Structures of the Class D Carbapenemase OXA-24 from Acinetobacter baumannii in Complex with Doripenem

Abstract

The emergence of class D β-lactamases with carbapenemase activity presents an enormous challenge to health practitioners, particularly with regard to the treatment of infections caused by Gram negative pathogens such as Acinetobacter baumanii. Unfortunately, class D β-lactamases with carbapenemase activity are resistant to β-lactamase inhibitors. To better understand the details of the how these enzymes bind and hydrolyze carbapenems, we have determined the structures of two deacylation-deficient variants (K84D and V130D) of the class D carbapenemase OXA-24 with doripenem bound as a covalent acyl-enzyme intermediate. Doripenem adopts essentially the same configuration in both OXA-24 variant structures, but varies significantly when compared to the non-carbapenemase class D member OXA-1/doripenem complex. The alcohol of the 6α hydroxyethyl moiety is directed away from the general base carboxy-K84, with implications for activation of the deacylating water. The tunnel formed by the Y112/M223 bridge in the apo form of OXA-24 is largely unchanged by the binding of doripenem. The presence of this bridge, however, causes the distal pyrrolidine/sulfonamide group to bind in a drastically different conformation compared to doripenem bound to OXA-1. The resulting difference in the position of the side-chain bridge sulfur of doripenem is consistent with the hypothesis that the tautomeric state of the pyrroline ring contributes to the different carbapenem hydrolysis rates of OXA-1 and OXA-24. These findings represent a snapshot of a key step in the catalytic mechanism of an important class D enzyme, and may be useful for the design of novel inhibitors.

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…”

OXA β-Lactamases

“…Among the carbapenems, OXA-23 has a much higher turnover rate for imipenem than for meropenem, ertapenem, or doripenem (36). Despite the relatively low turnover rates for carbapenems displayed by these enzymes, it appears that they counter this through the presence of a "hydrophobic bridge" across the top of the active site, formed by phenylalanine 110 and methionine 221 (35), as the presence of a corresponding bridge in OXA-40 has been shown to be responsible for tight binding of the carbapenems (38,39). Recently, it was demonstrated that OXA-146 has an expanded hydrolytic spectrum that includes ceftazidime, which is not hydrolyzed by the other class D OXA-type carbapenemases, while retaining its activity against the carbapenems (35).…”

Hydrolytic Mechanism of OXA-58 Enzyme, a Carbapenem-hydrolyzing Class D β-Lactamase from Acinetobacter baumannii

“…The main acquired and most widely spread carbapenemases in A. baumannii are OXA-23, OXA-24, and OXA-58, which represent three different CHDL groups and are encoded as either chromosomal or plasmid-borne genes (8,14). Of these three group representatives, only the structure of OXA-24 has been elucidated (15)(16)(17). Structural analysis and mutagenesis studies of OXA-24 suggest that a hydrophobic cleft over the active site could be responsible for the widened substrate specificity of these enzymes; imipenem is hydrolyzed about 10-fold more efficiently than oxacillin (15).…”