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

ABSTRACT: Class D ?-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 ?-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 ?5 and ?6 (the ?5-?6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D ?-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the ?5-?6 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 ?5-?6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the ?5-?6 loop in the carbapenemase activity of class D ?-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.

SUBMITTER: De Luca F 

PROVIDER: S-EPMC3215043 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature