title: Evolution to carbapenem-hydrolyzing activity in noncarbapenemase class D β-lactamase OXA-10 by rational protein design.
abstract: Class D β-lactamases with carbapenemase activity are emerging as carbapenem-resistance determinants in gram-negative bacterial pathogens, mostly Acinetobacter baumannii and Klebsiella pneumoniae. Carbapenemase activity is an unusual feature among class D β-lactamases, and the structural elements responsible for this activity remain unclear. Based on structural and molecular dynamics data, we previously hypothesized a potential role of the residues located in the short-loop connecting strands β5 and β6 (the β5-β6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D β-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the β5-β6 loop with those present in three different lineages of class D carbapenemases (OXA-23, OXA-24, and OXA-48). Biological assays and kinetic measurements showed that all three OXA-10-derived hybrids acquired significant carbapenemase activity. Structural analysis of the OXA-10loop24 and OXA-10loop48 hybrids revealed no significant changes in the molecular fold of the enzyme, except for the orientation of the substituted β5-β6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the β5-β6 loop in the carbapenemase activity of class D β-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.
authors: Dipartimento di Biotecnologie, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100 Siena, Italy.
authors: Filomena
authors: F
authors: De Luca
authors: Manuela
authors: M
authors: Benvenuti
authors: Filippo
authors: F
authors: Carboni
authors: Cecilia
authors: C
authors: Pozzi
authors: Gian Maria
authors: GM
authors: Rossolini
authors: Stefano
authors: S
authors: Mangani
authors: Jean-Denis
authors: JD
authors: Docquier
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D015780:Carbapenems / Q000378:metabolism*; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006868:Hydrolysis; D007700:Kinetics; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015780:Carbapenems; D017931:DNA Primers; C425107:beta-lactamase OXA-10; D001618:beta-Lactamases