Poster (Scientific congresses and symposiums)
Characterisation of the sub-class B2 metallo-β-lactamase of Yersinia mollaretii Wauters
Mercuri, Paola; Blétard, Sylvie; Kerff, Frédéric et al.
201713th β-lactamase Meeting"
 

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Keywords :
metallo-beta-lactamase Yersinia
Abstract :
[en] Background. The sub class B2 metallo-β- lactamases are Zn2+-dependent enzymes that efficiently hydrolyzes only carbapenem antibiotics. We study the metallo-β-lactamase (MBL) produced by a soil bacteria named Yersinia mollaretii Wauters DMS 18520 (1). The enzyme exhibits two potential zinc-binding sites, the conserved residues Zn1 site (N116-H118-H196), and Zn2 site (D120-C221-H263), tipycals of the sub-class B2 metallo-β- lactamases. Compared to CphA, we found the similar α3 helix located near the active-site and three prolines of the "rich prolines loop" were conserved (2). Materials&methods. The gene coding for the metallo-β-lactamase was isolated by PCR from the genomic DNA as template. The MBL was produced in E. coli Rosetta (DE3) pLysS with the help of overexpression vector as pET26b. The enzyme was produced in TB medium at 18°C in presence of 100 µM IPTG. The metallo-β- lactamases was purified by three steps, an ion exchange chromatography (Sepharose SP-HP column), a Pentadentate Chelator (PDC) Zn2+ column, followed by a molecular sieve column (Superdex 75). pH dependence of activity was study. The enzymatic profile was studied on a representative numbers of carbapenems and measured in the presence of increasing concentrations of zinc. The influence of chelating agents on the β-lactamase activity was also assessed. Results. The MBL from Yersinia mollaretii was overproduced in E. coli Rosetta (DE3) pLysS and purified in three chromatography steps as described in Materials & Methods. The active fractions were pooled and concentrated. The enzyme showed the best activity in MES buffer at pH 6.0. The steady state kinetic parameters were determined for a representative set of carbapenems antibiotics. We showed that Imipenem was the best substrate among the tested substrates. Conclusion. Compared to CphA the new sub class B2 MBL shows a narrow antibiotics profile, a reduced susceptibility toward high zinc concentration and to zinc chelators.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Mercuri, Paola ;  Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Blétard, Sylvie
Kerff, Frédéric  ;  Université de Liège - ULiège > Département des sciences de la vie > Centre d'ingénierie des protéines
Galleni, Moreno ;  Université de Liège - ULiège > Département des sciences de la vie > Macromolécules biologiques
Language :
English
Title :
Characterisation of the sub-class B2 metallo-β-lactamase of Yersinia mollaretii Wauters
Publication date :
June 2017
Event name :
13th β-lactamase Meeting"
Event organizer :
University of L'Aquila, Prof. Perilli
Event place :
Santo Stefano di Sessanio, l'Aquila, Italy
Event date :
du 16 juin au 19 Juin 2017
Audience :
International
Available on ORBi :
since 19 December 2017

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