Structural and Dynamic Features of Acinetobacter baumannii OXA-66 β-Lactamase Explain Its Stability and Evolution of Novel Variants

Zachary L Klamer; Cynthia M June; Zdzislaw Wawrzak; Magdalena A Taracila; Joshua A Grey; Alyssa M I Benn; Christopher P Russell; Robert A Bonomo; Rachel A Powers; David A Leonard; Agnieszka Szarecka

doi:10.1016/j.jmb.2024.168603

Structural and Dynamic Features of Acinetobacter baumannii OXA-66 β-Lactamase Explain Its Stability and Evolution of Novel Variants

J Mol Biol. 2024 May 8;436(12):168603. doi: 10.1016/j.jmb.2024.168603. Online ahead of print.

Affiliations

¹ Department of Cell and Molecular Biology, Grand Valley State University, Allendale, MI, USA.
² Department of Chemistry, Grand Valley State University, Allendale, MI, USA.
³ Life Sciences Collaborative Access Team, Synchrotron Research Center, Northwestern University, Argonne, IL, USA.
⁴ Department of Medicine, Case Western Reserve University, Cleveland, OH, USA; Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, USA.
⁵ Department of Medicine, Case Western Reserve University, Cleveland, OH, USA; Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, OH, USA; Departments of Pharmacology, Biochemistry, and Molecular Biology and Microbiology, and Proteomics and Bioinformatics, Case Western Reserve University, Cleveland, OH, USA; CWRU-Cleveland VAMC Center for Antimicrobial Resistance and Epidemiology (Case VA CARES) Cleveland, OH, USA. Electronic address: Rab14@case.edu.
⁶ Department of Chemistry, Grand Valley State University, Allendale, MI, USA. Electronic address: powersra@gvsu.edu.
⁷ Department of Chemistry, Grand Valley State University, Allendale, MI, USA. Electronic address: leonardd@gvsu.edu.
⁸ Department of Cell and Molecular Biology, Grand Valley State University, Allendale, MI, USA. Electronic address: szarecka@gvsu.edu.

PMID: 38729259
DOI: 10.1016/j.jmb.2024.168603

Abstract

OXA-66 is a member of the OXA-51 subfamily of class D β-lactamases native to the Acinetobacter genus that includes Acinetobacter baumannii, one of the ESKAPE pathogens and a major cause of drug-resistant nosocomial infections. Although both wild type OXA-66 and OXA-51 have low catalytic activity, they are ubiquitous in the Acinetobacter genomes. OXA-51 is also remarkably thermostable. In addition, newly emerging, single and double amino acid variants show increased activity against carbapenems, indicating that the OXA-51 subfamily is growing and gaining clinical significance. In this study, we used molecular dynamics simulations, X-ray crystallography, and thermal denaturation data to examine and compare the dynamics of OXA-66 wt and its gain-of-function variants: I129L (OXA-83), L167V (OXA-82), P130Q (OXA-109), P130A, and W222L (OXA-234). Our data indicate that OXA-66 wt also has a high melting temperature, and its remarkable stability is due to an extensive and rigid hydrophobic bridge formed by a number of residues around the active site and harbored by the three loops, P, Ω, and β5-β6. Compared to the WT enzyme, the mutants exhibit higher flexibility only in the loop regions, and are more stable than other robust carbapenemases, such as OXA-23 and OXA-24/40. All the mutants show increased rotational flexibility of residues I129 and W222, which allows carbapenems to bind. Overall, our data support the hypothesis that structural features in OXA-51 and OXA-66 promote evolution of multiple highly stable variants with increased clinical relevance in A. baumannii.

Keywords: Antibiotic resistance; Carbapenem binding; OXA-109; OXA-82; Thermal stability.