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Abstract Bla2 from Bacillus Anthracis ABSTRACT SPECTROCOPIC AND MECHANISTIC STUDIES ON METALLO-β-LACTAMASE BLA2 FROM BACILLUS ANTHRACIS by Megan Hawk In an effort to probe the structure, mechanism, and biochemical properties of metallo-- lactamase (EC 3.5.2.6) Bla2 from Bacillus anthracis, the enzyme was over-expressed, purified, and characterized. Metal analyses demonstrated that recombinant Bla2 tightly binds 1 equivalent of Zn(II). Steady-state kinetic studies showed that mononuclear Zn(II)-containing Bla2 (1Zn-Bla2) had the highest activity, while the dinuclear Zn(II)- containing Bla2 (ZnZn-Bla2) was unstable. However, dinuclear Co(II)-containing Bla2 (CoCo-Bla2) is more active than the mononuclear Co(II)-containing analog. UV-Vis, 1H NMR, EPR, and EXAFS spectroscopic studies were used to structurally characterize Bla2, and the resulting data show that Co(II) binding to Bla2 is cooperative, while Zn(II) binding is sequential. These spectroscopic studies were integral in determining which analog of Bla2 was used in our pre-steady state kinetic studies. 1Zn-Bla2 utilizes a two- step kinetic mechanism when nitrocefin is the substrate, while the enzyme uses a one-step kinetic mechanism when cefaclor or imipenem is used as the substrate. SPECTROCOPIC AND MECHANISTIC STUDIES ON METALLO-β-LACTAMASE BLA2 FROM BACILLUS ANTHRACIS A Thesis Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Masters of Science Department of Chemistry and Biochemistry by Megan June Hawk Miami University Oxford, Ohio 2008 Advisor __________________________ Dr. Michael Crowder Reader ___________________________ Dr. Ann Hagerman Reader ___________________________ Dr. Michael Kennedy Reader __________________________ Dr. Richard Taylor Table of Contents Chapter 1 Introduction 1.1 Introduction 1 1.2 Antibiotic Development 1 1.3 Prokaryotic Bacteria 3 1.4 β-Lactam-containing antibiotics 3 1.5 Antibiotic resistance 5 1.6 Classification of β-lactamases 11 1.7 Metallo-β-lactamases 12 1.7.1 Classification of metallo-β-lactamases 12 1.7.2 Structure of metallo-β-lactamases 12 1.7.3 Reaction mechanism of MβL 14 1.8 Bacillus anthracis and Bla2 16 1.9 Hypothesis and description of thesis 19 1.10 References 21 ii Chapter 2 Spectroscopic and mechanistic studies on metallo--lactamase Bla2 from Bacillus anthracis 2.1 Introduction 26 2.2 Experimental Procedures 29 2.2.1 Materials 29 2.2.2 Over-expression, purification, and biochemical 29 characterization of Bla2 2.2.3 Metal analyses 30 2.2.4 Steady-state kinetic studies 30 2.2.5 Preparation of apo-Bla2 31 2.2.6 UV-Vis spectrophotometry 31 2.2.7 1H NMR spectroscopy 31 2.2.8 EPR spectroscopy 32 2.2.9 EXAFS spectroscopy 32 2.2.10 Stopped-flow UV-Vis studies 33 2.3 Results 35 2.3.1 Over-expression, purification, and biochemical 35 characterization of Bla2 2.3.2 Steady-state kinetic studies on Bla2 35 2.3.3 UV-Vis spectroscopy 36 2.3.4 1H NMR spectroscopy 40 2.3.5 EPR spectroscopy 43 2.3.6 EXAFS spectroscopy 43 2.3.7 Stopped-flow UV-Vis kinetic studies 45 2.4 Discussion 49 2.5 References 61 iii Chapter 3 Conclusions 3.1 Conclusion 65 3.2 References 70 iv List of Tables 1-1: The historic development of classes of antibiotics 4 1-2: Characteristics of different metallo-β-lactamase subgroups 13 2-1: Best fits to Co(II) and Zn(II) Bla2 EXAFS. α 34 2-2: Steady-state kinetic parametersa for nitrocefin, imipenem, cefaclor, and 38 meropenem hydrolysis by Bla2 containing 1 equivalent of Zn(II) 2-3: Steady-state kinetic parameters for Bla2 containing 1 or 2 equivalents 39 of Zn(II) or Co(II) 2-4: Kinetic constants used in KINSIM simulations 50 v List of Figures 1-1: Structures of common β-lactam antibiotics 6 1-2: Structure of D-alanyl-D-alanine 7 1-3: Cross-linking of the peptidoglycan cell 8 1-4: Hydrolysis of nitrocefin 10 1-5: Crystal structures from each of the representative metallo-β-lactamase 15 subgroups 1-6: Proposed mechanisms for MβLs 17 1-7: The crystal structure of BcII 18 2-1: SDS-PAGE gel of purification of recombinant Bla2 37 2-2: UV-Vis difference spectrum of apo-Bla2 titrated with increasing 41 amounts of Co(II) 1 2-3: H NMR spectra of 2Co(II)-Bla2 in 10% D2O and 90% D2O 42 2-4: EPR spectra from Co(II)-containing Bla2 44 2-5: Fourier transformed EXAFS spectra of Co(II)-substituted Bla2 46 2-6: Fourier transformed EXAFS spectra of Zn(II)-substituted Bla2 47 2-7: Progress curves of the reaction of nitrocefin and Bla2 containing 51 1 eq. Zn(II) at 4 oC 2-8: Progress curves of the reaction of imipenem and Bla2 containing 52 1 eq. Zn(II) at 4 oC 2-9: Progress curves of the reaction of cefaclor and Bla2 containing 53 1 eq. Zn(II) at 4 oC 2-10: The proposed active site of Bla2 after the addition of 1 or 2 59 equivalents of Zn(II) or Co(II) to apo-Bla2. 