RESUMO
The evolution of multidrug resistance in Acinetobacter spp. increases the risk of our best antibiotics losing their efficacy. From a clinical perspective, the carbapenem-hydrolyzing class D ß-lactamase subfamily present in Acinetobacter spp. is particularly concerning because of its ability to confer resistance to carbapenems. The kinetic profiles of class D ß-lactamases exhibit variability in carbapenem hydrolysis, suggesting functional differences. To better understand the structure-function relationship between the carbapenem-hydrolyzing class D ß-lactamase OXA-24/40 found in Acinetobacter baumannii and carbapenem substrates, we analyzed steady-state kinetics with the carbapenem antibiotics meropenem and ertapenem and determined the structures of complexes of OXA-24/40 bound to imipenem, meropenem, doripenem, and ertapenem, as well as the expanded-spectrum cephalosporin cefotaxime, using X-ray crystallography. We show that OXA-24/40 exhibits a preference for ertapenem compared with meropenem, imipenem, and doripenem, with an increase in catalytic efficiency of up to fourfold. We suggest that superposition of the nine OXA-24/40 complexes will better inform future inhibitor design efforts by providing insight into the complicated and varying ways in which carbapenems are selected and bound by class D ß-lactamases.
Assuntos
Proteínas de Bactérias , Carbapenêmicos , beta-Lactamases , Acinetobacter baumannii/enzimologia , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carbapenêmicos/química , Carbapenêmicos/metabolismo , Hidrólise , Testes de Sensibilidade Microbiana , Conformação Proteica , Especificidade por Substrato , beta-Lactamases/química , beta-Lactamases/metabolismoRESUMO
P99 cephalosporinase is a class C ß-lactamase that is responsible in part for the widespread bacterial resistance to ß-lactam antibiotics. Mutations of the conserved active-site residue Asn152 of the enzyme have been shown to alter ß-lactam substrate specificity in vivo. Mutation of Asn152 to a glycine is notable in that it exhibits in vivo substrate-selectivity switching. In order to better understand the structural basis for this observed switch, the X-ray crystal structure of the apo Asn152Gly mutant of P99 was determined to 1.95 Å resolution. Unexpectedly, the artificial C-terminal His(6) tag of a symmetrically-related molecule was observed bound in the active site. The His(6) tag makes several interactions with key active-site residues, as well as with several sulfate ions. Additionally, the overall C-terminus occupies the space left vacant upon the mutation of Asn152 to glycine.
