Please use this identifier to cite or link to this item: http://hdl.handle.net/20.500.12779/5947
Title: Evolution to carbapenem -hydrolyzing activity in noncarbapenemase class D beta-lactamase OXA-10 by rational protein design
Authors: DE LUCA, Filomena
Benvenuti, Manuela 
Carboni, F
Pozzi, Cecilia 
Rossolini, GIAN MARIA
Mangani, Stefano 
Docquier, JEAN DENIS
Keywords: antibiotic resistance; protein engineering; protein evolution; X-ray structure
Issue Date: 2011
Project: None 
Journal: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.
Description: 
21851
URI: http://hdl.handle.net/20.500.12779/5947
ISSN: 0027-8424
DOI: 10.1073/pnas.1110530108
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