Through mutations and horizontal transfer, bacteria has been able to acquire different forms of antibiotic resistance. In particular, bacteria that produce β-lactamases as a result of mutated or acquired genes have the ability to break down the β-lactam ring structure in many common classes of antibiotics (penicillins, carbapenems, monobactams, and cephalosporins). Metallo-β-lactamases, enzymes that confer β-lactam antibiotic resistance, have shown an increased presence in the clinical setting and therefore have become a more significant issue. Structure determination for these enzymes helps future research regarding the search for inhibitors to be paired with antibiotics in new clinical regimens, disabling the enzyme’s contribution to β-lactam antibiotic resistance. New Delhi Metallo-β-lactamase (NDMs), a member of the B1 subclass of metallo-β-lactamases, has been gaining recognition due to its prevalence in the clinic, yet the field requires more research on its structure. This study focuses on the variant NDM-4. Activity assays were performed with NDM-4 to determine activity prior to any modifications. A new NDM-4 construct, NDM-4_6-Aldolase, was developed and was studied for the purpose of using cryo-EM to solve the structure of NDM-4. The tetramer of aldolase (PDB code 1ZAH, Fructose-1,6-bisphosphate aldolase) used in these constructs was chosen for its large size and symmetrical tetrameric structure. These constructs fused NDM-4 and aldolase to produce a construct amenable for Cryo-EM. Activity assays and a cryo-EM structure were successfully obtained. NDM-4_6-Aldolase exhibited a best-fit Km of 128 µM. Cryo-EM will be performed to resolve the structure of this new construct. Future work with dipicolinic acid (DPA) as an inhibitor of NDM-4_6-Aldolase is planned.
Authors: S. Kei Brown, Nastia Evstifeeva, & Richard C. Page, Ph.D.
Advisor(s): Richard Page, Chemistry & Biochemistry
Nastia Evstifeeva, Chemistry & Biochemistry
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