Mutational Effects in Carbapenem Hydrolysis of YEM-1, a New Sub-Class B2 Metallo-beta-Lactamase from Yersinia mollaretii

Background The genome of Yersinia mollaretii ATCC43969 was totally sequenced (NCBI NZ_AALD0200 0006.1). Its analysis indicated the presence of two β-lactamase genes, namely blaB and yem-1 coding for an AmpC-like and a metallo β-lactamase respectively. Up to date, many studies on BlaB were performed (1,2). Our work aimed to characterize the kinetic profile of YEM-1 and the residues essential for its activity. Methods YEM-1 was produced in E. coli Rosetta and purified to homogeneity. A survey of its kinetic properties was performed against different β-lactam antibiotics. A model of the three-dimensional structure of YEM-1 was build using the known structure of Aeromonas hydrophila CphA as template. Its analysis highlighted the major residues substitution between CphA and YEM-1. These residues were mutated by site-directed mutagenesis. The kinetic properties of the different mutants were analyzed. Results We noted that, at 37°C, YEM-1 is produced as inclusion bodies. We obtained a soluble form of YEM-1 when the culture was grown at 18°C in presence of IPTG 0.1 mM. The MBL was purified in three chromatographic steps that include a HiTRAP SP, an IMAC and a molecular sieve. The kinetic analysis of the YEM-1 showed that only imipenem was hydrolyzed efficiently. We confirmed that it belongs to the subclass B2 MBL family. As expected, the structure of YEM-1 has a similar fold than CphA. We found that the main differences between CphA and YEM-1 were the residues in position 67, 156 and 236 (BBL numbering) respectively. By site-directed mutagenesis, we made the YEM-1 single mutants (Y67V, T156F, S236F), the double mutant T156F-S236F and the triple mutant (Y67T156FS236F) in order to design the active site of CphA in YEM-1. The substitution Y67V yielded a global increase of the catalytic efficiency to carbapenems, with the exception of the imipenem. The mutations of T156 and/or S236 in phenylalanine globally decrease the enzymatic activity of the enzyme. Finally the double mutant and triple mutants did not increase the catalytic efficiencies of YEM-1 against carbapenems. Conclusions Despite a high sequence's similarity between CphA and YEM-1 (57%), we showed that the Yersinia MBL was less effective than CphA against carbapemens. We also highlighted the substitution Y67V in YEM-1 that increased its efficiency against all the carbapemen tested and in particular ertapenem and meropenem.