[en] Class D β-lactamases with carbapenemase activity are emerging as carbapenem-resistance determinants in Gram-negative bacterial pathogens, mostly Acinetobacter baumannii and Klebsiella pneumoniae. Carbapenemase activity is an unusual feature among class D β-lactamases, and the structural elements responsible for this activity remain unclear. Based on structural and molecular dynamics data, we previously hypothesized a potential role of the residues located in the short-loop connecting strands β5 and β6 (the β5-β6 loop) in conferring the carbapenemase activity of the OXA-48 enzyme. In this work, the narrow-spectrum OXA-10 class D β-lactamase, which is unable to hydrolyze carbapenems, was used as a model to investigate the possibility of evolving carbapenemase activity by replacement of the β5-β6 loop with those present in three different lineages of class D carbapenemases (OXA-23, OXA-24, and OXA-48). Biological assays and kinetic measurements showed that all three OXA-10-derived hybrids acquired significant carbapenemase activity. Structural analysis of the OXA-10loop24 and OXA-10loop48 hybrids revealed no significant changes in the molecular fold of the enzyme, except for the orientation of the substituted β5-β6 loops, which was reminiscent of that found in their parental enzymes. These results demonstrate the crucial role of the β5-β6 loop in the carbapenemase activity of class D β-lactamases, and provide previously unexplored insights into the mechanism by which these enzymes can evolve carbapenemase activity.
De Luca, F.; Dipartimento di Biotecnologie, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100 Siena, Italy
Benvenuti, M.; Dipartimento di Chimica, Università di Siena, I-53100 Siena, Italy
Carboni, F.; Dipartimento di Chimica, Università di Siena, I-53100 Siena, Italy
Pozzi, C.; Dipartimento di Chimica, Università di Siena, I-53100 Siena, Italy
Rossolini, G. M.; Dipartimento di Biotecnologie, Laboratorio di Fisiologia e Biotecnologia dei Microrganismi, Università di Siena, I-53100 Siena, Italy, Unità Operativa Complessa di Microbiologia e Virologia, Azienda Ospedaliera-Universitaria Senese, I-53100 Siena, Italy
Mangani, S.; Dipartimento di Chimica, Università di Siena, I-53100 Siena, Italy, Centro di Ricerca di Risonanze Magnetiche, Università di Firenze, I-50019 Sesto Fiorentino, Italy
Docquier, Jean-Denis ; Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Language :
English
Title :
Evolution to carbapenem-hydrolyzing activity in noncarbapenemase class D β-lactamase OXA-10 by rational protein design
Publication date :
2011
Journal title :
Proceedings of the National Academy of Sciences of the United States of America
ISSN :
0027-8424
eISSN :
1091-6490
Publisher :
National Academy of Sciences, Washington, United States - District of Columbia
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