Abstract
Colistin is a cationic polymyxin peptide antibiotic, used as a last resort treatment for multi-drug resistant Gram-negative bacterial (GNB) infections. This antibiotic associates with and disrupts the outer membrane of GNBs, acting as a detergent and displacing cell surface cations, leading to cell lysis and death.Phosphoethanolamine transferase enzymes are able to chemically modify the outer membrane of GNBs in order to confer resistance to colistin. Plasmid-encoded mobile colistin resistance (MCR) enzymes can, where expressed, transfer phosphoethanolamine onto lipid A of lipopolysaccharide (LPS), the primary component of GNB outer membranes. This target modification resistance mechanism reduces the negative charge of the GNB surface, reducing the affinity and therefore efficacy of colistin.
This thesis presents a mechanism by which MCR enzymes catalyse the phosphoethanolamine transfer reaction, using a range of experimental and computational techniques. A mechanism is proposed whereby MCR enzymes use one Zn²⁺ ion for the first step in the catalytic cycle, where phosphoethanolamine binds conserved catalytic residue Thr285, but requires two Zn²⁺ ions for the second step, where the phosphoethanolamine moiety is donated to lipid A. This mechanism is supported by observations in crystallo, in vitro and in silico of metal stoichiometry under different stages of substrate binding.
Molecular dynamics simulations are used to understand the relationship between phosphorylation/substrate binding and metal binding to the catalytic domain of MCR-1, both in the absence and presence of the transmembrane domain. X-ray crystallography alongside site-directed mutagenesis is used to model phosphorylated and unphosphorylated states of the enzyme in order to understand the structural relationship between phosphorylation and metal binding. Biochemical assays, including chromogenic and fluorescence-based assays, are used to define metal stoichiometry in solution, as well as determine binding constants for Zn²⁺ ions to the catalytic domain of MCR-1.
Finally, the results are combined with data from recently published quantum mechanical cluster models in Suardíaz et al. 2021 to propose a mechanism for both steps of the MCR-1 reaction, as well as more detailed information about global and local dynamics and substrate binding interactions.
| Date of Award | 6 Dec 2022 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Jim Spencer (Supervisor) & Adrian J Mulholland (Supervisor) |