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CHARACTERIZATION of IMIS, the METALLO-Β-LACTAMASE from AEROMONAS VERONII Bv

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CHARACTERIZATION of IMIS, the METALLO-Β-LACTAMASE from AEROMONAS VERONII Bv MIAMI UNIVERSITY – THE GRADUATE SCHOOL CERTIFICATE FOR APPROVING THE DISSERTATION We hereby approve the Dissertation of Patrick A. Crawford Candidate for Degree: Doctor of Philosophy _______________________________________________ Dr. Michael W. Crowder, Director _______________________________________________ Dr. Gilbert Gordon, Reader _______________________________________________ Dr. Gary Lorigan, Reader _______________________________________________ Dr. Christopher A. Makaroff, Reader _______________________________________________ Dr. Kenneth Wilson, Graduate School Representative ABSTRACT CHARACTERIZATION OF IMIS, THE METALLO-β-LACTAMASE FROM AEROMONAS VERONII bv. SOBRIA by Patrick Anthony Crawford Zinc-containing metallo-β-lactamases are an emerging class of enzymes that render bacteria resistant to β-lactam-containing antibiotics. In an effort to better understand the structure and function of the metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria, spectroscopic and mechanistic studies were performed. ImiS was over-expressed in E. coli and purified as a 25.2 kDa monomer, containing 0.48 equivalents of Zn(II). The purified enzyme -1 exhibited substrate selectivity toward carbapenems, hydrolyzing imipenem with a kcat of 233 s and KM of 154 µM. The presence of a second equivalent of Zn(II) resulted in the loss of enzymatic activity. For spectroscopic characterization the native and spectroscopically silent Zn(II) was replaced with Co(II). UV/Vis, NMR, and EPR spectroscopies were all gathered on the Co(II)-substituted ImiS samples and EXAFS data were collected on the Zn(II)-ImiS. The Co(II) in Co(II)-ImiS is 4-coordinate, with 1 cysteine, 1 histidine, and presumably 1 aspartic acid and 1 water serving as metal ligands. Proton inventory studies were inconclusive, not clearly indicating one or more than one proton being transferred during the rate-liming step. pH Dependence studies revealed the presence of a single pKa of 5.6, which was assigned to a Zn(II)- bound water. Rapid scanning and stopped-flow experiments revealed a possible reaction mechanism consistent with that seen for β-lactamase II. Taken together, this dissertation offers, for the first time, models for the metal binding site and for the reaction mechanism of ImiS. These data, along with previous results on the other metallo-β-lactamases, can be integrated and used to guide further rational inhibitor design efforts. CHARACTERIZARTION OF ImiS, THE METALLO-β-LACTAMASE FROM Aeromonas veronii bv. sobria A DISSERTATION Submitted to the Faculty of Miami University in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Chemistry and Biochemistry by Patrick A. Crawford Miami University Oxford, Ohio 2003 Dissertation Director: Dr. Michael Crowder TABLE OF CONTENTS Chapter 1 Introduction 1.1 Antibiotics 2 1.2 β-lactam Containing Antibiotics 2 1.3 Antibiotic Resistance 8 1.4 Mechanism of Resistance 9 1.5 β-Lactamases 10 1.5.1 Serine-β-Lactamases 10 1.5.2 Metallo-β-Lactamases 12 1.5.2.1 Zinc Metallo-Hydrolase Family of Proteins 16 1.6 Aeromonads 18 1.7 Antibiotic Resistance in Aeromonas veronii bv. sobria 20 1.8 Introduction to Dissertation 23 1.8.1 Rational Drug Design 23 1.8.2 Sections of the Dissertation 24 1.9 References 25 Chapter 2 Over-expression, Purification, and Characterization of Recombinant ImiS 2.1 Introduction to Chapter 2 30 ii 2.2 Materials and Methods 32 2.2.1 Materials 32 2.2.2 Methods 34 2.2.2.1 Plasmid Construction 34 2.2.2.2 Over-Expression and Purification of ImiS 35 2.2.2.3 Determination of a Molar Extinction Coefficient 37 for ImiS 2.2.2.4 Metal Analyses 38 2.2.2.5 Steady-State Kinetics 38 2.2.2.6 Gel-Filtration Chromatography 39 2.2.2.7 MALDI-TOF Spectrometry 40 2.2.2.8 CD Spectroscopy 40 2.2.2.9 N-terminal Amino Acid Sequencing 40 2.3 Results and Discussion 40 2.3.1 Over-Expression and Purification of Recombinant ImiS 40 2.3.2 Determination of Molecular Extinction Coefficient for 42 Recombinant ImiS 2.3.3 Physical Properties of Recombinant ImiS 44 2.3.4 Metal Analyses 45 2.3.5 CD Spectroscopy 50 2.3.6 Steady-State Kinetics 50 2.3.7 Comparison to ImiS Isolated Directly from Aeromonas 53 2.4 Conclusions 55 iii 2.5 References 59 Chapter 3 Spectroscopic Characterization of Recombinant ImiS 3.1 Introduction 62 3.1.1 Zn(II) Containing Metalloproteins 62 3.1.2 Structural Characterization