Mentor: Richard Page, Ph.D.

The discovery of antibiotics revolutionized medicine, but their misuse has driven antimicrobial resistance (AMR). Antibiotic exposure creates selective pressure, which enables bacteria to develop resistance through multiple mechanisms. Our lab focuses on enzymes that hydrolyze antibiotics. β-Lactam antibiotics are widely used for their affordability, low toxicity, and effectiveness. There are three classes of β-lactam antibiotics, which are penicillin, cephalosporins, and carbapenems. They all consist of a β-lactam ring, which is three carbons and one nitrogen, but differ in their side chains. Resistance occurs when enzymes called β-lactamases hydrolyze, or break, this ring. β-Lactamases can be classified as serine β-lactamases (SBLs) or metallo-β-lactamases (MBLs). Our lab focuses MBLs in particular. MBLs have zinc ions in their active site and are less researched than SBLs. MBLs are highly effective in driving antibiotic resistance because they hydrolyze almost all β-lactams, and SBL inhibitors do not work to inhibit MBLs. A particular strain of MBLs called the New Delhi metallo-β-lactamase (NDM) was first identified in 2008 and now has many strains. NDM has two zincs in its active its strains are evolving to be effective under limited zinc concentrations. Because MBLs hydrolyze substrates too quickly for traditional crystallography, our lab designed NDM-X, a mono-zinc variant of NDM-4 (Figure 1). Comparisons between NDM-4 and NDM-X by SDS-Page confirmed after anion exchange and size exclusion columns, pure protein at a size about 25 kDa was obtained. Differential scanning fluorimetry (DSF) revealed NDM-X has a melting temperature (Tm ) of 42.45 ºC and NDM-4 has a Tm of 44.90 ºC for their first unfolding. NDM-4 also has a Tm of 56.72 ºC for it’s second unfolding . Nitrocefin activity assays showed NDM-X had a Km value of 205.5 µM and NDM-4 had a Km value of 5.53 µM. This data gives us a greater depth of knowledge on the characteristics of NDM-X.

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