Abstract
Carbapenem-resistant Enterobacteriaceae are a significant threat to public health, and a major resistance determinant that promotes this phenotype is the production of the OXA-48 carbapenemase. The activity of OXA-48 towards carbapenems is a puzzling phenotype as its hydrolytic activity against doripenem is non-detectable. To probe the mechanistic basis for this observation, we determined the 1.5 Å resolution crystal structure of the deacylation deficient K73A variant of OXA-48 in complex with doripenem. Doripenem is observed in the Δ1R and Δ1S tautomeric states covalently attached to the catalytic S70 residue. Likely due to positioning of residue Y211, the carboxylate moiety of doripenem is making fewer hydrogen bonding/salt-bridge interactions with R250 compared to previously determined carbapenem OXA structures. Moreover, the hydroxyethyl side chain of doripenem is making van der Waals interactions with a key V120 residue, which likely affects the deacylation rate of doripenem. We hypothesize that positions V120 and Y211 play important roles in the carbapenemase profile of OXA-48. Herein, we provide insights for the further development of the carbapenem class of antibiotics that could render them less effective to hydrolysis by or even inhibit OXA carbapenemases.
Highlights
Carbapenems are a part of a broader class of antibiotics known as β-lactams and they are considered “last resort” antibiotics
The crystal structure of OXA-48 K73A in complex with doripenem was determined to probe the lack of observed catalytic activity of this carbapenemase towards doripenem
There appears to be a delicate balance in the positioning of select active site residues (V120 and Y211) as well as the interactions of these residues with carbapenem moieties that influence whether these drugs are hydrolyzed
Summary
Carbapenems are a part of a broader class of antibiotics known as β-lactams and they are considered “last resort” antibiotics. The OXA family of carbapenem-hydrolyzing metallo- or serine-based β-lactamase enzymes. OXA-48 most efficiently hydrolyzes imipenem with a kcat /Km value of 370 mM−1 s−1 , whereas hydrolysis of meropenem and ertapenem are appreciably less at 6 mM−1 s−1 and 1 mM−1 s−1 , respectively and doripenem turnover is not detectable [17,20,21,24]. The major active site residues in OXA-48 that contribute to β-lactam hydrolysis are the catalytic serine residue, S70 that forms a covalent/acyl bond with the substrates, and K73, which needs to be carbamylated in OXA-β-lactamases to aid in the deacylation reaction involving a water molecule [14,17]. Identifying the factor(s) that hinders doripenem catalysis by OXA-48 will provide medicinal chemists with pivotal insights needed to design novel therapeutics
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