Disarming β-Lactamases: A Structure Based Approach to Addressing Antibiotic Resistance

Research Team

Background

Aims

Methods

Results and Discussion

Conclusions and Future Work

References

Acknowledgements

NACE Career Readiness Competencies

Research Team

Kendal Rivera

Chemistry Major, Pre-Medical Studies Co-Major & Spanish Minor

Anastasiia Evstifeeva

PhD Candidate Department of Chemistry and Biochemistry

Richard C. Page PhD

Department of Chemistry and Biochemistry

Background

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.

Structure of NDM-X depicting one zinc in the active site.

Figure 1: Structure of NDM-4 (cyan) and of NDM-X (pink).

Aims

Our goal is to create crystal structures of the mono-zinc NDM-4 variant, NDM-X. Visualizing and understanding these β-lactamases when in their substrate-bound state before hydrolysis to a product can help to provide a foundation for creating structure-based inhibitors.

Methods

Cloning, Expression, and Purification of NDM-4 and NDM-X
Nitrocefin Activity Assay
Differential Scanning Fluorimetry

Results and Discussion

gel image of an SDS-page of NDM-4 after an anion exchange column. Thick, dark bands can be seen in the factions corresponding to a size of about 25 kDa.

Gel image of an SDS-page of NDM-4 after a size exclusion column. There are less other bands present. Still have thick, dark bands corresponding to about 25 kDa.

Figure 2: SDS-Page of NDM-4 after anion exchange column (left) and size exclusion column (right).

gel image of SDS-page of NDM-X after an anion exchange column. Can see some impurities but also thick, dark bands corresponding to about 25 kDa.

gel image of an SDS-page of NDM-X after a size exclusion column. Much more pure with thick, dark bands corresponding to about 25 kDa size still.

Figure 3: SDS-Page of NDM-X after anion exchange column (left) and size exclusion column (right).

DSF data plotted as the first derivative of the relative fluorescence units vs temperature in ºC. This corresponds to the first zinc leaving the active site of NDM-X (green) and NDM-4 (red)

Figure 4: First unfolding of NDM-4 (red) and NDM-X (green) plotted as the first derivative of relative fluorescence units (RFU) versus temperature (ºC).

The DSF data for the second unfolding of only NDM-4 as NDM-X does not have two zinc in its active site. Plotted as RFU vs temperature (ºC).

Figure 5: Second unfolding of NDM-4 (red) plotted as the first derivative of relative fluorescence units (RFU) versus temperature (ºC).

Michaelis-Menten plot of NDM-4. Shows that the Km is 5.53 micromolar.

Figure 6: Michaelis-Menten plot of NDM-4 plotted as Rate [μM/s] versus concentration of nitrocefin [μM].

Michaelis-Menten plot of NDM-X. Km value is 205.5 micro molar.

Figure 7: Michaelis-Menten plot of NDM-X plotted as rate [μM/s] versus concentration of nitrocefin [μM].

Protein purity increases after a size exclusion column is ran. The size of the dark bands is at roughly 25 kDa. The molecular weight of NDM-4 is 24.3 kDa (Figure 2 & Figure 3). The Tm for NDM-4 is 44.90 ºC while for NDM-X it is 42.45 ºC (Figure 4). This shows NDM-X is slightly less thermodynamically stable than NDM-4. The second unfolding Tm for NDM-4 is 56.72 ºC (Figure 5). NDM-X does not have second unfolding data because it has one zinc in its active site, while NDM-4 has two. The Michaelis-Menten plots of NDM-4 and NDM-X yielded a Km value of 5.53 µM and 205.5 µM, respectively (Figure 6 & Figure 7). These results suggest that the loss of the second zinc ion significantly impairs substrate binding and decreases the overall catalytic efficiency of the enzyme.

