β-lactamase enzymes catalyze the irreversible hydrolysis of the four-membered cyclic amide ring characteristic of β-lactam antibiotics rendering them inactive and useless against pathogenic bacteria. Understanding structure activity relationships between β-lactam antibiotics and β-lactamases is important for designing novel β-lactams, β-lactamase inhibitors, and β-lactam-based fluorescent probes for rapid diagnosis of β-lactam antibiotic resistant infections. The first half of this study focuses on the class A β-lactamase BlaC from Mycobacterium tuberculosis (Mtb) and addresses intermolecular interactions between BlaC and substrates, inhibitors, and biosensors that influence their kinetic parameters with BlaC and activities against Mtb. The substrate structure activity relationship explained the molecular basis for differential innate resistance of Mtb to faropenem, biapenem, and tebipenem by showing the interactions between BlaC and the lactams that govern differential acyl-intermediate stability and affinity. The inhibitor structure activity relationship revealed features of the BlaC active site that can be exploited to enhance binding and inhibition of BlaC by benzoxaboroles, and demonstrates their utility as potentiators of β-lactam antibiotic activity against Mtb. BlaC-specific β-lactam based fluorescent probes were designed and optimized for Mtb detection. Their utility was demonstrated by detecting down to 10 colony forming units of bacillus Mycobacterium bovis Calmette–Guérin (BCG) in human sputum. The second half of this study focuses on the New Delhi Metallo-β-lactamase-1 (NDM-1), which is rapidly generating bacterial resistance to nearly all β-lactams. The NDM-1 gene encodes a class B1 metallo-β-lactamase enzyme. Purified recombinant NDM-1 was biochemically and biophysically characterized. The crystal structures of apo and monometalated NDM-1 provided structural insight into metal binding and the promiscuous enzymatic activity of NDM-1. Mechanistic details of the NMD-1 reaction were examined by comparing crystal structures of NDM-1 in complex with an unhydrolyzed β-lactam substrate and with hydrolyzed products. These structures were used for quantum mechanics / molecular mechanics simulations to estimate the free energy along the β-lactamase reaction coordinate. The results suggest that NDM-1 uses bulk water as the nucleophile that attacks the β-lactam ring, and a coordinated hydroxide ion or water molecule as the catalytic base depending on pH.

by Leung Wing-cheuk. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 118-133). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract (in English) --- p.ii -iii / Abstract (in Chinese) --- p.iv -v / Table of Contents --- p.vi / Contents of Chapter 1: Introduction --- p.vii -viii / Contents of Chapter 2: Materials & Methods --- p.ix / Contents of Chapter 3: Results --- p.x / Contents of Chapter 4: Discussion --- p.xi / List of Tables --- p.xii / List of Figures --- p.xiii / Chapter Chapter 1: --- Introduction --- p.1-40 / Chapter Chapter 2: --- Materials & Methods --- p.41-67 / Chapter Chapter 3: --- Results --- p.68-99 / Chapter Chapter 4: --- Discussion --- p.100-117 / References --- p.118-133

