Evolution to carbapenem-hydrolyzing activity in noncarbapenemase class D β-lactamase OXA-10 by rational protein design

Filomena De Luca, Manuela Benvenuti, Filippo Carboni, Cecilia Pozzi, Gian Maria Rossolini, Stefano Mangani, Jean Denis Docquier

Research output: Contribution to journalArticle

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.

Original languageEnglish
Pages (from-to)18424-18429
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Issue number45
DOIs
Publication statusPublished - Nov 8 2011

Keywords

  • Antibiotic resistance
  • Protein engineering
  • Protein evolution
  • X-ray structure

ASJC Scopus subject areas

  • General

Fingerprint Dive into the research topics of 'Evolution to carbapenem-hydrolyzing activity in noncarbapenemase class D β-lactamase OXA-10 by rational protein design'. Together they form a unique fingerprint.

  • Cite this