Journal
INTERNATIONAL JOURNAL OF ANTIMICROBIAL AGENTS
Volume 55, Issue 1, Pages -Publisher
ELSEVIER
DOI: 10.1016/j.ijantimicag.2019.10.005
Keywords
beta-Lactams; New Delhi metallo-beta-lactamase; Antimicrobial resistance; Carbapenems; Thermal stability; Substrate docking
Funding
- Department of Biotechnology, Government of India [BT/PR24255/NER/95/716/2017]
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New Delhi metallo-beta-lactamase (NDM) is of significant public-health concern due to its enormous potential to hydrolyse all of the major beta-lactams, including carbapenems. Previous reports indicate that amino acid substitutions affect NDM activity despite being located outside of the active site. In this study, we attempted to identify specific mutations in loops near the active site that can influence the function of NDM-7. Overall, six substitutions were performed through site-directed mutagenesis near the active-site of NDM-7 and the change in antimicrobial resistance was subsequently monitored by expressing each mutant in a suitable bacterial host. Among the six mutants, serine at position 191 (S191) and glutamic acid at position 152 (E152) were identified as the most influencing residues for NDM-7. beta-Lactam resistance of NDM-7 was remarkably affected by substitution of both residues with alanine, and the results were in accordance with the changes in kinetic parameters. Purified NDM-7 ordinarily hydrolyses beta-lactams efficiently, but purified NDM-7_E152A, NDM-7_S191A and the double mutant NDM7_E152A+S191A had lost their ability to hydrolyse the beta-lactams tested, especially penicillins and carbapenems. Although the substitutions did not affect the overall folding pattern of the NDM-7 enzyme, substantial differences in thermal stability were observed. Therefore, we hypothesise that the residues S191 and E152 together play a crucial role in conferring the beta-lactamase character of NDM-7. (C) 2019 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.
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