In many dsDNA viruses, a single-strand-annealing homologous recombination (SSA) reaction is catalyzed by a pair of proteins. In phage lambda, this system is called Red, and is composed of lambda exonuclease (Lambda-Exo, a 5' to 3' dsDNA exonuclease), and Red-Beta (a ssDNA binding protein and annealase). I examined the physical and mechanistic coupling of Lambda-Exo and Red-Beta and confirmed that these proteins form a complex with a 1:1 subunit stoichiometry. The size of this complex was shown to be close to one MDa, possibly composed of 12 Lambda-Exo and 12 Red-Beta monomers. Red-Beta decreased the extent of digestion of dsDNA by Lambda-Exo, possibly by preventing its rebinding. The processivity of Lambda-Exo was not affected by Red-Beta, but the dwell-time of Lambda-Exo was significantly increased by Red-Beta. These effects of Red-Beta on Lambda-Exo may have important roles in controlling the SSA reaction by preventing hyper-resection of DNA, and/or by stabilizing Lambda-Exo/DNA complexes. The previous observations that Red-Beta protects ssDNA from nucleases and that SSB inhibits Red-Beta assembly onto ssDNA were confirmed and strengthened by our results. We determined that Red-Beta inhibits SSB binding to nascent ssDNA generated by Lambda-Exo. This strongly suggests that generation of nascent ssDNA by Lambda-Exo is coupled to assembly of Red-Beta onto nascent ssDNA. We describe two models for this coupled assembly: Model One suggests that Lambda-Exo loads Red-Beta on nascent ssDNA providing a kinetic advantage over SSB, and Model Two proposes that the complex of Lambda-Exo and Red-Beta feeds ssDNA directly onto the dodecameric Red-Beta ring. It was suggested that Lambda-Exo forms a topological link with nascent ssDNA, thereby making digestion highly processive. We challenged this model by removing the nascent ssDNA with ExoI during a Lambda-Exo digestion reaction. We observed that the nascent ssDNA was a major contributor to the processivity of Lambda-Exo since removal of nascent ssDNA resulted in a drastic decrease in the processivity of Lambda-Exo. This is the first demonstration that DNA is a processivity factor, strengthening the view that processive DNA processing enzymes should be thought of as nucleoprotein complexes that must be kinetically treated as both substrate and enzyme.

Introduction: The New Delhi metallo-β-lactamase (NDM-1) is an enzyme synthesized by bacterium which hydrolyses β-lactam antibiotics and inhibits their antibiotic effects. Its active site is highly diverse and the hydrolysis mechanism is not yet fully understood. So, there is currently no clinically useful inhibitor for this enzyme. Due to this β-lactam resistant characteristics and the ease of horizontal gene transfer between bacteria, NDM-1 producing bacteria poses a great threat to public health. Objective: Structural analysis on Zn-Zn distance in the active site of NDM-1 was carried out in order to gain a better insight on its hydrolysis mechanism. Method: Structures of NDM-1 were obtained from PDB database and were viewed on PyMOL. Out of 22 NDM-1 structures searched, 12 of them fulfilled the inclusion criterion and were included in this study. Zn-Zn distances were compared with different factors including pH and temperature during crystallization, presence of water in the active site and nature of ligand bound to active site. Results: A trend was observed when Zn-Zn distances were compared with pH. There was also a correlation between Zn-Zn distance and nature of ligand. Distance between the 2 zinc ions in the active site increased in acidic pH value of 6.5 or below and it favours substrate hydrolysis while neutral or alkaline pH shortened the distance between zinc ions leading to hydrolysis inhibition. Discussion: One NDM-1 structure can hydrolyse the substrate in the absence of water molecule. It suggested that there might be another alternative mechanism which does not involve water molecule as nucleophile. Future study can be carried out to verify this hypothesis. On the other hand, it was also suggested that a lower pH, likely to be a pH value of 6.5 or below, could lead to a relatively higher Zn-Zn distance, likely to be 4.00 Å or above. A Zn-Zn distance of 4.00 Å or above would facilitate the hydrolysis mechanism. In other words, a pH value lower than 6.5 might also promote the hydrolysis action against β-lactams. These give us a deeper understanding into the hydrolysis mechanism of NDM-1 and might be useful in future development of clinical inhibitor to tackle the public health threat brought by NDM-1. / published_or_final_version / Physiology / Master / Master of Medical Sciences

