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

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

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

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

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

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

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.

4-6: Stopped-flow kinetic experiments with 1.4 µM ImiS and various 129 concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to the Michaelis-Menton mechanism with KINSIM varying k3.

4-7: Stopped-flow kinetic experiments with 1.4 µM ImiS and various 131 concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to the L1/CcrA, buildup of intermediate mechanism with KINSIM.

viii

4-8: Stopped-flow kinetic experiments with 1.4 µM ImiS and various 133 concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to the β-lactamase II branched mechanism with KINSIM.

4-9: Rapid-scanning of Co(II)-substituted ImiS with imipenem as substrate. 134

4-10: Proposed mechanism for the hydrolysis of imipenem by ImiS. 138

LIST OF TABLES

1-1: Classification schemes for β-lactamases. 13

1-2: Bush group 3 metallo-β-lactamase sub-grouping scheme. 15

2-1: Metal content for ImiS in moles of metal ions per mole of ImiS. 46

2-2: Kinetic constants for ImiS revealing carbapenemase activity. 58

3-1: EXAFS data for Zn(II)-ImiS. 99

4-1: Data of stopped-flow traces fitted to a double exponential equation 125 with the Sigma Plot data analysis program.

Acknowledgements

I would like to sincerely thank my advisor, Dr. Michael Crowder. His tireless efforts have been greatly appreciated. His guidance, support, and friendship were invaluable to the completion of this effort. It has been a privilege and an honor to work for and study under such a dedicated person.

I would also like to thank my committee, Drs. Makaroff, Gordon, Lorigan, and Wilson for their support of my studies at Miami University. Additionally I would like to thank Daniel Sobieski, Adam Jablonski, Kelly Aston, Kimberly Markert, and Priya Gursahaney for their efforts in this endeavor.

Finally, I would like to dedicate this dissertation to my loving family who have given me the courage, strength, and support necessary to succeed.

Chapter 1

Introduction

Even before the 1800’s and Pasteur’s discovery of a connection between bacteria and

disease, a few people had worked diligently in an attempt to make life better by minimizing the

effects of microorganisms. However, this work was almost universally ignored. In the late

1800’s, Joseph Lister became the first surgeon to insist on using phenol to sterilize his surgical

instruments, significantly reducing the number of deaths resulting from infections acquired

during surgery [1]. Even as early 1896, a French medical student, Ernest Duchesne, showed that

a substance produced by mold was able to inhibit the growth of bacteria [1]. Few physicians

held high opinion of this work, and it was soon forgotten. It was not until 1928 and Alexander

Fleming’s accidental discovery of penicillin, due to its antibacterial activities, that a change in

attitude toward the treatment of disease occurred [2]. The World Wars and the proliferation of

death from sepsis, and not from the wound itself, no doubt aided this change in attitude. The only problem with Fleming’s discovery and isolation of penicillin was that his substance was very unstable and difficult to obtain pure in large quantities, not allowing for clinical use. It was not until 1940 with Florey, Chain, and Heatley that penicillin was utilized in its first human clinical trials, just in time for the Second World War [3]. For this work Sir Alexander Fleming,

Sir Howard Walter Florey, and Ernst Boris Chain were awarded the 1945 Nobel Prize in

Physiology or Medicine [http://www.nobel.se/medicine/laureates/1945/].

1.1 Antibiotics

With Fleming’s discovery of penicillin, the age of antibiotics began. Antibiotic means

“against life”, and effective antibiotics can inhibit microbial growth (bacteriostatic) or kill the microbe (bacteriocidal). The majority of antibiotics are natural products because they were isolated from bacteria or fungi [4]. The next largest class of antibiotics is semi-synthetic— natural products that have been slightly altered, by addition or subtraction of substituents for example, to improve efficacy or lower toxicity. Only about 10% of the antibiotics in clinical use today are completely synthetic, and most of these compounds were designed to inhibit a process that is unique and essential to the bacterium [4]. In the more than half century since penicillin entered the clinical realm, the pharmaceutical industry has developed more than 150 antibiotics, with over 50 additional analogues of the 3rd generation cephalosporins and quinolones since they were first introduced in the early 1980’s [4, 5]. These antibiotics target a variety of sites within a bacterial cell including the inhibition of cell wall biosynthesis, protein synthesis, and nucleic acid function (Figure 1-1) [5]. The overwhelming majority, greater than 50%, of all antibiotics in use contain a β-lactam ring (also known as β-lactams) [6].

1.2 β-Lactam Containing Antibiotics

β-lactams are the family of antibiotics whose structures are based on penicillin (Figure 1-

2). This large family has a single common structural characteristic, the four-member β-lactam ring. Members of this family include penicillins, cephalosporins, carbapenems, and

“nonclassical” β-lactams such as monobactams and the inhibitor clavulanic acid, (Figure 1-2)

[7]. Penicillins, cephalosporins, carbapenems, and other β- lactams exhibit bacteriocidal activity because they are able to disrupt bacterial cell wall synthesis [6-10].

Cell wall synthesis Vancomycin Cell membrane Monobactams Penicillins Cephalosporins Carbapenems Cell Wall

DNA synthesis Methotrexate DNA Processing DNA Ciproflozacin Nucleotides

Ribosomes

50 50 50 Protein syntheis (50S) 30 30 30 Erythromycin Chloramphenical

Periplasmic spaces Pre-protein synthesis β-lactamases Linezolid Amylglycoside- modifying enzymes Protein synthesis (30S) Tetracycline streptomycin

Figure 1-1: There are multiple sites for antibiotic attack in a bacterial cell.

The normal bacterial peptidoglycan cell wall is a large, cross-linked polymer net, upon

which the structural integrity of the cell depends [11]. The polymer consists of monomeric units

of disaccharides, N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc), bound

to a pentapeptide stem. The pentapeptide chain is formed from L-alanine, D-glutamate, L-lysine, and the dipeptide D-alanyl-D-alanine. The disaccharides are linked directly, in an action

performed by an enzyme called transglycosidase . Neighboring pentapeptides are then

crosslinked by transpeptidase providing the cell wall’s stability; a pentaglycine unit is inserted

between the L-lysine on one pentapeptide and the penultimate D-alanyl on an adjacent chain

(Figure 1-3). In this process, a lysine-pentaglycine unit acts as a nucleophile attacking the

carbonyl of the terminal D-alanyl residue, resulting in the cleavage of the terminal D-alanine

(Figure 1-4).

Different β-lactams have different targets within the cell, but the primary bacteriocidal

act of a β-lactam is the interference of normal cell wall biosynthesis [5, 7, 12]. The β-lactam is a

structural analogue of the D-alanyl-D-alanine terminal dipeptide of a normal bacterial cell wall

(Figure 1-4). Without a properly cross-linked cell wall, the bacteria can not withstand the high

osmotic pressure within the cell, and the cell lyses.

Carbapenems, such as the clinically relevant imipenem and meropenem, are a fairly new

generation of β-lactam antibiotics. While they were developed in the 1970’s, only during the last

decade have they become an option in the clinical setting, especially in Asia [6, 13, 14]. Unlike

penicillins and cephalosporins, β-lactams that are often prescribed for even minor infections,

carbapenems are used to treat serious infections, when the infection does not respond to other

antibiotics. While they are not considered the treatment of last resort, such as vancomycin,

resistance to carbapenems seriously limits clinical options.

ROCH S S ROCH

N N O CH R' - O 2 CO2 - CO2 penicillins cephalosporins

'R R N O - CO2

carbapenems

Figure 1-2: Core structures of common β-lactam containing antibiotics.

HO HO R O O O O O O HO R OH

O O O O

O L A l a R D G l u

O L L y s

D A l a L Ala D A l a X D Glu L Lys X = Gly5

D Ala

Figure 1-3: Crosslinking of building blocks of peptidoglycan layer.

Chain 1 O R C HN S HN CH CH3 C CN ONCH CH3 - H O COO C O O- Penicillin D-Ala D-Alanine

CH3 Transpeptidase + H N CH 3 O COO- R C HN S D-Ala Chain 1 CHN - O COO Chain 2 Transpeptidase HN CH CH3 O C L-Lys (Gly) O 4 C Transpeptidase Penicillin-Enzyme CH2 NH2 Complex

Transpeptidase

Chain 2 Chain 1 O

L-Lys (Gly)4 C HN CH CH3

CH2 NH C O

Successful Crosslinking

Figure 1-4: Cell wall biosynthesis, showing the mode of inhibition for β-lactam antibiotics.

1.3 Antibiotic Resistance

With the emergence of penicillin, later cephalosporins, and other new semi-synthetic β-

lactams, Ehrlich’s dream of a “magic bullet” appeared possible, if not realized. However, the

broad use of β-lactams since their introduction into the clinical setting in the 1940’s has provided

a selective pressure for resistant phenotypes. Soon after allied soldiers were first administered

penicillin during World War II, evidence of bacterial resistance emerged. In 1941 virtually all

Staphalococcus aureus infections were susceptible to penicillin G; by 1944, over half of the

Staph. strains were resistant to penicillin [5]. Currently, greater than 95 percent of S. aereus

organisms are resistant to penicillin as well as to most cephalosporins [5]. There is clinical

resistance to all known antibiotics, regardless of whether the antibiotic is a natural product, semi-

synthetic, or completely synthetic. For example in 2000, the FDA approved linezolid

(ZyvoxTM), which is a new, totally-synthetic antibiotic from Pharmacia Upjohn. Linezolid is an oxazolidinone, a class of antibiotics that prevents initiation of protein synthesis in the bacterium, which is an antibacterial target that had never before been utilized. By June of 2001, less than one year after reaching the clinical setting, patients were suffering from infections that did not respond to Zyvox . The misuse and overuse of antibiotics has provided a Darwinian bacterial selection where the susceptible (weak) organisms are killed off and the resistant (fittest) ones

survive and flourish [15].