of Metallo-β-Lactamases 64 3.1.3 Structural Characterization of Bush Group 3b 66 β-Lactamases 3.1.4 Co(II)-Substitution 68 3.1.4.1 Spectroscopically Silent Zn(II) 68 3.1.4.2 Co(II)-Substitution 69 3.1.5 Summary of Chapter 3 70 3.2 Materials and Methods 71 3.2.1 Materials 71 3.2.2 Methods 72 3.2.2.1 Preparation of Apo-ImiS 72 3.2.2.2 Preparation of Co(II)-substituted ImiS 73 3.2.2.3 Spectroscopic Characterization of Co(II)-ImiS 73 3.2.2.3.1 Electronic Spectroscopy 73 3.2.2.3.2 1H-NMR Spectroscopy 74 3.2.2.3.3 EPR Spectroscopy 75 3.2.2.3.4 EXAFS Spectroscopy 75 3.3 Results and Discussion 78 iv 3.3.1 Co(II)-Substitution of ImiS 78 3.3.1.1 Generating Apo-ImiS 78 3.3.1.2 Addition of Co(II) to Apo-ImiS 79 3.3.2 Electronic Spectra 82 3.3.3 1H-NMR Spectra 86 3.3.4 EPR Spectra 88 3.3.5 EXAFS Spectra 92 3.4 Conclusions 100 3.5 References 104 Chapter 4 Mechanistic Characterization of Recombinant ImiS 4.1 Introduction 109 4.2 Materials and Methods 110 4.2.1 Materials 110 4.2.2 Methods 110 4.2.2.1 Steady-State Kinetic Studies 110 4.2.2.2 Solvent Isotope Effect Studies 111 4.2.2.3 pH Dependence Studies 111 4.2.2.4 Presteady-State Kinetic Studies 112 4.2.2.5 Rapid-Scanning of Co(II)-ImiS 112 4.3 Results and Discussion 113 4.3.1 Steady-State Kinetics 113 v 4.3.2 Solvent Isotope Effects 114 4.3.3 pH Dependence 118 4.3.4 Presteady-State Kinetics 122 4.3.5 Rapid-scanning Studies of Co(II)-Substituted ImiS 132 4.4 Conclusions: ImiS’ Mechanism of β-Lactam Hydrolysis 135 4.5 References 139 Chapter 5 Conclusions: ImiS in Context 5.1 Antibiotic Resistance in Context 141 5.2 ImiS Conclusions 141 5.3 ImiS in Context 144 5.3.1 Inhibitor Design 144 5.3.2 Regulation of β-Lactamases 145 5.3.3 Metal Requirements 145 5.4 References 149 vi LIST OF FIGURES 1-1: There are multiple sites for antibiotic attack in a bacterial cell. 3 1-2: Core structures of common β-lactam containing antibiotics. 5 1-3: Crosslinking of building blocks of the peptidoglycan layer 6 1-4: Cell wall biosynthesis showing the mode of inhibition for β-lactam 7 antibiotics. 1-5: Hydrolysis of Imipenem. 11 1-6: Amino acid comparison of conserved segments of the zinc metallo- 17 hydrolase family of enymes. 2-1: Sequence comparision of Bce-569H: β-lactamase II from B. cereus [2], 31 Bfr-CfiA: CfiA from B. fragilis [3], Ahy: CphA from A. hydrophila [4], Asb-ImiS: ImiS from A. sobria [5], Stm-L1: L1 from S. maltophilia [6], and Cms-BlaB: BlaB from Chryseobacterium meningosepticum [7, 8]. 2-2: Construction of pET26bimiS plasmid used to produce active ImiS. 36 2-3: SDS-PAGE gel representing protein over-expression and purification. 43 2-4: MALDI-TOF spectrum of recombinant ImiS. 47 2-5: MALDI-TOF spectrum of native ImiS from A. sobria. 48 2-6: CD spectrum of recombinant ImiS and native ImiS from A. sobria. 49 2-7: Michaelis-Menton plot for the hydrolysis of imipenem by ImiS. 51 2-8: Relative kinetic activity of ImiS with increasing equivalents of zinc. 54 3-1: Structural picture of the active site of CcrA showing the two Zn(II) ions 67 (maroon spheres), the two solvent water/hydroxide molecules (blue spheres), and the metal binding amino acids. 3-2: CD spectra of as-isolated Zn(II)-ImiS (solid line), apo-ImiS 80 (large dashed line), and Co(II)-ImiS (small dashed line). 3-3: Electronic difference spectra of Co(II)0.5-, Co(II)1-, and Co(II)2-ImiS. 84 vii 1 3-4: H-NMR of Co(II)1-ImiS (A) and Co(II)2-ImiS (B) showing one and 89 three Co(II)-His resonances, respectively, indicated by an asterisk (*). 1 3-5: H-NMR spectrum of Co(II)1-ImiS showing the Co(II)-EDTA 90 resonance at 129 ppm and the Co(II)-His resonance at 63 ppm. 3-6: EPR spectrum of Co(II)2-ImiS (top curve) and a Co(II)1-ImiS 93 (bottom curve). 3-7: Temperature dependence at 10 mW of Co(II)2-ImiS. 94 3-8: Fourier transform of Zn(II)1-ImiS (A) and Zn (II)2-ImiS (B). 98 3-9: Proposed metal-binding site for ImiS. 103 4-1: Michaelis-Menton plot of the velocity of reaction versus substrate, 115 imipenem, concentration exhibiting substrate inhibition. 4-2: Proton inventory for ImiS at pH 7.0 using imipenem as a reporter 117 substrate. 4-3: The pH-dependence of kcat and kcat/KM for recombinant ImiS 121 hydrolyzing imipenem in MTEN buffer. 4-4: Stopped-flow kinetic experiments with 1.4 µM ImiS and various 123 concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to a double exponential equation. 4-5: Stopped-flow kinetic experiments with 1.4 µM ImiS and various 127 concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to the Michaelis-Menton mechanism with KINSIM varying k2.
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