Role of active site loop 3 in substrate recognition of the lactamase NDM-1

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

Nueva Orleans

Congreso; ASM Microbe 2017; 2017

American Society for Microbiology

Background. B1 Metallo-b-Lactamases (MBLs) have an αb/bα fold sandwich in which the binuclear Zn(II)-containing active site is surrounded by two protruding loops. ASL-3 is proposed to have an important role on substrate recognition by MBLs. 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 possibly 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 b-lactams, we characterized a series of ASL-3 mutants of NDM-1. Methods. NDM constructs were engineered to produce variants in which the ASL-3 was replaced by those of IMP-1 (N-IMP; with more charged residues) and VIM-2 (N-VIM; one residue shorter), and a third one in which the conserved Pro residue at the base of the ASL-3 (N-Pro) was introduced. Constructs were cloned into pMBLe and pET28 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 NDM-1-expressing E. coli, variants showed a significant decrease in activity against cefepime and imipenem (e.g. MICs= 128 and 2 mg/L for NDM-1 vs. 8 and 0.5 mg/L for N-Pro, 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 NDM-1. Pre-steady state kinetics assays revealed that N-VIM and N-Pro accumulated fewer or no intermediate during imipenem hydrolysis, as compared to the NDM-1 and N-IMP. Variants were less stable to thermal denaturation, and also differed from the NDM-1 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 ASL-3 in NDM-1 has an important function in the catalytic mechanism, enzyme stability, and indirectly affects metal uptake and active site conformation. This is an unprecedented finding in the structure-function relationship of MBLs.