The dwindling effect penicillin and other antibiotics have toward once simple bacterial

infections can be linked to one general cause: misuses of antibiotics [5, 16-19]. Misuse of

antibiotics include the prescription of antibiotics for non bacterial infections, over-prescription of

specific antibiotics, patient abuse such as not taking the full course of medication, self

prescription, and the false impression that antibacterial agents in household items such as soap, cleansers, toys, and clothing will improve health [5, 16, 20, 21].

Antibiotic resistance is not linked to human use alone; between 40 and 50 percent of all antibiotics produced in the U.S. are used to treat sick animals and encourage growth in livestock and poultry [5, 17]. Recently, Ashley Mulroy, a Wheeling, West Virginia high school student, probed for penicillin, tetracycline, and vancomycin in the town’s drinking water and in the Ohio

River [22]. Disturbingly, she found low concentrations, parts per trillion, of all three antibiotics in both water sources. The highest antibiotic concentrations were found in the water samples taken from sites near livestock and dairy farms.

1.4 Mechanisms of Resistance

There are three major mechanisms that contribute to the inactivation of antibiotics and the emergence of antibiotic resistance: (1) prevention of access to the target, (2) alteration of the target site, and (3) destruction or modification of the antibiotic [5]. Alteration of antibiotic efflux and permeability has rendered β-lactams, aminoglycosides, and tetracyclines ineffective against many bacteria, including Pseudomonas aeruginosa where the loss of a 54 kDa porin (OprD) renders it carbapenem resistant [5, 13]. The bacterial synthesis of modified D-D peptidases drastically reduces the effectiveness of β-lactams, and single amino acid changes in an enzyme can alter a bacteria’s sensitivity to β-lactams, macrolides, and folate synthesis antagonists [5].

Finally, destruction or modification of the antibiotic can occur through many pathways including aminoglycoside-inactivating enzymes and through the action of β-lactamases [5]. The production of one or multiple β-lactamases is the most common cause of antibiotic resistance in bacteria [5, 9].

1.5 β-Lactamases

β-lactamases are enzymes that hydrolyze the carbon-nitrogen bond in the β-lactam ring of the β-

lactams (Figure 1-5). These enzymes are especially significant since β-lactams account for over

50% of the world’s antibiotic arsenal [6]. Presently, over 340 individual β-lactamases have been

isolated and identified [23]. With new β-lactams being produced by pharmaceutical companies

and entering the clinical realm, more diverse and virulent β-lactamases can be found in an ever-

increasing number of pathogenic bacteria [5, 16, 17]. This process is known as the “β-lactamase cycle” [6, 23, 24]. New β-lactams lead to new β-lactamases and the spreading of resistance.

Resistance is further spread through horizontal gene transfer [25].

β-lactamases are extracellular, membrane-associated enzymes in Gram-positive bacteria and periplasmic proteins in Gram-negative bacteria. The cellular localization of β-lactamases enables the enzymes to interact with and hydrolyze the β-lactam containing antibiotics before the antibiotics can come into contact with transpeptidase (Figure 1-4).

1.5.1 Serine-β-lactamases

The majority of β-lactamases produced by bacteria contain a mechanistically significant serine at the active site [8, 24]. These serine-β-lactamases generally consist of two distinct domains: an all α-helical region and an α/β domain. The active site is situated in the cleft between these two

domains, and a serine residue is activated to be a nucleophile and positioned to attack the

carbonyl on β-lactams. There is strong evidence that serine-β-lactamases’ mechanism of

hydrolysis involves the formation of a relatively unstable acyl-enzyme intermediate [8, 24].

While more than 90% of all clinically known β-lactamases are serine β-

H HO HH H C S 3 N NH N H O COO-

-1 -1 ε300 = - 9000 M cm H HO HH H C S 3 N NH HN H O O- COO-

Figure 1-5: Hydrolysis of Imipenem.

lactamases, most bacterial strains harboring one of these β-lactamases can be circumvented by current β-lactams [26]. Serine-β-lactamases do not hydrolyze all known, clinically used antibiotics, such as carbapenems. The β-lactamase inhibitors, when used in combination with already available antibiotics, prove to be a common and powerful weapon against antibiotic resistant bacteria that produce a serine-β-lactamase.

1.5.2 Metallo-β-lactamases

The discovery of β-lactamase II, a zinc-containing enzyme from Bacillus cereus, in 1966, revealed that there was a small class of β-lactamases that require metal ions at their active sites

[26, 27]. Metallo-β-lactamases are generally broad-spectrum β-lactamases, which are able to hydrolyze β-lactams from all chemical classes except monobactams [26]. However, metallo-β- lactamases were not considered a significant clinical threat until they were discovered in more clinically relevant bacteria, such as Stenotrophomonas maltophilia (L1) and Bacteroides fragilis

(CcrA) [26]. Adding to the problem of antibiotic resistance conferred by metallo-β-lactamase is that any clinically useful β-lactamase inhibitor, such as clavulanic acid, does not inhibit them.

Metallo-β-lactamases are usually plasmid-encoded having the ability to spread resistance rapidly

[5, 25]; yet, no major infection epidemic has been traced to the production of a metallo-β- lactamase. All crystallographically characterized metallo-β-lactamases have an αββα fold, a fold that is now called the β-lactamase fold [28].

As more β-lactamases were identified and studied, classification schemes evolved, (Table

1-1). The first classification scheme, proposed by Richmond and Sykes in 1973, divided the β- lactamases from Gram-negative bacteria into five classes [29]. Sykes and Matthews improved

Bush-Jacoby- Ambler Inhibited by Medeiros molecular Preferred substrates group [34] class [31] CAa EDTAb 1 C Cephalosporins No No 2a A Penicillins Yes No 2b A Penicillins, cephalosporins Yes No

2be A Penicillins, narrow and Yes No extended spectrum cephalosporins

2br A Penicillins Yes/no No 2c A Penicillins, carbenicillin Yes No

2d D Penicillins, cloxacillin Yes/no No

2f A Penicillins, carbapenems, Yes No cephalosporins

3 B Most β-lactams No Yes 4 - Penicillins No ?

Table 1-1: Classification schemes for β-lactamases.

a. CA = Cavulanic Acid b. EDTA = Ethylenediaminetetraacetic acid

this scheme by including isoelectric focusing comparisons in 1976 [30]. However, the

Richmond-Sykes scheme soon proved to be too limited, not even accounting for metallo-β- lactamases. Ambler classified β-lactamases according to their molecular structure; serine-β- lactamases as class A and metallo-β-lactamases as class B [31]. The Ambler classification scheme was updated by Jaurin and Grundstrom [32] and Medeiros [33] adding class C and class

D and subdividing the serine-β-lactamases based upon their substrate specificity [8, 24]. Most recently, Bush et al. have published a classification scheme for β-lactamases, the Bush-Jacoby-

Medeiros groups, based upon biochemical characteristics and then subdivided within a group according to their substrate and inhibitor profiles [14, 34-36]. Bush groups 1, 2, and 4 β- lactamases are all serine-β-lactamases. Bush group 3 β-lactamases are metallo-β-lactamases

(Table 1-2) [37]. Subgroup 3a contains metallo-β-lactamases that hydrolyze a wide spectrum of antibiotics, with most showing a preference toward penicillins and to a lesser extent, cephalosporins. Metallo-β-lactamases in Bush group 3a include β-lactamase II from B. cereus and CcrA from B. fragilis. Subgroup 3b contains metallo-β-lactamases that preferentially hydrolyze carbapenems and exhibit poor activity against cephalosporins and penicillins.

Subgroup 3b is also distinct because its members require only one Zn(II) ion per protein for full activity, whereas other metallo-β-lactamases require two Zn(II) ions per protein for full activity

[38, 39]. This subgroup contains metallo-β-lactamases from Aeromonas spp., such as ImiS from

A. sobria, CphA from A. hydrophila, and PCM-1 from Bacillus cepacia. Subgroup 3c, currently the smallest subgroup, consists of metallo-β-lactamases that target ampicillin and cephaloridine specifically while poorly hydrolyzing carbapenems. This subgroup currently contains only the metallo-β-lactamase from Legionella gormanii and L1 from S. maltophilia [39]. As metallo-β-

Bush subgroup Substrate specificity Example 3a Penicillins and to a lesser extent β-Lactamase II cephalosporins CcrA L1 3b Carbapenems ImiS CphA 3c Penicillins, cephalosporins, cephamycins Metallo-β-lactamase from Legionella gormanii

Table 1-2: Bush group 3 metallo-β-lactamase sub-grouping scheme.

lactamases are further studied and more new β-lactams are introduced in the clinic, this classification scheme will continue to be modified and expanded.

