Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity

Abstract

Class D β-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this β-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various β-lactams were modeled and the potential active site residues that may interact with various β-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the β-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type β-lactamases.

The l,d-Transpeptidase LdtAb from Acinetobacter baumannii Is Poorly Inhibited by Carbapenems and Has a Unique Structural Architecture

“…In all cases, we also observed a small amount of a species that was 44 Da smaller than the acyl-enzyme complex. This could result from loss of either carbon dioxide at the C3 position or the hydroxyethyl group at the C6 position of the carbapenem, as was previously reported for their interaction with LDTs from other bacteria and class A, C, and D β-lactamases. ,− Since acylation was the most efficient with ertapenem, we used this antibiotic to measure the apparent acylation rate constant k 2 , which was found to be 0.22 ± 0.01 h –1 (Figure ); this would result in one acylation event every four and a half hours. These results demonstrated that Ldt Ab is acylated in vitro only by carbapenems, albeit at a very slow rate.…”

Discovery and molecular basis of chloride as an allosteric activator and catalytic inhibitor for Class-D β-lactamases

“…Active site residue R250 has been proposed to play an important role in catalysis by binding to the carboxylate of the substrates 47 . We have observed clear binding of Br − or I − ion interacting with R250 in our halide-soaked OXA-48 WT and OXA-48 AcK73 -imipenem-Br − structures ( Fig.…”

An Ion-Pair Induced Intermediate Complex Captured in Class D Carbapenemase Reveals Chloride Ion as a Janus Effector Modulating Activity

“…R250 has been proposed to play an important role in catalysis by binding to the carboxylate of the substrates. 34 To further explore whether the binding of halides at R250 is a key driving force for the formation of the inactive acyl intermediate, we performed ITC analysis and showed that the OXA-48 R250A variant no longer responds to the presence of chloride with biphasic features but the nonconserved OXA-48 R214A variant still does (Figure 1h, Figure S8). These data indicate that the binding of chloride to R250 is essential for chloride-induced biphasic kinetics.…”