A novel and effective mechanism of forming resistance to beta-lactam antibiotics by producing hybrid CTX-M-type beta-lactamases through genetic recombination was reported recently, albeit sporadically. Its primary introductory factor is the horizontal resistant gene transfer that occurs within the 876 bp 〖bla〗_(CTX-M) gene in Escherichia coli isolates, followed by the extensive application of beta-lactam antimicrobials. 〖bla〗_(CTX-M) originated from the nonpathogenic commensal Kluyvera spp., but it has become widely distributed in pathogenic organisms by plasmid-mediated gene transfer. Beta-lactams are effective against both gram-positive and gram-negative bacteria. However, misusing or overusing of beta-lactams has caused a globally corresponding emergence of antimicrobial resistant strains because of the selective pressure. This study included a total of 1,441 E. coli isolates, including 1,101 from six categories of animals and 340 from humans of both sexes. In total 612 strains were detected as extended spectrum beta-lactamase (ESBL)-positive. By using molecular tests on 248 ESBL-producers, 14 non-duplicate strains harbouring hybrid CTX-M-type beta-lactamases were found in a subset of 170 animal strains. A total of three alleles for hybrid CTX-M-type beta-lactamases (n = 14) were found, namely, CTX-M-64 (n = 6), CTX-M-123 (n = 6), and CTX-M-132 (n = 2). Six 〖bla〗_(CTX-M-64) and one 〖bla〗_(CTX-M-132) genes were located on approximately 70 kb IncI2 plasmids, and five 〖bla 〗_(CTX-M-123) genes were carried by 110 kb IncI1 (plasmid ST108) plasmids. All the 〖bla〗_(hybrid CTX-M) genes were placed between ISEcp1 followed by a 45 bp spacer and a 47 bp spacer followed by a 344 bp orf477delta gene. Among the six CTX-M-64-harbouring isolates detected, three of them were ST1011, phylogroup E, from chickens; the remaining three were ST224, phylogroup B1; ST93 CC168, phylogroup A; and ST117, phylogroup F. Every CTX-M-64-harbouring isolate had the co-resistance trait against both ciprofloxacin and tetracycline. The CTX-M-123-harbouring isolates were ST1485, phylogroup E; ST2732, phylogroup D; ST10, phylogroup A; ST457, phylogroup F; and ST 1196, phylogroup B1. All six CTX-M-123-harbouring strains were co-resistant to trimethoprim-sulphamethoxazole and tetracycline, and all of their transconjugants or transformants inherited the ability to resist fosfomycin. Both CTX-M-132-harbouring strains came from phylogroup A. One strain was ST3103, and the other one with a non-transferable plasmid was ST746. This study unravels the molecular epidemiology of hybrid CTX-M-type beta-lactamase-harbouring E. coli isolates. It also compared the prevalence of these isolates among both animal and human strains. Although hybrid CTX-Mtype beta-lactamases were not found in 340 clinical urinary isolates, 14 hybrid CTX-M-type beta-lactamases were identified in 14 animals in just one year. Hence, this study sets off alarm bells for the public to become more concerned about the antimicrobial resistance situation among animals. Furthermore, the discovery of hybrid CTX-M-type beta-lactamase-harbouring isolates also indicates a novel mechanism of antimicrobial resistance, i.e., homologous recombination, and shows a new way to study antimicrobial resistance. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Thesis (Ph. D.)--University of Wisconsin--Madison, 1982. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Proefschrift--Utrecht. / "Stellingen": [4] p. inserted. Includes bibliographies.

Halle, Wittenberg, Universiẗat, Diss., 2004.

Köln, University, Diss., 2004.

This thesis is devoted to some aspects of the theory of the weak continuous gamma radiation (often called 'Internal Bremsstrahlung') which accompanies beta processes, i.e. negative and positive electron emission and orbital electron capture. Whenever a beta process is accompanied by this gamma radiation, it will be called a "radiative" beta process, otherwise "radiationless". The results presented in this thesis go beyond those obtained by other authors in two respects. In the first place, the radiative beta emission probability is calculated for an allowed transition taking into account an arbitrary mixture of all the five beta interactions. Previously, only the theory for the case of pure interactions has been carried out. In the calculations, as in previous ones, Coulomb effects have been neglected. In the second place, the radiative K capture probability is calculated for an all owed transition taking into account again an arbitrary mixture of the five beta interactions, and, in addition, Coulomb effects. Previously, only the case of pure interactions with the neglection of Coulomb effects has been considered. In order to take Coulomb effects into account, a "semi-relativistic" approximation for the solutions to the Dirac equation with a Coulomb potential has been developed. It turns out that taking Coulomb effects into account reduces the probability of radiative K capture by an order of magnitude. / Science, Faculty of / Physics and Astronomy, Department of / Graduate