Structural Basis for Substrate Specificity and Carbapenemase Activity of OXA-48 Class D β-Lactamase

Abstract

Carbapenem-hydrolyzing class D β-lactamases (CHDLs) are a diverse family of enzymes that are rapidly becoming the predominant cause of bacterial resistance against βlactam antibiotics in many regions of the world. OXA-48, an atypical member of CHDLs, is one of the most frequently observed in the clinic and exhibits a unique substrate profile. We applied Xray crystallography to OXA-48 complexes with multiple β-lactam antibiotics to elucidate this enzyme's carbapenemase activity and its preference of imipenem over meropenem and other substrates such as cefotaxime. In particular, we obtained acyl-enzyme complexes of OXA-48 with imipenem, meropenem, faropenem, cefotaxime, and cefoxitin, and a product complex with imipenem. Importantly, the product complex captures a key reaction milestone with the newly generated carboxylate group still in the oxyanion hole, and represents the first such complex with a wild-type serine β-lactamase. A potential hydrogen bond is observed between the two carboxylate groups from the product and the carbamylated Lys73, representing the stage immediately after the breakage of the acyl-enzyme bond where the product carboxylate would be neutral. The placement of the product carboxylate also illustrates the approximate transient location of the deacylation water that has long eluded structural characterization in class D β-lactamases. Additionally, comparing the product complex with the acyl-enzyme intermediates provides new insights into the various mechanisms by which specific side chain groups hinder the access of the deacylation water to the acyl-enzyme linkage, especially in meropenem. Taken together, these data offer valuable information on the substrate specificity of OXA-48 and the catalytic mechanism of CHDLs.

KPC-2 β-lactamase enables carbapenem antibiotic resistance through fast deacylation of the covalent intermediate

“…In all structures, the C3 carboxylate participates in hydrogen bonds with the Ser130, Thr235 and Thr237 side-chain hydroxyl groups. The placement of the newly generated carboxylate is similar to the recently determined imipenem product complex with OXA-48 (49). There appears to be a hydrogen bond between the product carboxylate group and the Glu166 side chain, likely due to the low pH of the ammonium sulfate crystallization conditions and the short soaking time during complex crystal preparation.…”

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

“…To investigate the structural basis of the observed β-lactone versus hydrolysis product ratios, we analyzed crystallographically observed AECs derived from the reactions of class D SBLs with carbapenems (PDB entry 1H5X ) ( 18 , 19 , 20 , 24 , 25 , 26 , 27 , 28 , 29 ). The analyses reveal three different conformations (conformations I, II, and III) that are adopted by the C-6 hydroxyethyl side chain in the AECs ( Fig.…”

“…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.…”

Antimicrobial Resistance Conferred by OXA-48 β-Lactamases: Towards a Detailed Mechanistic Understanding

“…Carbapenemase activity The first carbapenem acylenzyme structure of OXA-48 (with imipenem) was released in 2018 (PDB ID 5QB4) alongside multiple structures with small inhibitor fragments. 47 From 2019 onward, further acylenzyme structures have been deposited with imipenem (PDB IDs 6P97, 6PTU, and 7KH9), 27,39,48 meropenem (PDB IDs 6P98, 6PT1, and 7KHQ), 27,39,48 doripenem (PDB IDs 6P9C and 6PXX), 27,49 ertapenem (PDB ID 6P99), 27 and faropenem (PDB ID 6PSG) 48 . Additionally, two acylenzyme structures of inactivated OXA-163 (K73A) with imipenem and meropenem are available (PDB IDs 7KHZ and 7KHY, respectively) 39 .…”

“…cefalotin and cefotaxime are inactivated readily, whereas minimal (or no) activity is measured against ceftazidime and cefepime. Further enzyme kinetic data for OXA- 48 and key variants are collated in the Supporting Information (spreadsheet).…”