RAMAN SPECTROSCOPIC STUDIES OF INHIBITOR REACTIONS IN CLASS A, B AND D β-LACTAMASES by TAO CHE Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Adviser: Dr. Paul R. Carey Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May, 2015 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Tao Che candidate for the Ph.D. degree*. (signed) Focco van den Akker (chair of the committee) Paul Carey Robert Bonomo Menachem Shoham Marion Skalweit (date) December, 2014 *We also certify that written approval has been obtained for any proprietary material contained therein. i TABLE OF CONTENTS LIST OF TABLES ........................................................................................................... V LIST OF SCHEMES ...................................................................................................... VI LIST OF ABBREVIATIONS ....................................................................................... XV ABSTRACT .................................................................................................................. XVI CHAPTER I: INTRODUCTION .................................................................................... 1 I-1 Bacterial resistance to β-lactam antibiotics .............................................................. 5 I-2 β-Lactamases ............................................................................................................... 9 I-3 Raman techniques for following enzyme-inhibitor reactions ............................... 19 CHAPTER II: RAMAN SPECTRA OF INTERCHANGING Β-LACTAMASE INHIBITOR INTERMEDIATES ON THE MILLISECOND TIME SCALE ........ 26 II.1 INTRODUCTION ................................................................................................... 27 II.2 MATERIALS AND METHODS ............................................................................ 29 II.3 RESULTS AND DISCUSSION .............................................................................. 31 II.3.1 Tazobactam reactions with SHV-1 in single crystals ........................................ 31 II.3.2 Freeze-drying does not affect protein secondary struture and catalytic property. ....................................................................................................................................... 33 II.3.3 Tazobactam reactions with SHV-1 in solution forms stable trans-enamine. .. 35 II.4 CONCLUSIONS ...................................................................................................... 39 CHAPTER III: DETECTING A QUASI-STABLE IMINE SPECIES ON THE REACTION PATHWAY OF SHV-1 Β-LACTAMASE AND 6Β- (HYDROXYMETHYL)PENICILLANIC ACID SULFONE .................................... 41 III.1 INTRODUCTION .................................................................................................. 42 III.2 MATERIALS AND METHODS........................................................................... 44 III.3 RESULTS ................................................................................................................ 51 ii III.3.1 Kinetic parameters ............................................................................................ 51 III.3.2 Susceptibility testing ......................................................................................... 52 III.3.3 Base hydrolysis ................................................................................................. 53 III.3.4 Raman studies of reactions with SHV-1 in solution ....................................... 58 III.3.5 X-ray and Raman analysis of single crystals ................................................... 62 III.3.6 Weak antibiotic activity..................................................................................... 66 III.4 DISCUSSION ......................................................................................................... 68 III.5 CONCLUSIONS..................................................................................................... 71 CHAPTER IV: RAMAN STUDIES OF A CLINICALLY IMPORTANT CLASS B Β-LACTAMASE, NDM-1, REACTING WITH A MONOBACTAM, AZTREONAM. ............................................................................................................... 72 IV.1 INTRODUCTION .................................................................................................. 72 IV.2 MATERIALS AND METHODS ........................................................................... 74 IV.3 RESULTS AND DISCUSSION ............................................................................. 76 IV.3.1 Base hydrolysis .................................................................................................. 77 IV.3.3 Aztreonam reactions with PBP5 in solution .................................................... 80 CHAPTER V: CARBOXYLATION AND DECARBOXYLATION OF ACTIVE SITE LYS 84 CONTROLS THE ACTIVITY OF OXA-24 Β-LACTAMASE OF ACINETOBACTER BAUMANNII: RAMAN CRYSTALLOGRAPHIC AND SOLUTION EVIDENCE .............................................................................................. 90 V.1 INTRODUCTION .................................................................................................... 91 V.2 MATERIALS AND METHODS ............................................................................ 96 V.3 RESULTS AND DISCUSSION .............................................................................. 98 V.3.1 The presence of PEG can decelerate the OXA-24 β-lactamase reaction with SA-1-204 inhibitor ........................................................................................................ 98 V.3.2 The reaction of SA-1-204 in OXA-24 crystals undergoes only one cycle ...... 103 V.3.3 Recarboxylation of the lysine residue 84 in the active site restores OXA-24's hydrolytic activity towards nitrocefin in solution ...................................................... 112 V.4 CONCLUSIONS .................................................................................................... 119 iii CHAPTER VI : THE DIFFERENT INHIBITION MECHANISMS OF OXA-1 AND OXA-24 Β-LACTAMASES ARE DETERMINED BY THE STABILITY OF ACTIVE-SITE CARBOXYLATED LYSINE ........................................................... 120 VI.1 INTRODUCTION ................................................................................................ 121 VI.2 MATERIALS AND METHODS ......................................................................... 123 VI.3 RESULTS .............................................................................................................. 129 VI.3.1 Kinetic data for penem 1 and 3 reacting with OXA-1 and OXA-24 ............. 129 VI.3.2 Spectroscopic evidence for different reaction schemes in OXA-1 and OXA-24 ..................................................................................................................................... 132 VI.3 DISCUSSION ........................................................................................................ 150 VI.4 CONCLUSIONS ................................................................................................... 154 APPENDIX .................................................................................................................... 156 Chapter III: Detecting a Quasi Stable Imine Species on the Reaction Pathway of SHV-1 β-Lactamase and 6β-(Hydroxymethyl)penicillanic Acid Sulfone ................ 156 Chapter V: Carboxylation and Decarboxylation of Active Site Lys 84 Controls the Activity of OXA-24 β-Lactamase of Acinetobacter baumannii: Raman Crystallographic and Solution Evidence .................................................................... 159 BIBLIOGRAPHY ......................................................................................................... 164 iv List of Tables Table I. 1 Classifications of class A β-lactamases. ........................................................... 12 Table II. 1 Raman peak assignments for the major peaks in tazobactam/SHV-1 difference spectra shown in Figure II.3.............................................................................................. 38 Table III. 1 X-ray data collection and refinement statistics. ............................................. 50 Table III. 2 Kinetic parameters of SHV-1 β-lactamase with various inhibitors. .............. 52 Table III. 3 Disc diffusion assays. .................................................................................... 53 Table III. 4 Peak assignment derived from Gaussian calculations. .................................. 56 Table V. 1 Peak assignment for unreacted SA-1-204 from Gaussian calculations. ....... 105 Table V. 2 Peak assignment for proposed early intermediate X from Gaussian calculations. .................................................................................................................... 107 Table V. 3 Peak assignment for proposed late intermediate Z from Gaussian calculations. ......................................................................................................................................... 111 Table VI. 1 Primer sequences. ........................................................................................ 125 Table VI. 2 Minimum inhibitory concentration of Laboratory Isolates. ........................ 131 Table VI. 3 Kinetic parameters of inhibition. ................................................................. 132 Table