Graduation Year
2022
Document Type
Dissertation
Degree
Ph.D.
Degree Name
Doctor of Philosophy (Ph.D.)
Degree Granting Department
Chemistry
Major Professor
Ioannis Gelis, Ph.D.
Committee Member
Theresa Evans-Nguyen, Ph.D.
Committee Member
Jianfeng Cai, Ph.D.
Committee Member
Yu Chen, Ph.D.
Keywords
Catalysis of Cefotaximase, Protein Backbone, Sequential Assignment, pKa, NMR
Abstract
Extended-spectrum β-lactamases (ESBLs) of different Enterobacteriaceae (e.g. Escherichia coli, Klebsiella pneumoniae) have developed significant resistance mechanisms against antibiotics. ESBLs, like class A CTX-Ms (cefotaximases) enzymes hydrolyze the β-lactam ring of different antibiotics such as penicillins and third-generation oxyimino-cephalosporins. Although CTX-Ms are repeatedly found in many clinical and community settings, molecular-level information on their function/activity is currently limited to structural studies performed by X-ray crystallography. In order to gain further insights into the structure and functions of this class of proteins, it is also essential to fully understand their dynamic and chemical properties in solution. In order to better understand the protein structure from the solution state NMR perspective, we have first completed the sequential assignments of the amide backbone of the two CTX-Ms (CTX-M-9 and CTX-M-16) reported in chapter 2. These are the first of the class A CTX-Ms NMR sequential assignments to our best knowledge. Moreover, in chapter 3 we investigated the protein dynamics of CTX-M-9 and CTX-M-16 in a solution state. The 15N relaxation experiments (R1, R2, and heteronuclear NOEs) were performed in multiple fields, and data were analyzed using the Lipari and Szabo model-free approaches. The site-specific dynamic behaviors of CTX-M β-lactamases from very fast (sub nanoseconds) to slow time scales (seconds) are studied here to provide a valuable insight to further validate and enrich the future computational simulation study of the molecular dynamics of CTX-Ms. As the generalized order parameter, S2 calculated from NMR and molecular dynamics (MD) can be compared and more refined reasoning can be obtained about the amide backbone dynamics in the active site region as well as in any flexible region such as in loops. The MD simulations can be focused further on the flexible region in order to obtain S2 accurately for loops. In that case, a reference point of the global tumbling time (τm) is needed to adjust the trajectory length of the simulations. The S2 and τm from the NMR study reported here can aid for such conditions in MD simulation. Moreover, the overestimation scenario in MD simulations where some of the local motions that are coordinated with a frequency comparable to the simulations trajectory length (often in ns time scale) could be more rapid in reality. In such a case, the S2 calculated from our NMR study can be used as a reference point. To compare both S2 from MD simulation and NMR a recoupling of the global tumbling timescale is required. For such a case, the MD simulation can be aided by the NMR-derived global tumbling time (τm) of the protein. The hydrolysis reaction of β-lactam ring-containing drugs involves the formation of an acyl-enzyme complex by the CTX-M enzymes. The active site residues in the complex play a pivotal role in catalysis. It is important to track the protonation state of catalytic residues to fully understand the mechanism behind the acylation-deacylation process. Besides sequential assignment and protein dynamics study, here, we present NMR evidence showing that binding of a non-β-lactam ring containing antibiotic named avibactam, results in a significantly lower pKa of the catalytic lysine (K73) of CTX-M-9 when compared to that with free lysine. This altered pKa provides a novel, important mechanistic understanding of the catalytic properties of these enzymes and thus their inhibition. The NMR study reported here was utilized with X-ray crystallography data (not reported here) to gain insight into the proton transfer process during the acylation reaction with active site residues and explain the vital role of K73 in the process. Therefore, we believe that the sequential assignments, protein dynamics and the role of an active site residue (K73) reported here can be useful for a better understanding of the structure-function relationships of CTX-Ms and also advance the systematic designing of novel antibiotics/inhibitors in future.
Scholar Commons Citation
Noor, Radwan Ebna, "An Investigation into the Protein Dynamics and Proton Transfer Mechanism of Class-A β-lactamase (CTX-Ms) by NMR Spectroscopy" (2022). USF Tampa Graduate Theses and Dissertations.
https://digitalcommons.usf.edu/etd/10334
