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Volume 168, Issue 6

Deciphering the role of residues in the loops nearing the active site of OXA-58 in imparting beta-lactamase activity Free

Abstract

The existence of OXA-58 carbapenemase alone or in combination with other beta-lactam resistance factors poses significant beta-lactam resistance. The exact mechanism of action of OXA type beta-lactamases is debatable due to the involvement of multiple residues within or outside the active site. In the present work, we have elucidated the relative role of residues present in the putative omega (W169, L170, K171) and β6-β7 (A226 and D228) loops on the activity of OXA-58 by substituting into alanine (and aspartate for A226) through site-directed mutagenesis. cells harbouring OXA-58, substituted at the putative omega loop, manifest a significant decrease in the beta-lactam resistance profile than that of the cells expressing OXA-58. Further, a reduction in the catalytic efficiency is observed for the purified variants of OXA-58 carrying individual substitutions in the putative omega loop than that of OXA-58. However, the addition of NaHCO (for carbamylation of K86) increases catalytic efficiency of the individual protein as revealed by nitrocefin hydrolysis assay and steady state kinetics. Moreover, W169A and K171A substitutions show significant effects on the thermal stability of OXA-58. Therefore, we conclude that the putative omega loop residues W169, L170 and K171, individually, have significant role in the activity and stability of OXA-58, mostly by stabilising carbamylated lysine of active site.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antimicrobial resistance , beta-lactamase , omega loop , OXA beta-lactamase and thermal stability
Funding
This study was supported by the:
  • Department of Biotechnology, Ministry of Science and Technology, India (Award BT/PR24255/NER/95/716/2017)
    • Principle Award Recipient: AnindyaS Ghosh
  • Council of Scientific and Industrial Research (CSIR), Government of India (Award 27(0367)/20/EMR-II)
    • Principle Award Recipient: AnindyaS Ghosh
© 2022 The Authors
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2022-06-29
2024-05-04
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References