3-1: A penicillin derivative inhibitor for metallo--lactamases with a 69 phosphinate group at the -lactam carbonyl position. vi List of Schemes 2-1: Proposed mechanism for nitrocefin 48 2-2: Proposed mechanism for imipenem and cefaclor 49 vii Acknowledgements I would like to thank Dr. Michael Crowder for allowing me to be a part of his group. My experience at Miami University has helped me identify my weaknesses and strengths. I learned that it is important to understand why an experiment is performed and to look at past journal articles to guide you in explaining your current work. I would like to thank my group members who helped me learn proper lab and instrumentation techniques. I would also like to thank Christine Hajdin and Katie Bender who were great assets during the characterization of Bla2. My experience at Miami University gave me an opportunity to work for the Center for Chemical Education (CCE). My mentors Mickey Sarquis, Lynn Hogue, Dr. Susan Hershberger, and Ed Smith have helped me grow and become comfortable with public speaking. The work at the Center has helped me understand the importance in working as a team to achieve greatness. I hope the Center’s contributions to education will inspire the youth in Ohio and increase the number of students who focus in science. Finally, I thank my parents who have allowed me to choose my own path in life. I appreciate their love and support throughout the years. viii Chapter 1 Introduction 1.1 Introduction The 20th century marked an age of discovery through luck and human ingenuity. While suffering from a sinus infection in 1922, Alexander Fleming, a bacteriologist, cultured secretions from his nose. When Fleming examined his culture plate, he allowed a tear to fall on the petri dish. The next day, Fleming observed a cleared space where the tear had landed. Fleming concluded that the tear was toxic to bacteria and produced a type of antibiotic. The tear contained an enzyme called lysozyme, which breaks down bacterial cell walls and kills certain types of bacteria. The “body’s own antibiotic,” lysozyme, was found to be of little clinical importance since this enzyme could not kill potent types of bacteria. In 1928, Fleming, returned from a vacation to find a unique type of fungus growing on his culture plate. The fungus had a ring around it where bacteria did not grow. Since the fungus on the contaminated plate was from the Penicillium family, Fleming named the substance produced from the mold penicillin and found that penicillin was toxic to many strains of bacteria. Fleming repeatedly tried to isolate penicillin; however, he was not successful and concluded that penicillin could not be used as a clinical therapeutic. A few years later, Howard Florey and Ernst Chain developed a procedure to isolate and concentrate penicillin, and penicillin was subsequently shown to have medicinal purposes, particularly in fighting bacterial infections in wounded World War II soldiers. Based on this work, Fleming, Florey, and Chain were awarded the 1945 Nobel Prize in Physiology or Medicine. With the use of penicillin, the age of modern antibiotics commenced, and the penicillin family of antibiotics, which includes cephalosporins and carbapenems, is the largest class of effective and inexpensive antimicrobial agents (1) . 1.2 Antibiotic development After the discovery and clinical use of penicillin, many other antibiotics were marketed by pharmaceutical companies. For example, Eli Lilly & Co. developed and marketed antibiotics such as erythromycin, vancomycin, and cephalosporins. By the late 1 1960’s, there were numerous antibiotics that could be used in the clinic, and the U.S. Surgeon general, William H. Stewart, asserted that we should “close the book on infectious disease (2).” Over the past 40 years, only two new classes of antibiotics have emerged: one in 2000 called the oxazolidinones and the other in 2003 called the lipopeptides (Table 1-1) (3). Unfortunately from the 1980’s until the present, infectious diseases have become the 3rd leading cause of death in the world. In addition, the emergence of bacteria that are resistant to most or all known clinical antibiotics has exacerbated the problem (4). Large pharmaceutical companies like Eli Lilly & Co. lost interest in developing new antibiotics in the 1980’s and 1990’s due to low profit margins. The large pharmaceutical companies have not responded well to the reduction of useful antibiotics. The development of a new antibiotics takes an average of ten years and costs $800 million before the antibiotic enters the market (4). Once the drug is introduced into the clinic, the lifetime of the drug is very short.
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