1.5.2.1 Zinc Metallo-Hydrolase Family of Proteins

With the recent accumulation of sequence and structural data, comparisons of many proteins have revealed extensive similarities among certain proteins. The zinc metallo hydrolase family of proteins, which are structural relatives of the class B β- lactamases, is growing quite rapidly

[28]. Members of this family of proteins participate is a wide variety of biological functions distributed over all three kingdoms of the living organisms. While, these proteins are characterized by a conserved folding pattern and structural motifs, they are highly divergent in their amino acid sequences. In fact, not every member of this family is a zinc protein. All of the metallo-β-lactamases, from which the family is defined, are zinc enzymes [39]. Glyoxylase 2 has been shown to bind either two zincs or a combination of iron, zinc, and manganese [28].

Additionally, rubredoxin oxygen:oxidoreductase (ROO) binds two irons [28]. Members of the zinc metallo hydrolase family exhibit the two-fold, tandem repeat αββα structural fold, the aforementioned β-lactamase fold, as seen in all metallo-β-lactamases thus far characterized by

X-ray crystallography and in other members of the family (glyoxylase, ROO, etc.) [28]. They also contain the conserved structural motifs characterized by the metallo-β-lactamases metal- binding sequence, the amino acid sequence HXHXD…H…C…H (Figure 1-6). These six amino acids, in conjunction with exogenous solvent molecules, usually water, bind to the metal ions that are required for optimal activity of metallo-β-lactamases. The first metal ion binding site,

Zn1 (the sites are labeled Zn because zinc is the preferential metal ion utilized by metallo-β- lactamases), consists of His116 (using the consensus amino acid sequence [40]), His118, His196,

BL II R V T D V I I T H A H A D R I G G ... PGKGH TE... ILVGGC LVKS... A V V P G H G CcrA K V T T F I P N H W H G D C I G G ... LGGGH AT... ILFGGC MLKD... Y V V P G H G ImiS P V L E V I N T N Y H T D R A G G ... LGPAH TP... VLYGNC ILKE... T V V G G H D L1 D L R L I L L S H A H A D H A G P ... FMAGH TP... IAYADS LSAP... VL L T P H P Glx 2-2 K I K F V L T T H H H W D H A D G ... TP -CH TK... AVFTGD TLFV... Q V Y C G H G ROO K I D Y L V I Q H L E L D H A G A ... TRMLH WP... VL I SND IFGQ ... F I C P D H G

Figure 1-6: Amino acid comparison conserved segments of the zinc metallo-hydrolase family of enymes (modified from [28] and [39]). Sequence comparision of β-lactamase II (BLII) from B. cereus [41], CcrA from B. fragilis [42], ImiS from A. sobria [43], L1 from S. maltophilia [44], glyoxalase II (Glx2-2) from A. thaliana [45], and rubredoxin oxygen:oxidoreductase (ROO) from D. gigas [46].

and a bridging water/hydroxide [39]. The second metal ion-binding site, Zn2, consists of

Asp120, His263, Cys221, a bridging hydroxide/water, and a terminally bound water [39]. The

exceptions to this consensus metal-binding sequence are (1) Bush group 3b metallo-β-

lactamases, which have a single amino acid point difference changing the amino acids in the Zn1 binding site to N116, H118, H196 (Figure 1-6), and (2) L1, which has an Asp in place of Cys221 and therefore uses His121 as another metal binding ligand to Zn2.

1.6 Aeromonads

Since 1891 when Aeromonads were first described as pathogens for warm- and cold-blooded animals, their presence in drinking water has been long known [47]. However, it was not until the 1960’s, that an Aeromonad was shown to be involved in human infections. With the knowledge that these environmental microorganisms are responsible for human infections, there was a desire to better understand Aeromonads.

Aeromonads are anaerobic, Gram-negative bacilli that are ubiquitous to aquatic environments [48, 49]. They have been isolated from virtually all known surfaces, fresh and marine aquatic environments including lakes, rivers, reservoirs and even from treated drinking water [48-50]. The only water source in which they are not often found is well-protected underground water. Their presence in most sources of water is due to their ability to grow in a wide range of temperatures, the optimal temperature being 22 to 28 °C, and to their requirement of only minimal amount of nutrients.

The main risks of acquiring Aeromonas associated diseases are through contact between contaminated water and an open wound, drinking of contaminated water, and eating contaminated food. It is not surprising with the nearly ubiquitous presence of Aeromonads in

water sources that there are many reports of the presence of these organisms in food, such as raw meats (especially seafood) and untreated milk [51].

Aeromonas strains, while pathogenic, are fairly innocuous, and most often lead to gastrointestinal problems. Extreme cases in very young or old and immuno-compromised patients can lead to diarrhea. Aeromonas-associated infections are most common in the summer months or in warm climate countries; environments that are optimal for maximal levels of bacteria in the water sources. Aeromonad strains have also been linked to wound and enteric extra-intestinal infections [48, 51].

Not all Aeromonads are pathogenic. Of the 13 characterized species of Aeromonas, nine are clinical specimens, while the rest have only been found in environmental settings [48, 49,

51]. Aeromonas hydrophila, Aeromonas veronii bv. sobria, and Aeromonas caviae pose the greatest public health risk, accounting for greater than 80% of clinical isolates [51]. Aeromonas veronii bv. sobria is the most common species found in lakes, reservoirs, and treated drinking water. Aeromonas veronii bv. sobria and A. caviae are the most common species found in intestinal infections, and A. veronii bv. sobria and A. hydrophila are the most common species found in extra-intestinal sources [48, 51].

Aeromonas species pose an even greater health risk due to their ability to produce up to three inducible, chromosomally encoded β-lactamases [52-54]. The first is a serine active-site,

Group 1 cephalosporinase. The second is a serine active-site, Group 2 penicillinase. The third is a group 3 (class 3b), metal-containing carbapenemase. β-Lactamase activity has been reported for six of the Aeromonas species, while the production of all three β-lactamases has only been found in Aeromonas veronii bv. sobria and Aeromonas hydrophila [51, 52, 55, 56]. In addition

to the production of β-lactamases, there is evidence of horizontal gene transfer between the species [48, 49, 51].

The metallo-β-lactamase produced by A. hydrophila and A. veronii bv. sobria are of particular interest and clinically relevant because of their ability to hydrolyze carbapenems.

They do not only have carbapenemase activity; they are selective toward carbapenems, exhibiting poor or no activity against other β-lactams [13, 54, 57]. The metallo-β-lactamases produced by A. hydrophila and A. veronii bv. sobria are very similar, differing in their amino acid sequences at just 7 residues [43]. They are quite divergent at the sequence level from other metallo-β-lactamases; however, the putative metal-binding residues are conserved, except the previously mentioned H to N point difference at amino acid 116 [43]. This divergence at the primary level leads to the unique functional and structural properties of the group 3b metallo-β- lactamases. First these enzymes are strict carbapenemases; second these enzyme’s optimal activity is achieved through the binding of just one equivalent of metal, not two like the other sub-classes of metallo-β-lactamases [38]. In fact binding of a second equivalent of metal is inhibitory.

1.7 Antibiotic Resistance in Aeromonas veronii bv. sobria

Aeromonas veronii bv sobria (then only classified as A. sobria) strain 163a was isolated from a patient at the Hammersmith Hospital, London, England [52]. The clinical isolate exhibited low-level antibiotic resistance to penicillins and cephalosporins. The mechanism of antibiotic resistance in strain 163a was explored, and β-lactamase production was found to be inducible in the presence of ampicillin, cefoxitin, and imipenem [52]. Extracts from these

induced cells all exhibited the same activity profile, hydrolyzing penicillins, cephalosporins, and

carbapenems.

From the strain 163a, a derepressed mutant, 163a-M, exhibiting strong hydrolytic activity

against all three major classes of β-lactams, was isolated. Hydrolytic activity against

carbapenems of the derepressed mutant, like that of the induced wild-type isolate, was 95%

inhibited by 10 mM EDTA [52]. The hydrolytic activities against penicillins and cephalosporins

of both the derepressed mutant and the induced wild-type isolate were not significantly affected by the presence of 10 mM EDTA. Carbapenemase activity could be restored by addition of

Zn(II) after exhaustive dialysis to remove the EDTA [52].

To study further the antibiotic resistance of the clinical isolate, the β-lactamase genes were cloned from 163a chromosomal DNA [52]. This cloning resulted in the recovery of two genes. The first gene isolated, amps, encoded a penicillinase, which exhibited activity against penicillins, weak activity against cephalosporins, and no activity against carbapenems. The second isolated gene, cepS, encoded a β-lactamase that exhibited activity against cephalosporins, while showing no detectable activity against penicillins or carbapenems. Both AmpS and CepS readily hydrolyzed nitrocefin. Neither AmpS nor CepS were inhibited by EDTA. Sequence analysis of AmpS and CepS showed that both were highly similar to serine-β-lactamases [53]; further analysis showed that AmpS is a Bush group 2d serine-β-lactamase and CepS is a Bush

group 1 serine β-lactamase.

Neither AmpS or CepS could account for the carbapenemase activity or the metal

sensitivity of the cell extracts containing Aeromonas veronii bv. sobria strain 163a. In another

Aeromonad, A. hydrophila, a metallo-β-lactamase, CphA, which exhibited carbapenemase activity, poor nitrocefin activity, sensitivity to EDTA, and restoration of activity by Zn2+ was

reported [58]. Later, Rossolini and co-workers determined that sequences similar to the cphA gene were found in other tested A. hydrophila, A. veronii (both the veronii and sobria biotypes),

and A. jandaei strains but not in any other tested Aeromonas species [59]. Carbapenemase

activity was detected in 83% of the strains testing positive for cphA homologues, all of which

were inhibited by EDTA.

