Rol of Active Site Loop 3 in Substrate Recognition of the Lactamase NDM-1.

MAGDALENA TARACILA; MARIA FERNANDA MOJICA; LETICIA IRENE LLARRULL; ESTEFANÍA GIANNINI; ALEJANDRO JOSÉ VILA; ROBERT BONOMO; ANTONELA ROCÍO PALACIOS; CHRISTIAN BETHEL; JAMES SPENCER

New Orleans

Congreso; ASM Microbe 2017; 2017

American Society for Microbiology

Rol of active site loop 3 in substrate recognition of the lactamase NDM-1.PALACIOS, A.R.1, MOJICA, M.F.2,3, TARACILA, M.A. 2,3, BETHEL, C.R. 3, GIANNINI, E. 1, LLARRULL, L.L. 1, SPENCER, J.4 BONOMO, R.A. 2,3, VILA, A.J. 1 1IBR-CONICET, Argentina. 2CWRU, 3Cleveland VAMC, 4University of Bristol, UKBackground. B1 Metallo-β-Lactamases (MβLs) have an αβ/βα fold sandwich in which the binuclear Zn(II)- containing active site is surrounded by 5 loops. It has been proposed that the active site loop 3 (ASL3) has an important role on substrate recognition on MβLs. Comparison of crystal structures of NDM-1, VIM-2 and IMP-1 revealed that in NDM-1 the ASL-3 lacks a conserved Pro residue at its C-terminus leading to a more flexible loop and a wider active site. To test the hypothesis that these features account for the higher affinity of this enzyme for several β-lactams, we characterized a series of ASL-3 mutants of NDM-1.Methods. NDM constructs were custom synthesized (Celtek Genes) to produce variants in which the ASL-3 was replaced by those of IMP-1 (NIMP; more charged) and VIM-2 (NVIM; one residue shorter), and a third one in which the conserved Pro residue at the base of the ASL-3 (NPro) was introduced. Constructs were cloned into pMBLe and pET24 vectors and transformed into E. coli DH10B and BL21 (DE3) cells, respectively. Minimal inhibitory concentrations (MICs) were determined using CLSI approved methods. Biochemical characterization performed on purified enzymes included steady state and pre steady state activity, thermal stability and active site coordination changes based on Co(II) derivatives. Results. Compared to the NDMwt-expressing E. coli, mutants showed a significant decrease against cefepime and imipenem (e.g. 128 and 2 mg/L for NDMwt vs. 8 and 0.5 mg/L for NPro, respectively). Steady-state kinetics showed that the variants were active against all the antibiotics tested but their catalytic efficiency was considerably lower than that of NDMwt. Pre-steady state kinetics assays showed that NVIM and NPro accumulate fewer or no intermediate during imipenem hydrolysis, as compared to the NDMWT and NIMP. Variants are less stable to thermal denaturation, and also differ from the NDMWT in their Zn(II) affinities and binding mode (cooperative vs. non-cooperative). Importantly, changes in the active site coordination, specifically in the Zn2 site, were noted. Conclusions. In addition to its role in substrate recognition, we showed that ASL3 in NDM-1 has an important function in enzyme stability, and indirectly affects metal uptake and active site conformation. This is an unprecedented finding in the structure-function relationship of MβLs.