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    NDM-3: A Study of the Expression, Purification, and Characterisation of a B1-type metallo β-lactamase

    Phelan, Emer (2023) NDM-3: A Study of the Expression, Purification, and Characterisation of a B1-type metallo β-lactamase. Masters thesis, National University of Ireland Maynooth.

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    Antibiotic resistance is a prevalent and global problem. It is one of the biggest, if not the biggest, health care challenge we face this century. Metallo-beta-lactamases (MBLs) are a group of enzymes that play a pivotal role in the spread of resistance. There are many contributing factors for the success of MBLs at overcoming current antibiotics, and why they evade and evolve to overcome potential new antibiotics before said antibiotics would make it to market. One of these reasons is the sheer number of MBLs and more specifically, the number of mutants of these MBLs. There are as many as four subgroups in the MBL family, labelled B1 to B4. B1 is the best-studied subgroup, and the one that is most prevalent in pathogenic microorganisms. Members of this subgroup employ two metal ions (generally Zn2+) in their active sites for hydrolysis of antibiotics. NDM (New Delhi MBL) is a prominent example that has spread quickly and poses a real threat to society’s use of antibiotics. Known as a “superbug,” NDM-1 has made use of various tools to spread rapidly (horizontal gene transfer), evade inhibition, and evolve catalytic efficiency. The introduction of specific mutations has led to the emergence of at least 15 known variants of this enzyme that vary in their substrate preference and catalytic properties. NDM-3 is one such variant and is the topic of this research project, which aims to determine and characterise its catalytic properties and compare those to corresponding parameters reported for the original NDM variant discovered, NDM-1. What was discovered during this thesis was the optimised process to isolate, express, and purify NDM-3. This was completed with two constructs of NDM-3: NDM-3+Ub, and NDM-3(NC) – NDM-3(NC) did not have the ubiquitin tag attached. Purification of both of these constructs were completed using hexahistidine IMAC chromatography. NDM-3+Ub then underwent cleavage using USP2cc protease, however no activity was ever witnessed on this cleaved enzyme. Activity assays were performed on both the uncleaved NDM-3+Ub, and NDM-3(NC). These results were compared with the NDM-3(NC) showing stronger activity than the construct with the ubiquitin tag. Overall, NDM-3(NC) has lower activity than NDM-1. From these works it is shown that like NDM-1, NDM-3(NC) was shown to have activity across a wide range of substrates but NDM-1 has superior hydrolytic activity against most β-lactam antibiotics tested. As an example, the kcat values of NDM-1 for the hydrolysis of ampicillin and penicillin are 182 s-1 and 142 s-1, respectively. The corresponding parameters for NDM-3(NC) are only 47 s-1 and 38 s-1, respectively, effectively showing only ~40% of NDM-1 activity. Similarly, NDM-3(NC) underwent pH profile assays to determine the pH range it has activity against various beta lactam antibiotic substrates, and to determine the pH point that activity is the strongest. As would be expected for a virulent and pathogen that infects humans, NDM-3(NC) like NDM-1, has strongest activity at physiological pH points (i.e. around pH 7.5) and showing the strongest activity overall against ampicillin and meropenem across the pH ranges.

    Item Type: Thesis (Masters)
    Keywords: NDM-3; Expression; Purification; Characterisation; B1-type metallo; β-lactamase;
    Academic Unit: Faculty of Science and Engineering > Chemistry
    Item ID: 17272
    Depositing User: IR eTheses
    Date Deposited: 06 Jun 2023 10:16
      Use Licence: This item is available under a Creative Commons Attribution Non Commercial Share Alike Licence (CC BY-NC-SA). Details of this licence are available here

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