T.R. Walsh and co-workers were able to modify their search to reveal that Aeromonas

veronii bv. sobria strain 163a produced a third β-lactamase, which was sensitive to imipenem

[54]. Initial characterization of the resulting enzyme, ImiS, revealed an approximately 28 kDa

protein whose activity against imipenem was lost in the presence of EDTA. The addition of

excess Zn2+, at levels greater than 10 mM, resulted in the precipitation of the protein. The testing

of various β-lactams revealed that ImiS hydrolyzed the carbapenems efficiently and penicillins at

rates significantly slower than carbapenems while poorly hydrolyzing nitrocefin. ImiS did not

exhibit detectable hydrolysis of any other cephalosporin tested. These identical characteristics

were also reported for CphA, the metallo-β-lactamase from A. hydrophila [38]. Further analysis

of imiS revealed a 762 nucleotide open reading frame (ORF) sharing 94% identity with cphA

[43]. The sequence immediately downstream of the imiS ORF shares no homology with the

same sequence in cphA. Like most other β-lactamases imiS has a high G/C content (62%) with

greater than (85%) of the third nucleotide in a codon being a G/C. Translation of the imiS ORF resulted in the production of a 254 amino acid preprotein [43]. This peptide is then cleaved between two alanines at positions 27 and 28 resulting in a 25.25 kDa protein containing 227 amino acids [43]. Comparison of the ImiS amino acid sequence showed a high level of identity to CphA, with only 7 amino acids differing.

1.8 Introduction to the Dissertation

Inorganic elements are ubiquitous in nature and essential for life. Bioinorganic chemistry

involves the study of metal ions in biological systems. It is a relatively young, broad field of

scientific study that is rapidly growing, bringing together researchers from many diverse

disciplines. Biological systems are studied to determine the in vivo function of metals in their

naturally occurring environments, as probes, and as pharmaceutical agents [60, 61].

Metal ions are commonly found as natural constituents of proteins. With each passing

year, more and more enzymes, both newly discovered and those that have been known to exist

for a long time, are being shown to require metal ions for activity. These metalloproteins exploit

the physical properties of the metal ions to perform a wide variety of functions associated with

many life processes. While metal ions have been shown to be essential for biosystems, not all

metallo-species in biosystems are beneficial to humans. Metalloenzymes and the alteration of

normal metal ion homeostasis, due to mutation or gene deletion, have been linked to many

diseases [62]. In fact, metal ions in biological systems are generally bound in a ligated form, because free metal ions are extremely toxic even at low concentrations [63].

1.8.1 Rational Drug Design

The majority of pharmaceutical “drug design” in the past has relied heavily on chance discoveries, in a process often compared to finding the proverbial needle in a haystack. Novel compounds, often from exotic natural sources, would be isolated, purified, and structurally characterized. These known compounds would then either be isolated and purified from their natural sources or synthesized and purified in the lab. Once enough compounds were pure enough, they would be screened to determine their pharmaceutical activity.

Rational drug design allows for a more direct approach, in effect shrinking the size of the haystack. Instead of looking at compounds and trying to find a pharmaceutical target, rational drug design looks at a target and tries to design a pharmaceutical compound. This form of drug design is most often and most successfully applied to protein targets, where protein-substrate interactions can be studied, and novel inhibitors can then be designed based upon the protein- substrate studies.

In an effort to help combat antibiotic resistance, rational drug design research has been undertaken to identify and structurally characterize metallo-β-lactamases during the rate-limiting step of β-lactam hydrolysis. Hopefully, this information can then be used to develop potential metallo-β-lactamase inhibitors, which ideally would eventually be administered in combination with existing antibiotics.

1.8.2 Sections of the Dissertation

This dissertation describes the structural and kinetic characteristics of the metallo- β- lactamase ImiS from A. sobria. In Chapter 2, the over-expression, purification, and biochemical characterization of recombinant ImiS is presented, and the properties of the recombinant enzyme are compared to those of the other metallo-β-lactamases. In Chapter 3, the spectroscopic characteristics of Co(II)-substituted ImiS are presented, and a structural model of the active site is given. In Chapter 4, the kinetic properties of ImiS are presented, and a mechanistic model of

β-lactam hydrolysis is hypothesized. Chapter 5 discusses the work contained in this dissertation in the scope of the current understanding of metallo-β-lactamases, and even more generally β- lactamases, and antibiotic resistance in society.

1.9 References

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Chapter 2

Over-expression, Purification, and Characterization of Recombinant ImiS

2.1 Introduction to Chapter 2

The Group 3b metallo-β-lactamases are significantly different from other metallo-β-

lactamases. For example, CphA and ImiS require only 1 Zn(II) for full catalytic activity, CphA

and ImiS prefer carbapenems as substrates over other β-lactams, and CphA and ImiS do not

contain the consensus motif of the other metallo-β-lactamases (Ch 3 and Figure 2-1).

Although many attempts have been made to solve the crystal structures of ImiS and

CphA, no crystals yielding suitable resolution have been obtained (Dr. James Spencer,

University of Bristol, UK, personal communication). The only structural information yet

available on a Bush Group 3b β-lactamase is a recently reported Extended X-ray Absorption

Fine Structure (EXAFS) study by Meyer-Klaucke et. al. [1]. This EXAFS study predicted that

the metal binding ligands in CphA are a sulfur, a O/N donor, and two imidazole donors. This

predicted site could be accounted for by a combination of the metallo-β-lactamase Zn1 and Zn2 metal-binding sites; a configuration that is unprecedented any of the proteins containing a β- lactamase fold. In addition, this EXAFS study predicts a 4-coordinate Zn(II) that does not contain an open site for substrate unless the coordination number of Zn(II) is increased to 5; which is not a preferred coordination number for Zn(II). Because ImiS and CphA are not amenable to crystallographic characterization and since there are apparent problems with the predictions from the published EXAFS studies on CphA, other spectroscopic studies are needed to better understand the structure of these enzymes. However, spectroscopic techniques often

Bce-569H SQKVEKTVIKNETGTISISQLNKNVWVHTELGSFNGEA-VPSNGLVLNTSKGLVLVD Bfr-CfiA ------QKSVKISDDISITQLSDKVYTYVSLAEIEGWGMVPSNGMIVINNHQAALLD Sma-IMP1 ------AESLPDLKIEKLDEGVYVHTSFEEVNGWGVVPKHGLVVLVNAEAYLID Ahy-CphA ------AGMSLTQVSGPVYV------VEDNYYVQENSMVYFGAKGVTVVG Asb-ImiS ------AGMSLTQVSGPVYV------VEDNYYVQENSMVYFGAKGVTVVG Stm-L1 ------VDASWLQPMAPLQIADHTW------QIGTEDLTALLVQTPDGAVLLD Cms-BlaB ------QENPDVKIEKLKDNLYVYTTYNTFNGTK YAANAVYLVTDKGVVVID consensus * Q* V V *** **

Bce-569H SSWDDKLTKELIEMVEKKFQK RVTDVIITHAHADRIGGIKTLKER-GIKAHSTALT Bfr-CfiA TPINDAQTEMLVNWVTDSLHA KVTTFIPNHWHGDCIGGLGYLQRK-GVQSYANQMT Sma-IMP1 TPFTAKDTEKLVTWFVERGY KIKGSISSHFHSDSTGGIEWLNSR-SIPTYASELT Ahy-CphA ATWTPDTARELHKLIKRVSRK PVLEVINTNYHTDRAGGNAYWKSI-GAKVVSTRQT Asb-ImiS ATWTPDTARELHKLIKRVSRK PVLEVINTNYHTDRAGGNAYWKSI-GAKVVSTRQT Stm-L1 GGMPQMASHLLDNMKARGVTPRDLRLILLSHAHADHAGPVAELKRRTGAKVAANAES Cms-BlaB CPWGEDKFKSFTDEIYKKHGK KVIMNIATHSHDDRAGGLEYFGKI-GAKTYSTKMT consensus * L * I H H*D GG G

Bce-569H AELAKKN------GYEEPLGDLQTVTNLKFGNMKVETFYPGKGHTED Bfr-CfiA IDLAKEK------GLPVPEHGFTDSLTVSLDGMPLQCYYLGGGHATD Sma-IMP1 NELLKKD------GKVQATNSFS-GVNYWLVKNKIEVFYPGPGHTPD Ahy-CphA RDLMKSDWAEIVAFTEKGLPEYPDLPLVLPNVVHDGDFTLQEGKVRAFYAGPAHTPD Asb-ImiS RDLMKSDWAEIVAFTEKGLPEYPDLPLVLPNVVHEGDFTLQEGKLRAFYLGPAHTPD Stm-L1 AVLLARGGSD----DLHFGDGITYPPANADRIVMDGEVITVGGIVFTAHFMAGHTP- Cms-BlaB DSILAKE------NKPRAQYTFDNNKSFKVGKSEFQVYYPGKGHTAD consensus L K Y G *HT D

