Structure-fucntion relationship of the NDM-1 metallo-beta-lactamase: Importance of Trp-87 in enzyme structure, function and stability.

Background. NDM-1, an acquired subclass B1 metallo-beta-lactamase (MBL), represents a new worrisome global health issue, due to its rapid dissemination among clinical isolates in several countries and further aggravated by the unavailability of clinically-useful MBL inhibitors. NDM-1 thus represents an extremely relevant target for MBL inhibitors, and its structure-function relationship deserves investigation. In a previous study performed on VIM-2, the highly conserved Trp-87 residue was identified as an essential determinant for the enzyme stability and folding. In this work, we probed the impact of Trp-87 substitutions in NDM-1 on the enzyme properties and bacterial resistance. Methods. The role of Trp-87 of NDM-1 was investigated by saturation mutagenesis. A library of blaNDM-1 mutants was obtained by means of a mutagenic PCR, using plasmid pLBII-NDM-1 as the template, and the recombinant plasmids transformed in E. coli. The clones carrying the various blaNDM-1 mutants genes were subjected to sequence analysis and their antimicrobial susceptibility profile determined using the broth microdulition technique as recommended by CLSI. Various NDM-1 variants were also subjected by biochemical characterization using kinetic assays. Results. Antimicrobial susceptibility data obtained with E. coli strains producing the various Trp-87 NDM-1 variants consistently showed a significant reduction of the MIC values with several β-lactam antibiotics (e. g., the ceftazidime MIC values were decreased up to 128-fold, as compared to that shown by the strain producing the wild-type NDM-1). However, the substitution of Trp-87 did not compromise per se the activity of the purified enzyme, as revealed by kinetic assays. These observations are very similar to those previously obtained with the VIM-2 MBL, indicating that residue Trp-87, although not directly involved in catalysis, is a crucial determinant for conferring β-lactam resistance, likely due to its relevance in enzyme stability and proper in vivo folding. Conclusions. These data overall support and validate the hypothesis that the conserved Trp-87 of MBLs is an essential determinant for enzyme stability and in vivo folding and thus would represent an important structural determinant to consider for the development of rationally-designed MBL inhibitors.