Analysis of β-lactone formation by clinically observed carbapenemases informs on a novel antibiotic resistance mechanism

Abstract

An important mechanism of resistance to β-lactam antibiotics is via their β-lactamase-catalyzed hydrolysis. Recent work has shown that, in addition to the established hydrolysis products, the reaction of the class D nucleophilic serine β-lactamases (SBLs) with carbapenems also produces β-lactones. We report studies on the factors determining β-lactone formation by class D SBLs. We show that variations in hydrophobic residues at the active site of class D SBLs (i.e. Trp¹⁰⁵, Val¹²⁰, and Leu¹⁵⁸, using OXA-48 numbering) impact on the relative levels of β-lactones and hydrolysis products formed. Some variants, i.e. the OXA-48 V120L and OXA-23 V128L variants, catalyze increased β-lactone formation compared with the WT enzymes. The results of kinetic and product studies reveal that variations of residues other than those directly involved in catalysis, including those arising from clinically observed mutations, can alter the reaction outcome of class D SBL catalysis. NMR studies show that some class D SBL variants catalyze formation of β-lactones from all clinically relevant carbapenems regardless of the presence or absence of a 1β-methyl substituent. Analysis of reported crystal structures for carbapenem-derived acyl-enzyme complexes reveals preferred conformations for hydrolysis and β-lactone formation. The observation of increased β-lactone formation by class D SBL variants, including the clinically observed carbapenemase OXA-48 V120L, supports the proposal that class D SBL-catalyzed rearrangement of β-lactams to β-lactones is important as a resistance mechanism.

Keywords: antibiotic action; antibiotic resistance; antibiotics; carbapenem; carbapenemase; class D serine β-lactamase; enzyme kinetics; enzyme mechanism; β-lactone.

Figure 1

Reactions of carbapenems with class D serine β-lactamases.A, class D SBLs can convert 1β-methyl–substituted carbapenems to reversibly form β-lactone (red) and irreversibly form hydrolysis (blue) products. The C-6 hydroxyethyl side chain is in green. Note that there are different possible tautomeric states of the carbapenem-derived pyrroline ring, both in the AEC and for the products; in the case of both hydrolysis and lactone-forming reactions, the major nascent product is likely the enamine tautomer (Fig. S5) (15). B, crystallographically derived views of the active site of OXA-48 after reaction with imipenem (white) (PDB entry 5QB4) (18). The imipenem C-6 hydroxyethyl side chain (green) is positioned to interact with the side chains of Val¹²⁰, Leu¹⁵⁸, and Trp¹⁰⁵ (orange). Nucleophilic Ser⁷⁰ (gray) and carbamylated Lys⁷³ (KCX 73; gray) are also shown.

Figure 2

The identity of the hydrophobic residue at position 120 impacts on the extent of β-lactone formation.A, time-course analyses showing the products formed from meropenem in the presence of WT OXA-48, OXA-48 V120L (OXA-519), and OXA-48 V120I, as monitored by ¹H NMR (600 MHz). t = 0 refers to measurements made prior to enzyme addition. B, comparative analysis of β-lactones and hydrolysis products derived from imipenem, panipenem, and meropenem formed by OXA-48 V120L and WT OXA-48, measured by ¹H NMR spectroscopy (600 MHz). OXA-519 was observed to form β-lactones from all three tested carbapenems, whereas WT OXA-48 was only observed to produce β-lactones from the 1β-methyl–substituted carbapenem (meropenem). Note that the depletion of β-lactone levels at long time points is expected because the β-lactones bind reversibly with the class D SBLs and can undergo hydrolysis (14).

Figure 3

Crystallographically observed conformations of the carbapenem C-6 hydroxyethyl side chain.A, dihedral angles (Dh angles) of the hydroxyethyl side chain (O C-8 C-6 C-5) in the AECs of class D SBLs and carbapenems, in the presence and absence of a carbamylated lysine. The following structures were analyzed: OXA-1 with doripenem, pH 7.5 (PDB entry 3ISG) (19); OXA-23 F110A, M221A with imipenem, pH 7.0 (PDB entry 6N6X) (20); OXA-23 F110A, M221A with meropenem, pH 7.0 (PDB entry 6N6Y) (20); OXA-23 F110A, M221A with meropenem, pH 4.1 (PDB entry 6N6V) (20); OXA-23 F110A, M221A pH 4.1 with imipenem, (PDB entry 6N6U) (20); OXA-24/40 K84D with doripenem, pH 8.5 (PDB entry 3PAE) (24); OXA-24/40 V130D with doripenem, pH 8.5 (PDB entry 3PAG) (24); OXA-23 with meropenem, pH 4.1 (PDB entry 4JF4) (25); OXA-51 K83D, I129L with doripenem, pH 6.5 (PDB entry 5L2F) (26); OXA-13 with imipenem (PDB entry 1H5X) (28); OXA-48 with imipenem, pH 7.5 (1) (PDB entry 5QB4) (18); OXA-48 K73A with doripenem, pH 4.0 (PDB entry 6PXX) (30); OXA-48 with ertapenem, pH 4.0 (PDB entry 6P99) (32); OXA-48 with imipenem, pH 4.0 (3) (PDB entry 6P97) (32); OXA-48 with meropenem, pH 4.0 (1) (PDB entry 6P98) (32); OXA-48 with doripenem, pH 4.0 (PDB entry 6P9C) (32); OXA-48 with imipenem, pH 4.6 (2) (PDB entry 6PTU); OXA-48 with meropenem, pH 4.6 (2) (PDB entry 6PT1); OXA-239 K82D with doripenem, pH 4.2 (PDB entry 5WI7) (27); and OXA-239 K82D with imipenem, pH 4.2 (PDB entry 5WIB) (27). Numbers are used in cases in which there were more than one of the same enzyme: carba penem crystal structure. This analysis suggests that the carbamylated lysine is an important determinant of the C-6 hydroxyethyl side chain conformation. B, view of hydroxyethyl conformations in the OXA-1 active site with doripenem (conformations I and II) and the OXA-23 F110A/M221A active site with meropenem (conformation III), showing dihedral angles and interactions of the carbamylated lysine and surrounding residues with the C-6 hydroxyethyl side chain. Conformation I is proposed to most closely represent the conformation required for β-lactone formation, because the C-6 hydroxyethyl hydroxyl group is positioned in a suitable orientation (i.e. has a favorable Bürgi–Dunitz trajectory) relative to the AEC carbonyl. Note, Trp¹¹³/Val¹²⁸/Leu¹⁶⁶ in OXA-23 and Trp¹⁰²/Val¹¹⁷/Leu¹⁶¹ in OXA-1 are equivalent to Trp¹⁰⁵/Val¹²⁰/Leu¹⁵⁸ in OXA-48. KCX 70, carbamylated Lys⁷⁰.