Bce-569H NIVVWLPQY------NILVGGCLVKSTSAKDLGNVADAY-VNEWSTSIENVLKRYR Bfr-CfiA NIVVWLPTE------NILFGGCMLKDNQATSIGNISDAD-VTAWPKTLDKVKAKFP Sma-IMP1 NVVVWLPER------KILFGGCFIK---PYGLGNLGDAN-IEAWPKSAKLLKSKYG Ahy-CphA GIFVYFPDE------QVLYGNCILKEK----LGNLSFAD-VKAYPQTLERLKAMKL Asb-ImiS GIFVYFPDQ------QVLYGNCILKEK----LGNLSFAD-VKAYPQTLERLKAMKL Stm-L1 GSTAWTWTDTRNGKPVRIAYADSLSAPG-YQLQGNPRYPHLIEDYRRSFATVRA—-L Cms-BlaB NVVVWFPKE------KVLVGGCIIKSADSKDLGYIGEAY-VNDWTQSVHNIQQKFS consensus V P *L G C *K *GN* * * *

Bce-569H NINAVVPGHGE VGDKGLLLHTLDLLK Bfr-CfiA SARYVVPGHGK YGGTELIEHTKQIVNQYIESTSKP Sma-IMP1 KAKLVVPSHSE VGDASLLKLTLEQAVKGLNESKKPSKPSN Ahy-CphA PIKTVIGGHDSPLHGPELIDHYEALIKAAPQS Asb-ImiS PIKTVVGGHDSPLHGPELIDHYEALIKAASQS Stm-L1 PCDVLLTPHPGASNWDYAAGARAGAKALTCKAYADAAEQKFDGQLAKETAGAR Cms-BlaB GAQYVVAGHDD WKDQRSIQHTLDLINEYQQKQKASN consensus VV H

Figure 2-1: Sequence comparision of Bce-569H: β-lactamase II from B. cereus [2], 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]. Amino acids in red are metal binding residues. Residues in blue represents indicate point difference for ImiS and CphA from the consensus metal ligating amino acid sequence. Residues that are conserved in 4 out of 7 genes are shown as the consensus. This information is adapted from Crowder and Walsh [9].

require large quantitites of enzymes.

Currently, there are two published methods to obtain a Group 3b metallo-β-lactamase.

CphA has been cloned and over-expressed in E. coli, obtaining 30 µg/mL [10]. On the other

hand, ImiS has obtained directly from cultures of A. sobria. 8 L of A. sobria containing growth

culture that had been incubated for 18 hours, 37 ºC, were harvested by centrifugation. The

resulting A. sobria cell pellet was resuspended in 200 mL, and rendered into spheroplasts

following treatment with lysozyme. Multiple rounds of dialysis and column chromatography

were utilized to produce a pure ImiS protein [11]. To prevent exposure to a potential pathogen, a

means to obtain large quantities of recombinant ImiS from E. coli is desired.

The ultimate goal of the Crowder group is to design novel structure and mechanism based inhibitors of the metallo-β-lactamases. It is hoped that these inhibitors can be given in

combination with current β-lactams as a treatment for bacterial strains harboring a metallo-β-

lactamase. To accomplish this goal, the Crowder group is structurally and mechanistically

characterizing a metallo-β-lactamase from each of the subgroups (see Chapter 1) and searching

for common traits, shared by all metallo-β-lactamases, to which inhibitors can be targeted. This

chapter describes the subcloning, over-expression, purification, and biochemical characterization

of recombinant ImiS.

2.2 Materials and Methods

2.2.1 Materials

E. coli strains DH5α and BL21(DE3) were purchased from Gibco BRL (Gaithersburg,

MD) and Novagen (Madison, WI), respectively. The over-expression vector, pET26b, and

cloning vector, pUC19, were obtained from Novagen, while the gene, imiS, was a kind gift from

Dr. Timothy Walsh (University of Bristol, UK). Ligating imiS between the HindIII and NdeI

restriction sites of pET26b generated the ImiS over-expression plasmid, pET26bimiS. Primers

for DNA sequencing were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

The restriction enzymes (HindIII and NdeI), MgSO4, deoxynucleotides (dNTPs), thermopol buffer, and Deep Vent were obtained from New England Biolabs (Beverly, MA) or Promega

Corporation (Madison, WI). Polymerase chain reaction (PCR) was performed using the

Thermolyne Amplitron II from Barnstead (Dubuque, IA). DNA was purified using the QIAGEN

QuiQuick Gel Extraction kit or the plasmid purification kit with QIAGEN-tip 100 (Midi) columns (Valencia, CA). The Wizard Plus Miniprep kit was obtained from Promega

Corporation. Luria-Bertani (LB) media was made following published procedures [12] or from a pre-mix purchased from Fisher Scientific (Pittsburgh, PA). Isopropyl-β-D-thiogalactoside

(IPTG) was acquired from Gold Biotechnology (St. Louis, MO) or from Anatrace. Buffered solutions were prepared from enzyme grade salts purchased from Fisher Scientific. Buffers,

media, and all other solutions were made with distilled, deionized Barnstead NANOpure,

ultrapure water (Dubuque, IA). The solutions were rendered metal-free by treatment with

Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA) followed by filtration through a 0.22

micron filter membrane (Osmonics Inc.) Dialysis tubing was prepared from Spectro/Por

regenerated cellulose (RC) molecular porous membranes with a molecular weight cut-off

(MWCO) of 6000-8000 Daltons (Spectra/Por 1 Dialysis Membranes) (Spectrum Corporation,

Gardena, CA) and made metal-free according to Sambrook, et al. [12]. Columns and media for

Fast Performance Liquid Chromatography (FPLC) were obtained from Amersham Pharmacia

Biotech AB (Kalamazoo, MI). Nitrocefin was purchased from Becton Dickinson Microbiology

Systems (Cockeysville, MD), and solutions of nitrocefin were prepared as previously described

[9, 13]. Penicillin G was purchased from Fisher Scientific. Imipenem and meropenem were

kind gifts from Merck & Co., Inc. (Elkton, VA) and Astra-Zeneca, respectively. Circular

Dichroism (CD) spectra were collected on a Jasco J-810 Circular Dichroism spectropolarimeter.

2.2.2 Methods

2.2.2.1 Plasmid Construction

To over-express the 227 amino acid post-translationally modified form of ImiS, the imiS

containing plasmid, pET26bimiS, and the plasmid used for sequencing, pUC19, were digested

with NdeI and HindIII. The resulting products were gel purified on a 1% agarose gel, and the 2.4

kb pUC19 piece and the 800 bp imiS gene insert were gel extracted. The gel-purified pieces

were then ligated with T4 ligase, according to Sambrook [12], resulting in an 3.25 kb, ampicillin-

resistant vector, pUC19imiS, which was transformed by electroporation into DH5α E. coli cells and plated onto LB-AMP (100 µg/mL ampicillin) agar plates. The plates were incubated at 37

°C overnight. DNA minipreps were preformed on 5-10 of the resulting colonies. The plasmid

DNA was digested with HindIII and run on a 1 % agarose gel to identify which colonies contained plasmid DNA of the correct size. A large-scale DNA preparation was conducted on one of the colonies containing the properly sized DNA. DNA sequencing was performed by the

Biosynthesis and Sequencing Facility at Johns Hopkins University (Baltimore, MD).

For over-expression, the 850 bp imiS insert was ligated, with T4 ligase, into a 5.25 kb pET26b vector which had be digested with HindIII and NdeI (Figure 2-2). The resulting kanamycin-resistant plasmid, pET26bimiS, was transformed by electoporation into BL21 (DE3)

E. coli cells and plated onto LB-KAN (25 µg/mL Kanamycin) agar plates. DNA minipreps were preformed to identify colonies containing correct sized plasmids. A colony with the correct

plasmid was selected, grown overnight in 5 mL of LB containing 25 µg/mL kanamycin. The

overnight cultures were used to generate glycerol stocks according to Manniatis [12], and the

glycerol stocks were stored in the –80 °C Revco freezer.

2.2.2.2 Over-expression and Purification of ImiS

Recombinant ImiS was over-expressed using a procedure adapted from Iaconis and

Sanders and Walsh, et al [11, 14]. A stab was taken from a glycerol stock and used to inoculate

50 mL of LB containing 25 µg/mL kanamycin. This culture was allowed to shake at 37 °C overnight. After 15-18 hours 4 x 1L flasks of LB containing 25 µg/mL kanamycin and 100 µM

Zn(II) (200 µL of 500 mM ZnCl2 per 1L) were inoculated with 10 mL of the overnight

preculture. The full-scale growth cultures were allowed to grow with shaking at 37 °C until an

optical density of 0.6-0.8 (O.D.600nm) was reached. Protein production was induced by making

the cell cultures 1 mM in IPTG and the cells were shaken at 37 °C for 3 hours. The cells were collected by centrifugation for 15 minutes at 7,000 rpm and at 4 °C. The supernatant was discarded, and the cell pellet was resuspended in 30 mL of cold, 50 mM TRIS, pH 7.0, containing 500 mM NaCl (FPLC Buffer B). The cells were then lysed by 2 passages through a french press, under approximately 20,000 pounds per square inch of pressure. The cell debris was removed from the sample by centrifugation for 30 minutes at 15,000 rpm and at 4 °C. The crude protein solution was dialyzed overnight at 4 °C versus 2 L of 50 mM TRIS, pH 7.0 (FPLC

Buffer A).