  1. Ambler RP. The structure of beta-lactamases. Philos Trans R Soc Lond B Biol Sci 1980; 289:321–331 [View Article] [PubMed]
     [Google Scholar]
  2. Higgins PG, Dammhayn C, Hackel M, Seifert H. Global spread of carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother 2010; 65:233–238 [View Article] [PubMed]
     [Google Scholar]
  3. Walther-Rasmussen J, Høiby N. OXA-type carbapenemases. J Antimicrob Chemother 2006; 57:373–383 [View Article] [PubMed]
     [Google Scholar]
  4. Evans BA, Amyes SGB. OXA β-lactamases. Clin Microbiol Rev 2014; 27:241–263 [View Article] [PubMed]
     [Google Scholar]
  5. Poirel L, Naas T, Nordmann P. Diversity, epidemiology, and genetics of class D beta-lactamases. Antimicrob Agents Chemother 2010; 54:24–38 [View Article] [PubMed]
     [Google Scholar]
  6. Couture F, Lachapelle J, Levesque RC. Phylogeny of LCR-1 and OXA-5 with class A and class D beta-lactamases. Mol Microbiol 1992; 6:1693–1705 [View Article] [PubMed]
     [Google Scholar]
  7. Golemi D, Maveyraud L, Vakulenko S, Samama J-P, Mobashery S. Critical involvement of a carbamylated lysine in catalytic function of class D beta-lactamases. Proc Natl Acad Sci U S A 2001; 98:14280–14285 [View Article] [PubMed]
     [Google Scholar]
  8. Golemi D, Maveyraud L, Vakulenko S, Tranier S, Ishiwata A et al. The first structural and mechanistic insights for class D β-lactamases: evidence for a novel catalytic process for turnover of β-lactam antibiotics. J Am Chem Soc 2000; 122:6132–6133 [View Article]
     [Google Scholar]
  9. Schneider KD, Bethel CR, Distler AM, Hujer AM, Bonomo RA et al. Mutation of the active site carboxy-lysine (K70) of OXA-1 beta-lactamase results in a deacylation-deficient enzyme. Biochemistry 2009; 48:6136–6145 [View Article] [PubMed]
     [Google Scholar]
  10. Dabos L, Zavala A, Bonnin RA, Beckstein O, Retailleau P et al. Substrate specificity of OXA-48 after β5-β6 loop replacement. ACS Infect Dis 2020; 6:1032–1043 [View Article] [PubMed]
     [Google Scholar]
  11. Poirel L, Marqué S, Héritier C, Segonds C, Chabanon G et al. OXA-58, a novel class D {beta}-lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob Agents Chemother 2005; 49:202–208 [View Article] [PubMed]
     [Google Scholar]
  12. Bush K, Jacoby GA. Updated functional classification of beta-lactamases. Antimicrob Agents Chemother 2010; 54:969–976 [View Article] [PubMed]
     [Google Scholar]
  13. Smith CA, Antunes NT, Toth M, Vakulenko SB. Crystal structure of carbapenemase OXA-58 from Acinetobacter baumannii. Antimicrob Agents Chemother 2014; 58:2135–2143 [View Article] [PubMed]
     [Google Scholar]
  14. Verma V, Testero SA, Amini K, Wei W, Liu J et al. Hydrolytic mechanism of OXA-58 enzyme, a carbapenem-hydrolyzing class D β-lactamase from Acinetobacter baumannii. J Biol Chem 2011; 286:37292–37303 [View Article] [PubMed]
     [Google Scholar]
  15. Kumar G, Biswal S, Nathan S, Ghosh AS. Glutamate residues at positions 162 and 164 influence the beta-lactamase activity of SHV-14 obtained from Klebsiella pneumoniae. FEMS Microbiol Lett 2018; 365: [View Article] [PubMed]
     [Google Scholar]
  16. Kumar G, Issa B, Biswal S, Jain D, Bhattacharjee A et al. Glutamic acid at position 152 and serine at position 191 are key residues required for the metallo-β-lactamase activity of NDM-7. Int J Antimicrob Agents 2020; 55:105824 [View Article] [PubMed]
     [Google Scholar]
  17. Baurin S, Vercheval L, Bouillenne F, Falzone C, Brans A et al. Critical role of tryptophan 154 for the activity and stability of class D beta-lactamases. Biochemistry 2009; 48:11252–11263 [View Article] [PubMed]
     [Google Scholar]
  18. Hujer AM, Bethel CR, Bonomo RA. Antibody mapping of the linear epitopes of CMY-2 and SHV-1 beta-lactamases. Antimicrob Agents Chemother 2004; 48:3980–3988 [View Article]
     [Google Scholar]
  19. Egorov A, Rubtsova M, Grigorenko V, Uporov I, Veselovsky A. The role of the Ω-loop in regulation of the catalytic activity of TEM-Type β-lactamases. Biomolecules 2019; 9:E854 [View Article] [PubMed]
     [Google Scholar]
  20. Guzman L-M, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol 1995; 177:4121–4130 [View Article] [PubMed]
     [Google Scholar]
  21. Ghosh AS, Young KD. Sequences near the active site in chimeric penicillin binding proteins 5 and 6 affect uniform morphology of Escherichia coli. J Bacteriol 2003; 185:2178–2186 [View Article] [PubMed]
     [Google Scholar]
  22. Kumar G, Issa B, Kar D, Biswal S, Ghosh AS. E152A substitution drastically affects NDM-5 activity. FEMS Microbiol Lett 2017; 364: [View Article] [PubMed]
     [Google Scholar]
  23. Wayne P. Clinical and laboratory standards institute. Performance Standards for Antimicrobial Susceptibility Testing 2007; 17:
     [Google Scholar]
  24. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 2011; 7:539 [View Article] [PubMed]
     [Google Scholar]
  25. Sali A, Blundell T. Comparative protein modelling by satisfaction of spatial restraints. Protein structure by distance analysis 1994; 64:C86
     [Google Scholar]
  26. Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK et al. AutoDock4 and AutoDockTools4: automated docking with selective receptor flexibility. J Comput Chem 2009; 30:2785–2791 [View Article] [PubMed]
     [Google Scholar]
  27. Pratap S, Katiki M, Gill P, Kumar P, Golemi-Kotra D. Active-site plasticity Is essential to carbapenem hydrolysis by OXA-58 class D β-lactamase of Acinetobacter baumannii. Antimicrob Agents Chemother 2016; 60:75–86 [View Article] [PubMed]
     [Google Scholar]
  28. Docquier J-D, Calderone V, De Luca F, Benvenuti M, Giuliani F et al. Crystal structure of the OXA-48 beta-lactamase reveals mechanistic diversity among class D carbapenemases. Chem Biol 2009; 16:540–547 [View Article] [PubMed]
     [Google Scholar]
  29. Saino H, Sugiyabu T, Ueno G, Yamamoto M, Ishii Y et al. Crystal structure of OXA-58 with the substrate-binding cleft in a closed state: insights into the mobility and stability of the OXA-58 structure. PLoS One 2015; 10:e0145869 [View Article] [PubMed]
     [Google Scholar]
  30. Verma J, Jain D, Mallik D, Ghosh AS. Comparative insight into the roles of the non active-site residues E169 and N173 in imparting the beta-lactamase activity of CTX-M-15. FEMS Microbiol Lett 2022; 369:fnac018 [View Article] [PubMed]
     [Google Scholar]
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Deciphering the role of residues in the loops nearing the active site of OXA-58 in imparting beta-lactamase activity
Microbiology 168, 001203 (2022); https://doi.org/10.1099/mic.0.001203
/content/journal/micro/10.1099/mic.0.001203
/content/journal/micro/10.1099/mic.0.001203
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