Doctor of Philosophy (Ph.D.)
Degree Granting Department
Yu Chen, Ph.D.
Ioannis Gelis, Ph.D.
Gloria Ferriera, Ph.D.
James Leahy, Ph.D.
Xingmin Sun, Ph.D.
antibiotic resistance, beta-lactam antibiotics, enzyme mechanism, non-covalent inhibitions
The emergence of antibiotic resistance and spread of Gram negative bacteria poses a very real health threat to the public. The main mode of resistance within Gram negative bacteria is the production of β-lactamase enzymes that catalyze the breakdown of β-lactam antibiotics through a hydrolysis mechanism. Once the β-lactam ring is hydrolyzed and opened, the drug loses its efficacy, which allows for the bacteria to grow and proliferate uninhibited. These β-lactamase enzymes are organized into four categories based on the Ambler classification, with classes A, C and D being denoted as serine-based β-lactamase enzymes. Class B is composed of metalloenzymes that use zinc cofactors to catalyze the breakdown of β-lactam antibiotics. The focus of the work presented here today will concern serine β-lactamase catalysis with emphasis on the class A β-lactamase enzymes CTX-M-9, CTX-M-14 and CTX-M-27. This thesis will delve into the underlying mechanics of the class A β-lactamase hydrolysis by way of studying the specific stages of the catalytic mechanism as well as look at nuanced aspects of the active site that leans towards drug discovery and resistance mechanisms. The organization of the document focuses on looking at function analysis, inhibition and resistance of class A β-lactamase.
Understanding the nuances involved in the acid base catalysis of the β-lactamase enzymes will lead to better development of relevant antibiotics that may circumvent resistance mechanisms. With this in mind, I used various techniques can resolve proton locations to help elucidate potential occurrences of low barrier hydrogen bonding exhibited in the mechanism of β-lactamase previously reported during the formation of the pre-covalent complex prior to the acylation transition state. This was done through obtaining a 1.8 Å D2O exchange apo neutron crystal structure of CTX-M-9. This was followed up by a perdeuterated structure of CTX-M-9 in complex with the tetrazole scaffold 3GK that was shown to trap the enzyme in the pre-covalent conformation in earlier studies. The resolution of the joint neutron/x-ray structure was 1.7 Å for both neutron and x-ray data sets. The third project entailed using sub angstrom x-ray crystallography to obtain a crystal structure of the deacylation transition state at a resolution of 0.76 Å. The last section of chapter 2 looks at protein engineering, where disulfide bonds are instituted into the active site through mutating residue glutamate 166 to a cysteine. The premise is to selectively engineer perturbations to the microenvironment to help understand the impact on the formation of the low barrier hydrogen bond (LBHB) witnessed between Ser70 and Lys73 in the pre-covalent complex.
The third chapter concerning inhibition of CTX-M-14 and CTX-M-27 focuses on looking at the amide-heteroarene stacking interactions as potential facets for drug design. Different tetrazole-based scaffolds were modified with heteroarene substituents and analyzed for pi stacking interactions with the amide backbone of Gly238. Five crystal structures were obtained and all bound in the predicted fashion with exhibited heteroarene-amide pi stacking interactions, thus lending credence to use CTX-M as a model system.
The last project investigated resistance mechanisms within class A β-lactamase with focus on aspartate – aspartate interactions. Three crystal structures were obtained for this study with the WT CTX-M-14 apo crystal, the CTX-M-14 D233N mutant and the CTX-M-14 D233N J1X tetrazole complex. My study focused on the contribution of aspartate residues to the integrity of the active site with a partial focus on the importance of the 213-219 loop. My findings suggest that β-lactamases can potentially develop resistance against certain inhibitors by increasing its active site flexibility.
Scholar Commons Citation
Kemp, Michael Trent, "Mechanistic Insight into β-Lactamase Catalysis, Inhibitor Design and Resistance" (2021). USF Tampa Graduate Theses and Dissertations.