The dialyzed, crude protein solution was then centrifuged for 30 minutes at 15,000 rpm to remove any precipitated proteins or lipids, and the cleared supernatant was loaded onto a 16 x 40 mm SP-Sepharose column, equilibrated with 50 mM TRIS-HCl, pH 7.0. Bound proteins were

Figure 2-2: Construction of pET26bimiS plasmid used to produce active recombinant ImiS.

eluted from the column with a linear gradient of 0 to 500 mM NaCl in 50 mM TRIS-HCl, pH

7.0, at a flow rate of 2 mL per minute. The gradient increased at a rate of approximately 1% per

minute, and 8 mL fractions were collected. Fractions containing ImiS were identified by SDS-

PAGE, after staining the gels with Coomassie blue. Those fractions exhibiting bands at about 25

kDa with greater than 95 % purity were pooled and concentrated in an Amicon ultrafiltration cell

equipped with a YM-10 cellulose membrane.

2.2.2.3 Determination of a Molar Extinction Coefficient for ImiS

The molar extinction coefficient for ImiS was determined initially by bicinchoninic acid

(BCA) protein assay analysis, following manufacturer’s instructions and by using albumin as a protein standard. The molar extinction coefficient was more accurately determined using amino acid analyses. ImiS samples were prepared, their absorbance at 280 nm recorded, and a known quantity of enzyme was sent to Commonwealth Biotech Inc. (CBI, Richmond, VA) for amino acid analysis. CBI analyzed for each amino acid and reported the results in both nanomoles of amino acid and micrograms of amino acid for each amino acid except Cys and Trp. The total number of moles of each amino acid was divided by the number of each respective amino acid from the primary sequence [5] giving the number of moles of protein. The number of moles of protein could then be divided by the volume of the sample sent to CBI to give the concentration

(in mol/L or M ImiS). Finally, using Beer’s Law (A = εbc), the known absorbance at 280 nm for

the sample of ImiS sent to CBI, and the molar concentration of ImiS, the extinction coefficient at

280 nm was calculated to be 23,800 ± 200 M-1cm-1.

2.2.2.4 Metal Analyses

As isolated ImiS was quantitated by using the protein’s absorbance at 280 nm, and the

extinction coefficient previously calculated. ImiS was dialyzed versus 3 x 1L metal-free 50 mM

TRIS, pH 7.0, at 4 °C and was diluted with metal-free, 50 mM TRIS, pH 7.0, to a final

concentration of 5-10 µM. Metal contents of multiple ImiS samples were determined using a

Varian Inductively Coupled Plasma Spectrometer with an atomic emission spectroscopy

detection (ICP-AES). The final dialysis buffer was used as a reference blank. Calibration curves

for all metals tested were based on at least 3 standards, and all calibration curves had correlation

coefficients of 0.9950 or better. Emission lines at 213.856 nm, 238.892 nm, 324.754 nm,

259.940 nm, 257.610 nm, and 231.604 nm, the most intense emissions for zinc, cobalt, copper,

iron, manganese, and nickel, respectively, were used to determine metal content for each enzyme

preparation. Errors in metal content were reported as standard deviations (σn-1) of replicate

samples.

2.2.2.5 Steady-State Kinetics

The hydrolysis of imipenem by ImiS was monitored by following the loss of the substrate

at 300 nm. Hydrolysis of other antibiotics, meropenem, penicillin G, and nitrocefin, were

followed at 300 nm, 240 nm, and 485 nm, respectively. Absorbance data were converted into

concentration data using the previously reported extinction coefficients (imipenem, ε300 = -9,000

-1 -1 -1 -1 -1 -1 M cm ; meropenem, ε300 = -3,100 M cm ; penicillin G, ε235 = -936 M cm ; nitrocefin, ε485 =

17,400 M-1cm-1). Kinetic experiments were carried out in 50 mM TRIS buffer, pH 7.0, or in 15

mM cacodylate buffer, pH 6.5, on a Hewlett Packard 5480A UV-Vis spectrophotometer, using

an Isotemp circulator to maintain the reactions at 25 °C. Absorbance data were taken every

second for thirty seconds, corresponding to approximately the first 10% of the reactions.

Substrate concentrations were varied between 0.1 and 10 times the reported KM values, or until

significant substrate inhibition was observed. Concentrations of substrate disappearance per

second (velocity) data were plotted versus initial substrate concentration and fitted to the

Michaelis-Menton equation, using the computer program Curve Fit, in order to determine steady-

state kinetic constants KM and kcat. The errors are reported as standard deviations (σn-1) from multiple kinetic trials.

2.2.2.6 Gel Filtration Chromatography

Gel filtration chromatography was performed on an Amersham Pharmacia Biotech

Sephacryl S-200 column with a flow rate of 1 mL/min, according to the manufacturer’s instructions. The column had been equilibrated with and all sample solutions were prepared with

50 mM TRIS, pH = 7.0. Chymotrypsinogen A (25 kDa), ovalbumin (43 kDa), bovine serum albumin (BSA) (67 kDa), and ribonuclease A (13.7 kDa) were used as molecular weight standards, and Blue Dextran 2000 was used to measure the dead time. The retention time of the four standard proteins were measured relative to the elution of the Blue Dextran 2000 and used to generate a calibration curve. An ImiS sample, approximately 2 mL at 2 mg/mL, was run over the column, again utilizing Blue Dextran 2000 to measure the dead time. The retention time of

ImiS was then measured relative to the elution of Blue Dextran 2000 and correlated to the calibration curve of the standards to determine the molecular weight of ImiS.

2.2.2.7 MALDI-TOF Mass Spectrometry

Approximately 300 µL of 150 µM as-isolated ImiS, in a 50 mM TRIS buffer, pH = 7.0, was prepared and sealed. The sample was sent to Campus Chemical Instrumentation Center

(CCIC) at the Ohio State University for MALDI-TOF mass spectra of the recombinant ImiS.

2.2.2.8 CD Spectroscopy

Circular dichroism spectra were obtained on ImiS samples that were prepared by dialyzing the purified enzyme samples versus 3 x 2L of 5 mM phosphate buffer, pH 7.0, over six hours. The samples were then diluted with 5 mM phosphate buffer, pH 7.0, to a final concentration of approximately 75 µg/mL. Spectra were obtained on a JASCO J-810 CD spectropolarimeter, operating at 25 ºC. CD spectra were analyzed for secondary structural content using the CDSSTR simulation program at the DICHROWEB internet site[15-20].

2.2.2.9 N-terminal Amino Acid Sequencing

TRIS buffer was replaced by dialyzed each ImiS sample against 1 L of 50 mM HEPES, pH = 7.0, 4 ºC, for 4 hours. 100 µL of approximately 15 µM ImiS in HEPES buffer was sent to

Biosynthesis and Sequencing Facility in the Department of Physiology at Johns Hopkins

University Medical School for N-terminal amino acid sequencing.

2.3 Results and Discussion

2.3.1 Over-expression and Purification of Recombinant ImiS

By modifying the previously published over-expression and purification systems for recombinant β-lactamases [9], ImiS was produced in large quantities so that future structural and

mechanistic studies could be carried out. The ImiS producing gene, imiS, was ligated into pET26b that allows for over-expression of protein under the control of a strong promotor and kanamycin selection. The over-expression vector, pET26bimiS, was then transformed into

BL21(DE3) E. coli cells, which were used to produce soluble protein. Over-expression levels were optimized by using test cultures: it was determined that allowing the bacteria to grow to an optical density at 600 nm (O.D.600) of 0.6 to 0.8 before induction and allowing the cells to induce for three hours resulted in the greatest levels of over-expressed ImiS. The over-expressed ImiS was purified by using a procedure that was modified from that used by Walsh et al. [11] to purify

ImiS directly from Aeromonas. In the published procedure by Walsh et al. [11], crude ImiS was isolated from the spheroplasts, and the protein mixture was loaded onto a SP-Sepharose column equilibrated with 50 mM TRIS, pH 7.0. The soluble protein samples exhibiting β-lactamase activity were combined, concentrated, and loaded onto a second SP-Sepharose column equilibrated with 50 mM TRIS, pH 6.0. After this second SP-Sepharose column, ImiS was estimated to be 95% pure by SDS-PAGE. In contrast the recombinant ImiS was determined to be greater than 95% pure after the first SP-Sepharose, pH 7.0, column (Figure 2-3). In an effort to further purify recombinant ImiS, the fractions containing recombinant ImiS that were eluted from the first SP-Sepharose column (pH 7.0) were pooled and loaded onto a pH 6.0 SP-

Sepharose column. However, there was an approximate 30% loss in protein with no improvement in purity. Therefore, the second SP-Sepharose column was not used in any subsequent studies. The over-expression and purification protocol described above yielded approximately 50 mg of active, 95% pure protein per 4L of growth culture.

2.3.2 Determination of Molar Extinction Coefficient for Recombinant ImiS

In order to characterize ImiS, an accurate protein concentration must be known. Most

commonly, a protein solution’s absorbance at 280 nm, which is primarily due to the number of

tryptophans and tyrosines in the protein [21], is used with a predetermined molar extinction

coefficient and Beer’s law to determine the concentration of a protein. To determine a molar

extinction coefficient for a particular protein, one normally measures the absorbance at 280 nm

of the given protein solution, independently determines the concentration of the protein in

solution, and uses Beer’s law to determine ε280nm. Three different methods were used to

determine the concentration of ImiS solutions: (1) the Edelhoch method [21], (2) a BCA method, and (3) amino acid analysis. The molar extinction was initially determined by using the

Edelhoch method that allow for a calculated molar extinction coefficient, in M-1cm-1 [21]. This

method assumes that only tyrosines, tryptophans, and cystine disulfide bridges contribute

significantly to a protein’s absorbance at 280 nm and that each component’s contribution is given

by the following equation:

-1 -1 ε280 (M cm ) = (#Trp)(5,500) + (#Tyr)(1,490) + (#cystine)(125)

The Edelhoch method predicted an extinction coefficient of 29,900 M-1cm-1 for recombinant

ImiS.