Conclusions and Future Work

The SDS-Page analysis of NDM-4 and NDM-X demonstrates that after the protein is ran though an anion exchange column and size exclusion column, relatively pure protein is obtained. The molecular weight of NDM-4 is 24.3 kDa, and the dark bands in our SDS-Page gels are found at a size corresponding to about 25 kDa. While the DSF data shows NDM-X is slightly less thermodynamically stable than NDM-4, the nitrocefin activity assay shows NDM-X is far less catalytically efficient. This shows we succeeded in creating an NDM variant that will hydrolyze its substrate at a slower rate. In the future, 4-(2-pyridylazo)resorcinol (PAR) assays and isothermal titration calorimetry (ITC) can be done to compare NDM-X and NDM-4, allowing for an even greater depth of knowledge on the characteristics of NDM-X. The low catalytical efficiency of NDM-X also suggest it could be possible to obtain crystal structures of NDM-X with unhydrolyzed substrate. X-ray crystallography of NDM-X can be done in the future to obtain a better understand of the structure of NDM-X with substrate, and thus, allowing an insight to the structure of NDM variants and substrate.

The following is an image of poster presented at the 2026 Undergraduate Research Forum.

References

(1) Nimmana, B. K. K.; Nguyen, A. D. Antibiotic Resistance. https://www.ncbi.nlm.nih.gov/books/NBK513277/ (accessed 2026-03-10).

(2) Stewart, A. C.; Bethel, C. R.; VanPelt, J.; Bergstrom, A.; Cheng, Z.; Miller, C. G.; Williams, C.; Poth, R.; Morris, M.; Lahey, O.; Nix, J. C.; Tierney, D. L.; Page, R. C.; Crowder, M. W.; Bonomo, R. A.; Fast, W. Clinical Variants of New Delhi Metallo-β-Lactamase Are Evolving to Overcome Zinc Scarcity. ACS Infectious Diseases 2017, 3 (12), 927–940. DOI:10.1021/acsinfecdis.7b00128.

(3) Makena, A.; Brem, J.; Pfeffer, I.; Geffen, R. E.; Wilkins, S. E.; Tarhonskaya, H.; Flashman, E.; Phee, L. M.; Wareham, D. W.; Schofield, C. J. Biochemical Characterization of New Delhi Metallo-β-Lactamase Variants Reveals Differences in Protein Stability. Journal of Antimicrobial Chemotherapy 2014, 70 (2), 463–469. DOI:10.1093/jac/dku403.

(4) Evans, L. E.; Krishna, A.; Ma, Y.; Webb, T. E.; Marshall, D. C.; Tooke, C. L.; Spencer, J.; Clarke, T. B.; Armstrong, A.; Edwards, A. M. Exploitation of Antibiotic Resistance as a Novel Drug Target: Development of a β-Lactamase-Activated Antibacterial Prodrug. Journal of Medicinal Chemistry 2019, 62 (9), 4411–4425. DOI:10.1021/acs.jmedchem.8b01923.

(5) Pandey, N. Beta-Lactam Antibiotics. https://www.ncbi.nlm.nih.gov/books/NBK545311/ (accessed 2026-03-18).

(6) Coppo, E.; Del Bono, V.; Ventura, F.; Camera, M.; Orengo, G.; Viscoli, C.; Marchese, A. Identification of a New Delhi Metallo-β-Lactamase-4 (Ndm-4)-Producing Escherichia Coli in Italy. BMC Microbiology 2014, 14 (1). DOI:10.1186/1471-2180-14-148.

(7) (EN) - Qiaprep Spin Miniprep Kit (2015) - Contains Qiaprep 2.0 Spin Column. https://www.qiagen.com/us/resources/protocols/hb-1921-1091289-pcard-qiaprepspinmini-0215-ww (accessed 2026-04-03).

Acknowledgements

The work detailed in this poster was funded by the National Institute of Health through grant R35GM128595 to R.C. Page.

NACE Career Readiness Competencies

Career & Self-Development

Understanding the research process is a very important skill that many medical schools like to see. By participating in undergraduate research for 3.5 years, I demonstrated to medical schools that I have an understanding of what is necessary to conduct good research. Also, as a medical student and physician, it is very likely that I will be assisting with or conducting my own research. The skills I learned in this lab will help me be successful in any research I do later in my career.

Professionalism

During my time in my research lab, I have shown professionalism by being a dependable member, showing up consistently, even during busy semesters. I paid close attention to detail to ensure experiments were prepared and performed correctly.

Teamwork

During my time as an undergraduate research assistant, I have worked with other undergraduates, graduate students, and my principal investigator. I have built a good relationship with the graduate student who oversees my work, and I have helped train new undergraduate students in the lab. We all work together and share the responsibilities of the lab to ensure our research moves forward.

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