Characterisations of a hospital-acquired carbapenemase from enterobacteriaceae

Prombhul S1,2, Pedroso MM1,2, Lo AW1,2, Nouwens A1, Josh P1, Neo IYY1, Mcgeary RP1,2, Schembri MA1,2 and Schenk G1,2

  1. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Queensland 4072, Australia.
  2. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, Queensland 4072, Australia.

New Delhi metallo-β-lactamase (NDM) is a major health concern due to its efficient inactivation of antibiotics and its rapid spread across the globe [1]. This enzyme displays catalytic efficiency towards a broad spectrum of clinically used β-lactam drugs [2]. Here we constructed two variants of NDM-1, M27 and G36, as previously reported [1, 3-7], and probed the Zn2+ stoichiometry of the enzymes using mass spectrometry. The mass spectral data indicate that the M27 variant is predominantly present in bimetallic form, while for the G36 variant both bi- and monometallic as well as metal-free species are observed. This observed variation between the two variants suggests that the N-terminal domain may play a direct role in metal ion binding and possibly also in the protein solubility and stability. Moreover, we examined the effects of aztreonam (a monobactam derivative) on the catalytic efficiency of NDM-1; while it has an inhibitory effect for full-length NDM-1, the two variants with truncated N-terminal ends are not affected. Our results thus agree with previous findings that ascribed a role for the N-terminus of NDM-1 in substrate/inhibitor binding and specificity [8, 9]. Also, we recently presented evidence that copper may act as a sensitisation agent for clinical antibiotics [10]. Our results were consistent with a loss of NDM-1 activity in the presence of copper, indicating a noncompetitive mode of inhibition with an apparent inhibition constant (Ki) of ~10 μM. However, we observed that the enzymes were precipitated in the presence of CuCl2, this might be due to the occurrence of interprotein crosslinking between metal-ion bound and the accessible histidines, and leads to the formation of large and insoluble complexes. This is another step to explore the application of metal coordination complex as an attempt to replace predominant zinc metals of the enzyme and the importance of N-terminal sequence of protein. Furthermore, we currently explored the potential mode of binding of a compound 5 (N-(1-Benzyl-4,5-diphenyl-1H-imidazol-2-yl)benzamide) in the active sites of the enzyme to examine specificity of this inhibitor with respect to the two variants. Interestingly, the compound represented partial inhibitory activity towards the two variants of NDM-1 when penicillin G was used as substrate. The result showed a competitive mode of inhibition with an apparent inhibition constant (Kic) of 9.85 and 2.6 μM for M27 and G36, respectively. Further studies will focus more on investigating the inhibitory mechanism and visualising the binding mode(s) of the potential compounds to develop universal MBL inhibitors.