UBC Theses and Dissertations
Mutational scanning of metallo-β-lactamases to probe functional determinants, selection pressure dependence and homolog incompatibilities Chen, John Zhongshi
Enzymes known as metallo-β-lactamases (MBLs) are a major source of bacterial resistance against β-lactams, one of the most widely used classes of clinical antibiotics. MBLs degrade almost all classes of β-lactams with high efficacy, and can be transmitted on plasmids. Detailed understanding of MBLs in terms of their sequence-function relationships can be beneficial in the design of new antibiotics or inhibitors, understanding how mutations affect MBL function can help understand and predict their evolution, and give general knowledge on how mutations could be leveraged in protein engineering. Although there are numerous detailed studies of MBLs, our knowledge is limited to a handful of residues in a small number of sequences. A recently developed method known as deep mutational scanning (DMS) can enable us to construct comprehensive sequence-function maps of proteins. Using DMS, we comprehensively explored the sequence-function relationship of 2 representative MBL homologs, NDM-1 and VIM-2. The DMS data revealed the key functional requirements of the MBLs, including those on activity, stability and substrate specificity. Furthermore, DMS conducted across different antibiotic concentrations allows us to explore the concentration dependence of sequence-function relationships. Finally, comparisons between NDM-1 and VIM-2 datasets revealed prevalent epistasis, with further analysis highlighting the structural trends and interactions that underlie such observations.
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