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Structural Analysis of The OXA-48 Carbapenemase Bound to A "Poor" Carbapenem Substrate, Doripenem
Krisztina M Papp-Wallace1,2,3, Vijay Kumar4, Elise T Zeiser5
1Department of Biochemistry, Case Western Reserve University, Cleveland, OH 44106, USA. krisztina.papp@va.gov.
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.
Insights
OXA-48 carbapenemase, a threat in healthcare, shows low activity against doripenem. Structural analysis reveals key residues V120 and Y211 influence doripenem binding and hydrolysis, guiding new antibiotic development.
Area of Science:
- Microbiology
- Structural Biology
- Drug Discovery
Background:
- Carbapenem-resistant Enterobacteriaceae (CRE) pose a significant global health threat.
- OXA-48 carbapenemase is a major mechanism of carbapenem resistance.
- The low hydrolytic activity of OXA-48 against doripenem remains mechanistically unclear.
Purpose of the Study:
- To elucidate the structural basis for OXA-48's resistance to doripenem.
- To identify key residues involved in OXA-48's interaction with doripenem.
- To inform the design of novel carbapenem antibiotics less susceptible to OXA-48 hydrolysis.
Main Methods:
- Determined the crystal structure of a deacylation-deficient OXA-48 variant (K73A) complexed with doripenem at 1.5 Å resolution.
- Analyzed doripenem binding modes and interactions within the OXA-48 active site.
- Compared doripenem interactions to those in previously studied OXA-carbapenem complexes.
Main Results:
- Doripenem was observed covalently bound to the catalytic Ser70 residue in two tautomeric states.
- Residue Y211's positioning reduced interactions of doripenem's carboxylate moiety with R250.
- Residue V120 engaged in van der Waals interactions with doripenem's hydroxyethyl side chain, likely impacting deacylation.
Conclusions:
- Residues V120 and Y211 are hypothesized to be critical for OXA-48's specific carbapenemase profile.
- Structural insights provide a basis for developing carbapenem antibiotics resistant to OXA-48 hydrolysis.
- Findings may guide the development of inhibitors targeting OXA carbapenemases.