Title
Glutamic acid at position 152 and Serine at position 191 are the key residues required for the metallo-beta-lactamase activity of NDM-7
Author
Gaurav Kumar, Bagre Issa, Sarmistha Biswal, Diamond Jain, Amitabha Bhattacharjee, Anindya S. Ghosh
Year
2019
Journal
International Journal of Antimicrobial Agents
Abstract
New Delhi Metallo beta-lactamase (NDM) is of significant public health concern due to its enormous potential to hydrolyse all the major beta-lactams including carbapenems. Previous reports indicate that the amino acid substitutions affect NDM activity despite being located outside the active site. In this work, we attempted to identify specific mutationson loops near the active-site that can influence the function of NDM-7. Overall, six substitution mutations were done near the active-site of NDM-7 and subsequently, the change in antimicrobial resistance was monitored upon expressing each mutant in a suitable bacterial host cell. Among the six mutants, serine at position 191 and glutamic acid at position 152 were identified as the most influencing residues for NDM-7. The beta-lactam resistance of NDM-7 was remarkably affected upon the substitution of both with alanine and the results were in synchrony with the modulations in kinetic parameters. Normally, purified NDM-7 hydrolyzes beta-lactams efficiently, but purified NDM-7_E152A, NDM-7_S191A and their double mutant had lost its ability to hydrolyze beta-lactams tested, especially penicillins and carbapenems. Though, the substitutions did not affect the overall folding pattern of the NDM-7, substantial differences in thermal stabilities were observed. Therefore, we hypothesize that S191 and E152 together play the crucial role in exhibiting beta-lactamase nature of NDM-7.
Instrument
J-815
Keywords
Circular dichroism, Secondary structure, Protein folding, Thermal stability,