Active Site Loop 3 Defines Substrate Specificity in NDM-1

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

San Diego

Congreso; ICAAC/ICC 2015; 2015

American Society for Microbiology

BACKGROUND. The active site of class B1 metallo-beta-lactamases (MBL) is flanked by flexible Active Site Loops (ASL), ASL-3 (residues 57- 68) and ASL-10 (residues 220-240), which stabilize the substrate during catalysis. Compared to VIM-2 and IMP-1, the NDM-1?s ASL-3 is more open, flexible and hydrophobic. To test the hypothesis that these features account for the higher affinity of this enzyme for several beta-lactams, we characterize the substrate profile of a series of ASL-3 mutants of NDM-1.METHODS. NDM constructs were custom synthesized (Celtek Genes) to replace the ASL-3 of NDM-1 with those of IMP-1 (NDM_IMP-1; more charged) and VIM-2 (NDM_VIM-2; one residue shorter). Constructs were cloned into pHSG298 and pET24 vectors and transformed into E. coli DH10B and BL21 (DE3) cells, respectively. Minimal inhibitory concentrations (MICs) were determined using CLSI approved methods, immunoblotting was performed to determine amounts of beta-lactamase expression and steady-state kinetics assays were carried out with purified enzymes. RESULTS. Immunoblotting performed on crude extracts revealed that while NDM-1 and NDM_IMP-1 were expressed at similar levels, expression of NDM_VIM-2 was lower. Compared to NDM-1, NDM_VIM-2 conferred decreased resistance toward cefepime and imipenem (256 vs. 8 mg/L and 64 vs. 8 mg/L, respectively), whereas NDM_IMP-1 only affected imipenem MICs (64 vs. 8 mg/L). Imipenem hydrolysis curves showed differences in the catalytic efficiencies among the enzymes.CONCLUSION. Our preliminary data suggests that changes in ASL-3 alter the catalytic efficiency of NDM-1, supporting the importance of this loop in fine-tuning the substrate specificity of MBLs.