The second method used to determine protein concentration and ε280nm was the

Bicinchoninic Acid (BCA) method. The BCA assay is a colorimetric method in which protein residues reduce Cu2+ ions to Cu1+ [22]; the Cu1+ ions are chelated by bicinchoninic acid to form a

Cu-BCA complex that absorbs at 562 nm. The BCA assay predicted a molar exinction coefficient of 36,000 M-1cm-1 for recombinant ImiS.

The third method used to determine protein concentration was amino acid analysis. This

1 2 3 4 5 6 7

Figure 2-3: SDS-PAGE gel representing protein over-expression and purification. Lanes:

1: Perfect Protein molecular weight Markers. 2: Boiled cell fraction of resuspended cell pellet, pre-induction. 3: Boiled cell fraction of resuspended cell pellet, 3 hours post induction with 1mM IPTG. 4: Supernatant, post French press. 5: Supernatant, post overnight dialysis. 6: Eluent from SP-Sepharose FPLC column, post concentration. 7: Perfect Protein molecular weight Markers.

method quantitates the amounts of all of the amino acids in a sample, except cys, trp, and met.

The calculated extinction coefficient based on the amino acid analysis was 23,800 M-1cm-1.

In previous studies in the Crowder lab, it has been demonstrated that the BCA method is

the least accurate of the methods tested to determine protein concentration (Dr. Michael

Crowder, unpublished). The molar extinction coefficients determined by the Edelhoch method

and the amino acid analysis method were compared by determining the amount of Zn(II) that

ImiS isolated with and the kinetic constant, kcat, with both of these values (data not shown).

Neither could be shown to be more accurate. Therefore, the ε280 determined from the BCA method was not used, and the average ε280 from the Edelhoch and amino acid analyses, 26,850

M-1cm-1, value was used for all subsequent studies.

2.3.3 Physical Properties of Recombinant ImiS

The approximate size and purity of recombinant ImiS was established by utilizing SDS-

PAGE (Figure 2-3). ImiS migrated in SDS-PAGE consistent with a mass slightly less than 25 kDa. A sample of purified ImiS was passed over a Sephacryl S-200 column, and ImiS eluted off as a monomeric protein with a mass of approximately 25 kDa. MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectra were collected on several samples of ImiS to determine an exact molecular weight (Figure 2-4 and 2-5). Initially, the MALDI spectra collected at Miami University exhibited peaks at m/z values that did not correspond to the predicted mass for ImiS, based on the DNA sequence [5]. The size differences were disparate and not easily accounted for; such mass differences could result from missing a metal cofactor

(Zn(II)), post-translational modifications, or proteolytic cleavage of the protein. Walsh and coworkers have published that the imiS gene encodes for a 254 amino acid protein, and that ImiS

is post-translationally modified to a 227 amino acid protein. To rule out the possibility that the incorrect masses from MALDI-TOF MS were du[5]e to incorrect proteolytic cleavage of the N- terminus, a sample of ImiS was sent to Johns Hopkins University for N-terminal sequencing, which yielded an N-terminal sequence of AGMSL. This sequence is consistent with the published sequence for the modified 227 amino acid protein [5]. To rule out the possibility that

ImiS was losing varied amounts of Zn(II) during the ionization process used in MALDI-TOF, purified ImiS was made apo by dialyzing the protein versus EDTA. The resulting protein still yielded a MS with a m/z not consistent with the predicted ImiS protein. To rule out the possibility that the incorrect masses were not due to the operator/instrument error, a sample of recombinant ImiS was sent The Ohio State University for MALDI-TOF analysis. A mass spectrum resulted that showed a single peak with a m/z ratios of 25,277, which is exactly the mass predicted by the DNA sequence of the ImiS gene in pET26bimiS (Figure 2-4).

2.3.4 Metal Analyses

The metal content of recombinant ImiS samples were determined using ICP-AES.

Isolated ImiS samples were dialyzed versus 3 x 1L of metal-free, 50 mM TRIS, pH 7.0, at 4 °C, or metal-free, 15 mM cacodylate, pH 6.8, at 4 °C (the buffer utilized during metal substitution).

In all cases ImiS was shown to bind 0.54 ± 0.05 moles of Zn(II) ion per mole of protein and no other metal ions in appreciable amounts (Table 2-1). This is less than the one equivalent of

Zn(II) the as-isolated ImiS was expected bind. There are two logical possibilities for this result.

First, the molar extinction coefficient could be off by a factor of ~ 2. This would result in ImiS being only half as concentrated as originally thought and therefore the metal to protein stoichiometry would be approximately 1 to 1.

Metal Ion Concentration

Co < 0.005

Cu < 0.005

Fe 0.028 ± 0.002

Mn < 0.005

Ni < 0.005

Zn 0.54 ± 0.05

Table 2-1: Metal content for ImiS in moles of metal ions per mole of ImiS. ImiS samples were approximately 7 µM in concentration, with the 50 mM TRIS, pH = 7.0, buffer taken as a background measurement.

y Intensit

M/S

Figure 2-4: MALDI-TOF spectrum of recombinant ImiS.

y Intensit

M/S

Figure 2-5: MALDI-TOF spectrum of native ImiS from A. sobria.

) 30 g e

d 20 (m ty

i 10 tic 0 llip E

r 190 200 210 220 230 240 -10 la u c

le -20

Mo -30 Wavelength (nm)

Figure 2-6: CD spectrum of recombinant ImiS and native ImiS from A. sobria. Spectrum is of approximately 5 µM ImiS in 5 mM phosphate buffer, pH = 7.0, at 25 ºC.

The second possibility is that ImiS does not bind the Zn(II) tightly enough to ensure

saturation of the binding site. In support of this hypothesis are metal binding KD studies on

CphA; Valladares, et al reported that Zn(II) binds to the catalytic site with a KD of 1 µM [24].

With a KD this weak, it is possible that Zn(II) falls out during the chromatography steps because the chromatography buffers do not contain greater than trace amounts of Zn(II). Future studies are required to determine which one of these hypotheses is correct.

2.3.5 CD Spectroscopy

Circular dichroism (CD) spectra of ImiS were collected to ensure that the recombinant

ImiS was folding with secondary structural components similar to other metallo-β-lactamases.

Analysis of the CD spectrum (Figure 2-6) with CDSSTR method revealed that ImiS is primarily

an α/β protein, with calculated 20 % α helix, 48 % β sheet, and the remaining 32 %

unstructured. The spectrum is consistent with that published for CphA [23].

2.3.6 Steady-State Kinetics

Steady-state kinetic studies were performed on several preparations of ImiS to explore

substrate specificity and to compare the steady-state kinetic data for recombinant ImiS to those

previously published on ImiS isolated directly from Aeromonas. The steady-state kinetic

constants KM and kcat were determined by fitting kinetic data, velocity (concentration of substrate lost per second) versus initial substrate concentration, to the Michaelis-Menten equation (Figure

-1 -1 2-7). Recombinant ImiS exhibited kcat values of 233 s and 3000 s and KM values of 154 µM and 13.4 µM when using imipenem and meropenem as substrates, respectively (Table 2-2) when the reactions were conducted in 50 mM TRIS, pH 7.0, at 25 ºC (Table 2-2). When using

-7 8x10

6 e t

ra 4

rate (M/S) 2

0 50 100 150 200 Imipenem (µM)

Figure 2-7: Michaelis-Menton plot for the hydrolysis of imipenem by ImiS.

-1 nitrocefin as a substrate, ImiS exhibited a kcat value of 0.0017 s and a KM value of 51 µM. In addition, when a wide range of concentrations (from 10 µM to 1 mM) of penicillin G was utilized as substrate, the lower limit of absorbance was consistently greater than the upper limit of product formed. ImiS was unable to hydrolyze penicillin G, or any other penicillin attempted, at an appreciable rate, no matter how long the reaction was allowed to proceed or how much substrate was present in the cuvette. These data confirm that recombinant ImiS is a carbapenemase, belongs to Bush class 3b, and that recombinant ImiS is similar to the enzyme directly isolated from Aeromonas. In the previous research, ImiS isolated directly from

Aeromonas was quantitated using a molar extinction coefficient of 32500 M-1cm-1 (Dr. James

Spencer, personal communication) [11]. When the kcat determined using above calculated molar extinction coefficient was redetermined using the molar extinction coefficient from this work

-1 -1 (26,850 M cm ) the kcat for native and recombinant ImiS were shown to be within error of each other.

Previous studies on the other Group 3b β-lactamase, CphA, demonstrated that this enzyme tightly binds 1 Zn(II) and requires 1 Zn(II) for full catalytic activity [23]. The addition of excess Zn(II) presumably populates a second Zn(II) site and results in inhibition of carbapenemase activity. Because recombinant ImiS contains 0.54 ± 0.05 Zn(II) ions per protein, it was anticipated that addition of Zn(II) to recombinant ImiS would result in increased activity.

