Structural Basis for Carbapenemase Activity of the OXA-23 β-Lactamase from Acinetobacter baumannii

Abstract

SUMMARY Dissemination of Acinetobacter baumannii strains harboring class D β-lactamases producing resistance to carbapenem antibiotics severely limits our ability to treat deadly Acinetobacter infections. Susceptibility determination in the A. baumannii background and kinetic studies with a homogeneous preparation of OXA-23 β-lactamase, the major carbapenemase present in A. baumannii, document the ability of this enzyme in manifesting resistance to the last-resort carbapenem antibiotics. We also report three x-ray structures of OXA-23; apo OXA-23 at two different pH values, and wild-type OXA-23 in complex with meropenem, a carbapenem substrate. This represents the first structure of a wild-type class D carbapenemase with a carbapenem antibiotic in its active site. The structures and dynamics simulations reveal an important role for Leu166, whose motion regulates the access of a hydrolytic water molecule to the acyl-enzyme species in imparting carbapenemase activity.

A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

“…Furthermore, the hydroxyethyl hydroxy group can adopt an orientation suitable for nucleophilic attack onto the ester carbonyl (i.e., the Bürgi–Dunitz trajectory),14 with the carbamylated lysine apparently positioned to act as a general base 12, 13. Although this conformation of the hydroxyethyl side chain was not observed in related crystal structures (Figure S27), these structures depict enzymes in which the lysine is not carbamylated (e.g., due to low pH or mutations) 5, 15, 16…”

“…The classical mechanism of the serine carbapenemases proceeds through a two‐step pathway involving the formation of an acyl‐enzyme intermediate, which is then hydrolyzed by a nucleophilic water molecule 3. All clinically used carbapenem antibiotics have a 6‐hydroxyethyl side chain, the presence of which is proposed to disrupt the hydrolysis step 4, 5, 6. We show that the class D β‐lactamases employ a previously unidentified mechanism for carbapenem degradation in which a β‐lactone is formed (Figure 1 A).…”

OXA β-Lactamases

“…The discovery of several bla OXA-23-like genes (bla , bla , bla , bla , bla , and bla OXA-134 ) on the chromosome of Acinetobacter radioresistens isolates indicates that this species is the likely natural source of this enzyme group (30)(31)(32). Comparison of the GC con- (34)(35)(36). The kinetic properties determined vary considerably between studies, even for the same enzyme, making it difficult to make valid comparisons.…”

Dissemination and evolution of blaOXA-23 gene among carbapenem-resistant Acinetobacter baumannii isolates from central China

“…Until now, whether the prevalence of blaOXA-23 gene results from a previously established multidrug-resistant clonal lineage or whether the success of certain clonal lineages is due to blaOXA-23 gene that they acquired is not clear. [2,10] Although the alanine insertion results in 20-fold and 100-fold decreases in Km against cefotaxime and ceftriaxone respectively [12], isolates carrying blaOXA-146 or blaOXA-49 only occurred in three different cities across China, which indicates that the evolved antibiotic-resistant genes may not cause a serious prevalence status. The incidence of sign epistasis [14], compensatory mutations [15] as well as co-occurrence mutations [16] may cause the failure of a novel mutation to pass on in the evolutionary trajectory of blaOXA-23.…”

“…All of these four strains were isolated from sputum in four different clinical units, and they were resistant to ten commonly used antibiotics. According to previous studies, strains carrying OXA-146 or OXA-49 possess a duplication of A220, adding one residue in the β5-β6 loop and affect the hydrolytic activity toward antibiotics tremendously [12,13]. How other mutations (K266N, H41N and F245V) affect carbapenem resistance is not clear: they may affect kinetics, but mutations do not appear to directly influence the active site; further studies will be carried on in silico and in vitro.…”