Role of Active Site Loop-3 (ASL-3) in Substrate Recognition of the beta-lactamase NDM-1

PALACIOS, A.R.; MOJICA, M.F.; GIANNINI, E.; LLARRULL, L.I.; BONOMO, R.A.; VILA A. J.

San Miguel de Tucumán

Congreso; IIILAFeBS, IX IberoAmerican congress of Biophysics, XLV SAB Annual Meeting; 2016

Beta-lactams are the most important class of clinically available antibiotics used to treat bacterial infections. Metallo-b-Lactamases (MBLs) are metal-dependent enzymes with the ability to hydrolyze and confer resistance to virtually all classes of b-lactam antibiotics. Among MBLs, NDM-1 poses an increasing clinical concern since it is one of the most potent and widespread enzymes (1). Current evidence suggests that the active site loop 3 (ASL-3) of NDM plays an important role on substrate recognition in MbLs (2,3). Comparison of crystal structures of NDM-1 (4) to other clinically relevant MBLs such as VIM-2 (5) and IMP-1 (6) revealed that in NDM-1 the ASL-3 lacks a conserved Pro residue at its C-terminus. This significant difference is hypothesized to lead to a more flexible loop and a wider active site. To test the hypothesis that these features account for the higher affinity and catalytic activity of this enzyme for several b-lactams, we characterized the substrate profile of three ASL-3 variants of NDM-1: two in which the ASL-3 was replaced by those of IMP-1 (more charged) and VIM-2 (one residue shorter), and one in which we introduced the conserved Pro residue at the base of the ASL-3.