Contribution of asparagine 346 residue to the carbapenemase activity of CMY-2 β-lactamase

Authors

  • Sandrine Dahyot,

    1. Service de Bactériologie, Centre Hospitalo-Universitaire d'Amiens, Amiens, France
    2. Faculté de Médecine d'Amiens, Université de Picardie Jules Verne, Amiens, France
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  • Isabelle Broutin,

    1. Faculté de Pharmacie, Laboratoire de cristallographie et RMN biologiques, CNRS UMR8015, Université Paris Descartes, Paris, France
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  • Christophe de Champs,

    1. Service de Bactériologie-Virologie-Hygiène hospitalière, Centre Hospitalo-Universitaire de Reims, Hôpital Robert Debré, Reims, France
    2. Faculté de Médecine de Reims, Université Reims-Champagne-Ardennes, Reims, France
    3. Equipe d'accueil 4687 ERA - Résistance aux antibiotiques chez les entérobactéries, Université Reims-Champagne-Ardennes, Reims, France
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  • Hélène Guillon,

    1. Service de Bactériologie, Centre Hospitalo-Universitaire d'Amiens, Amiens, France
    2. Faculté de Médecine d'Amiens, Université de Picardie Jules Verne, Amiens, France
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  • Hedi Mammeri

    Corresponding author
    1. Faculté de Médecine d'Amiens, Université de Picardie Jules Verne, Amiens, France
    2. Equipe d'accueil 4687 ERA - Résistance aux antibiotiques chez les entérobactéries, Université Reims-Champagne-Ardennes, Reims, France
    • Service de Bactériologie, Centre Hospitalo-Universitaire d'Amiens, Amiens, France
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Correspondence: Hedi Mammeri, Service de Bactériologie, Centre hospitalo-Universitaire d'Amiens, Centre de Biologie Humaine, Hôpital sud (Salouël), avenue René Laënnec, 80054 Amiens, Cedex 1, France.

Tel.: +33 3 22 66 84 30;

fax: +33 3 22 66 84 98;

e-mail: mammeri.hedi@chu-amiens.fr

Abstract

Only a few plasmid-borne AmpC (pAmpC) β-lactamases, such as CMY-2, can account for carbapenem resistance in Enterobacteriaceae in combination with outer membrane impermeability. The aim of this study was to elucidate the contribution of Asn-346, which is well conserved among carbapenem-hydrolyzing pAmpCs, to the hydrolysis spectrum of CMY-2. Site-directed mutagenesis experiments were carried out to replace Asn-346 with glycine, alanine, valine, glutamate, aspartate, serine, threonine, glutamine, tyrosine, isoleucine, lysine, and histidine. The recombinant plasmids were transferred into wild-type and porin-deficient Escherichia coli strains. Asn-346 replacement reduced significantly the MICs of all β-lactams, except the Asn-346-Ile substitution that increased the MICs of cephalosporins, whereas it decreased those of carbapenems. The biochemical characterization, along with a molecular modeling study, showed that the size and the polarity of the side chain at position 346 assisted substrate binding and turnover. This study shows for the first time that the amino acid at position 346 contributes to the β-lactamase activity of cephalosporinases. Asparagine and isoleucine residues, which are well conserved at position 346 among AmpC-type enzymes, modulate their hydrolysis spectrum in an opposing sense. Ile-346 confers higher level of cephalosporins resistance, whereas Asn-346 confers carbapenem resistance in combination with outer membrane impermeability.

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