The 1.4 Å Crystal Structure of the Class D β-Lactamase OXA-1 Complexed with Doripenem

Abstract

The clinical efficacy of carbapenem antibiotics depends on their resistance to the hydrolytic action of β-lactamase enzymes. The structure of the class D β-lactamase OXA-1 as an acyl-complex with the carbapenem doripenem was determined to 1.4 Å resolution. Unlike most class A and class C carbapenem complexes, the acyl carbonyl oxygen in OXA-1/doripenem is bound in the oxyanion hole. Interestingly, no water molecules were observed in the vicinity of the acyl linkage, providing an explanation for why carbapenems inhibit OXA-1. The side-chain amine of K70 remains fully carboxylated in the acyl structure, and the resulting carbamate group hydrogen bonds to the alcohol of the 6α-hydroxyethyl moiety of doripenem. The carboxylate attached to the β-lactam ring of doripenem is stabilized by a salt-bridge to K212 and a hydrogen bond with T213, in lieu of the interaction with an arginine side-chain found in most other β-lactamase/β-lactam complexes (eg. R244 in the class A member TEM-1). This novel set of interactions with the carboxylate results in a major shift of the carbapenem's pyrroline ring compared to the structure of the same ring in meropenem bound to OXA-13. Additionally, bond angles of the pyrroline ring suggest that after acylation, doripenem adopts the Δ 1 tautomer. These findings provide important insights into the role that carbapenems may have in the inactivation process of class D β-lactamases.Carbapenems are broad spectrum β-lactam antibiotics that represent one of the last lines of defense against severe bacterial infections. Doripenem ( Figure 1A) was approved for use in the United States in 2007 with indications for urinary tract infections and complicated intraabdominal infections (1). Though a broad spectrum antibiotic, it has shown higher efficacy than imipenem and meropenem in troublesome Gram-negative species such as Pseudomonas aeruginosa and Acinetobacter baumannii (1).One of the defining features of carbapenems is the presence of a 6α-hydroxyethyl moiety in lieu of the more bulky 6β amide side-chains found in other β-lactam antibiotics. This unique structural element is thought to be responsible for resistance of these drugs to the hydrolytic action of some β-lactamases (2,3). Classes A, C and D β-lactamases use a serine-nucleophile based covalent catalysis strategy (4). Interestingly, carbapenems are able to bind in the active site of these enzymes and can even proceed to a covalent acyl-intermediate with the active site serine (2,3,5). In many of these β-lactamases however, the water-dependent deacylation process is impaired. The end result is that carbapenems serve as effective irreversible inhibitors of many β-lactamases.The interaction of carbapenems with β-lactamases has been studied extensively. X-ray crystallographic studies of imipenem and meropenem bound to class A (3,6-9), class C (2) and class D (5) enzymes have yielded a wealth of structural detail about how the acyl-intermediate interacts with active site elements. In the case of most class A and class C enzymes, acylatio...

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

“…Non‐covalent interaction plots indicate that the 1β‐methyl group may interact sterically with the hydroxyethyl group in the acyl‐enzyme complex, which is largely alleviated in the corresponding 1β‐proton system (Figure 2 B). Molecular dynamics simulations (100 ns) of the acyl‐enzyme complex derived from OXA‐1 and doripenem12 also suggest that the conformation of the hydroxyethyl group is influenced by the 1β‐substituent, with the system bearing a 1β‐proton showing more flexibility (Tables S21–S23, Figures S28–S31). Notably, in the MD simulations, the 1β‐methyl system initially alternates between two conformations (Figure 2 C), one of which appears to be consistent with that expected for β‐lactone formation (Figure S27).…”

“…Crystallographic studies of OXA‐1 with doripenem12 and of OXA‐58 with a 6α‐hydroxymethyl penicillin13 indicate that the catalytically important carbamylated lysine residue interacts with the hydroxyethyl side chain (Figure S27). 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…”

“…Reduced density gradient isosurface s =0.5, ρ ( r ) signλ 2 (e/au) color scale runs from −0.02 (blue) to 0.02 (red). C) Representations of two major conformations of the hydroxyethyl group observed during MD simulation of the OXA‐1 doripenem complex;12 the upper conformation appears to be more favorable for β‐lactone formation. Note that the doripenem thioether side chain and serine backbone are represented as methyl groups.…”

Clinical Variants of the Native Class D β-Lactamase of Acinetobacter baumannii Pose an Emerging Threat through Increased Hydrolytic Activity against Carbapenems

“…For most of these enzymes, structures have been determined with substrates or inhibitors bound in the active site (21)(22)(23)(24)(25)(26). Like class A and class C ␤-lactamases, members of class D make use of a serine-nucleophile acylation/deacylation double-displacement mechanism to hydrolyze the ␤-lactam ring (10).…”

“…Second, an active-site isoleucine (I129) projects its ␦ carbon into the active site, where it would likely clash with the carbapenem's hydroxyethyl group. This position is occupied by valine in nearly all other non-OXA-51-like class D ␤-lactamases, and structural studies show a very tight fit between the valine of those enzymes and bound carbapenem substrates (21)(22)(23)(24).…”