic activity of OXA-24 against carbapenems. When both amino acids are mutated, the MIC for imipenem and meropenem decreased and reached basal levels, respectively. Biochemical experiments also confirm the lower catalytic efficiency of the mutants for carbapenems.
Class D carbapenemases are usually resistant to commercial β-lactamase inhibitors, so new effective inhibitors are urgently needed. Three unique 6- alkylidene-2 ́- substituted penicillanic acid sulfones were synthesized and tested against OXA-24. The X-ray structures of OXA-24 in complex with these compounds reveal the formation of stable bicyclic aromatic intermediates with their carbonyl oxygen in the oxyanion hole. The position of the inhibitors was clearly defined in the active site through its conjugated acyl group covalently attached to the catalytic serine (S81). The conformation of the active site is very similar to that of the native enzyme, but several conformational changes can be observed in some residues involved directly in the accommodation of the inhibitor. A network of interactions contributes to stabilize the intermediate state. The stable acyl-enzyme complexes from the 6- alkylidene-2′-substituted penicillin sulfone inhibitors are formed by a sequence of events in which the initial nucleophilic attack of S81 Oγ on the carbonyl of the penicillin ring releases the β-lactam nitrogen lone pair, thus enabling the opening of the neighboring sulfone ring. Another rearrangement provided by the pyridyl nitrogen bonded to the former C5 of the resultant imine leads to the formation of the crystallographically observed indolizine system. These data provide us with the first structural evidence that 6-alkylidene-2 ́- substituted penicillin sulfones are effective mechanism-based inactivators of class D β-lactamases.
Description146 p.-77 fig.-18 tab.
Appears in Collections:(CIB) Tesis
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