Assays with enough Zn(II) in the buffer to ensure that the resulting enzyme had one mole Zn(II) yielded a highly active enzyme (Figure 2-8), when using imipenem as the substrate. Parallel assays with Zn(II) concentrations twice that of the enzyme yielded a less active protein (Figure

2-8), when using imipenem as the substrate. Finally, assays containing 100 mM ZnCl2, the

concentration of Zn(II) that has shown to be required to fully populate both Zn(II) sites [23], yielded activity exhibiting significant decreases in hydrolytic activity (Figure 2-8).

There are two logical explanations for the observed decrease in activity when there is more than one equivalent of Zn(II): (1) the coordination of more than one Zn(II) is inhibitory as has been suggested for group 3b β-lactamases, including CphA [23], or (2) the titration of the protein sample with additional Zn(II) causes a portion of the protein to precipitate. The first possibility is what is expected for a Bush Group 3b β-lactamase and has previously been shown to be true for CphA. The second possibility, protein precipitation was seen upon the addition of

Zn(II) to a concentrated apo-ImiS sample when attempts were made to generate hetereodimetallic, Co(II)Zn(II)- and Zn(II)Co(II)-, substituted derivatives of ImiS samples

(unpublished data). Protein precipitation could account for the minimal activity increase between the as-isolated ImiS (0.54 equivalents of Zn(II)) and the ImiS with 1 equivalent of

Zn(II) and the significant drop in activity as Zn(II) concentration is increased (Figure 2-8).

While the observed decrease in activity as Zn(II) equivalents increase beyond one is consistent with previous studies on CphA [24], ImiS precipitation as Zn(II) is added to the enzyme cannot be unequivocally ruled out as a possibility.

2.3.5 Comparison to ImiS isolated directly from Aeromonas

To test the integrity of recombinant ImiS, a sample of ImiS, which was isolated directly from Aeromonas, was obtained from Dr. Jim Spencer of the University of Bristol and was characterized as described above. The native ImiS contained 0.61 moles of Zn(II) per mole of protein and exhibited kinetic constants similar to those observed with recombinant ImiS (Table

2-2). On the other hand, MALDI-TOF MS revealed an increase of 30 m/z between native and

120

100 ty

vi 80 ti

t A 60 n e

r 40 e P 20

0 012345 Equivalents of Zn(II)

Figure 2-8: Relative kinetic activity of ImiS with increasing equivalents of zinc. Percent activity = (kcat n equivalents Zn(II) ÷ kcat 1 equivlant Zn(II)) * 100.

recombinant ImiS samples. After several attempts, DNA sequencing of the imiS gene in pET26bimiS revealed an alanine to threonine point mutation at position 76 (published sequence of imiS—EMBL accession number Y10415 [5]1). Given that this point mutation did not alter the kinetic characteristics or metal binding selectivity, recombinant ImiS with the A→T point mutation was used for all of the remaining studies in the dissertation.

2.4 Conclusions

Over-expressing and purifying enzymes in E. coli, rather than purifying them from their native sources, has proven to be a powerful tool in all aspects of enzymology. Recombinant technology allows for the generation of site-directed mutants to test the roles of individual amino acids in catalysis and metal binding. This technology also allows for the study of metal ion preferences, because the strict control of metal ion concentrations can be better controlled in E. coli. Lastly, recombinant technology allows for large quantities of the enzyme to be expressed and purified, without the need to grow large quantities of potentially harmful, pathogenic bacteria.

The most important consideration when using recombinant technology is that the recombinant protein of interest maintains its native structure and activity. Mechanistic or structural studies on a protein that is not biologically-relevant yield information that is meaningless. Data collected on the recombinant form of the enzyme can only be extrapolated to the native form of the enzyme and suppositions made about the behavior of the enzyme in its native organism if the native enzyme and the recombinant enzyme are the same. The data

1 It should be noted that the EMBL accession number actually published in this citation (Y01415) was not properly transcribed; the correct accession number is given (Y10415).

presented in this chapter indicate that ImiS can be over-expressed and purified with high yields from E. coli.

Kinetic studies have revealed that the recombinant ImiS utilized throughout this research maintains the carbapenemase hydrolytic profile that is expected for a Bush Group 3b β- lactamase and similar to published hydrolytic profile for native ImiS, isolated directly from the

Aeromonas bacterium, (Table 2-2) [11]. The point mutation, T76A, is believed to be at a non- integral site as it has not affected metal binding selectivity or the activity of the recombinant

ImiS. Information obtained from spectroscopic and mechanistic studies of the recombinant ImiS will be able to be extrapolated directly to ImiS and its behavior in the native location, the bacterium Aeromonas veronii bv. sobria.

Recombinant ImiS behaves in a manner very similar to the well-studied Bush Group 3b

β-lactamase from Aeromonas hydrophylia, CphA [10, 23-25]. Both are highly selectively carbapenemases, require one Zn(II) for optimal activity, and are inhibited by more than one

Zn(II). Interestingly, they both purify with less full complement of Zn(II) bound; ImiS is isolated with approximately 0.5 equivalents of Zn(II) and CphA closer to one equivalent [23].

This low Zn(II) concentration/enzyme ratio could be a result of the point difference (H116N) that group 3b β-lactamases have from the consensus metallo-β-lactamase metal-binding sequence.

Because this point difference occurs in the Zn1 site, the purported tight binding site for non- group 3b metallo-β-lactamases, the net effect should be a reduced affinity for the metal ion [23,

26]. No conclusions can be drawn about which o

The similarities between these two enzymes is not surprising, because they have such high homology, differing at only 7 amino acids; the high homology and subsequent similarities are most likely due to the natural phenomenon of bacterial gene swapping, where genes that aid a

bacterial cell in overcoming selective pressure, such as an antibiotic, are horizontally transferred

[5, 27-29]. This would suggest that the mechanistic and spectroscopic characterization for one should apply to the other. Unfortunately, little spectroscopic or mechanistic data is available for either enzyme; only basic activity studies and limited spectroscopy information have been reported [11, 23, 24]. Additionally, basic kinetic examination cannot fully elucidate possible differences between these two enzymes. To fully understand Bush Group 3b β-lactamses a thorough understanding of both ImiS and CphA is required.

Recombinant ImiS Native ImiSa -1 -1 Substrate KM (µM) kcat (s ) KM (µM) kcat (s )

Imipenem 154 ± 40 233 ± 17 180 160

Meropenem 13.4 ± 3000 330 1100

Nitrocefin 51 ± 12 0.017 ± 0.002 16 0.06

Penicillin G NDb NDb NR NR

Table 2-2: Kinetic constants for ImiS, revealing carbapenemase activity.

ND = No hydrolysis detected. NR = Not Reported a Native ImiS data taken from Walsh et al. [11]. No error was reported. A molar extinction coefficient of 32550 M-1cm-1 was used to quantitate native ImiS (Dr. James Spencer, personal communication). b Lower limit of absorption for the product formation was consistently greater than the upper limit of product formation.

2.5 References

1. Meyer-Klaucke, W., R.P. Soto, M.H. Balladares, H.-W. Adolph, H.-F. Nolting, J.-M. Frere and M. Zeppezauer, A Comparison of Bacillus Cereus and Aeromonas Hydrophilia Zn-β-lactamases. Journal of Synchrotron Radiation, 1999. 6: p. 400-402.

2. Hussain, M., A. Carlino, M.J. Madonna and J.O. Lampen, Cloning and sequencing of the metallothioprotein β-lactamase II gene of Bacillus cereus 569/H in Escherichia coli. J. Bacteriol., 1985. 164: p. 223-229.

3. Thompson, J.S. and M.H. Malamy, Sequencing and the gene for an imipenem-cefoxitin hydrolyzing enzyme (CfiA) from Bacteroides fragilis TAL2480 reveals strong similarity between CfiA and Bacillus cereus β-lactamase II. J. Bacteriol., 1990. 172(5): p. 2584- 2593.

4. Massida, O., G.M. Rossolini and G. Satta, The Aeromonas hydrophila cphA Gene: Molecular Heterogeneity among Class B Metallo-β-Lactamase. J. Bacteriol., 1991. 173(15): p. 4611-4617.

5. Walsh, T., W. Neville, M. Haran, D. Tolson, D. Payne, J. Bateson, A. MacGowan and P. Bennett, Nucleotide and Amino Acid Sequences of the Metallo-β-Lactamase, ImiS, from Aeromonas veronii bv. sobria. Antimicrob. Agents Chemother., 1998. 42(2): p. 436-439.

6. Walsh, T.R., L. Hall, S.J. Assinder, W.W. Nichols, S.J. Cartwright, A.P. MacGowan and P.M. Bennett, Sequence analysis of the L1 metallo-β-lactamase from Xanthomonas maltophilia. Biochim. Biophys. Acta, 1994. 1218: p. 199-201.

7. Rossolini, G., N. Franceschini, M. Riccio, P. Mercuri, M. Perilli, M. Galleni and J. Frere, Characterization and sequence of the Chryseobacterium(Flavobacterium) meningosepticum carbapenemase: a new molecular class B β-lactamases showing a broad substrate profile. Biochem. J., 1998. 332: p. 145-152.

8. Wommer, S., S. Rival, U. Heinz, M. Galleni, J.-M. Frere, N. Franceschini, G. Amicosante, B. Rasmussen, R. Bauer and H.-W. Adolph, Substrate-activated Zinc Binding of Metallo-β-lactamases. J. Bio. Chem., 2002. 277: p. 24142-24147.

9. Crowder, M.W., T.R. Walsh, L. Banovic, M. Pettit and J. Spencer, Overexpression, Purification, and Characterization of the Cloned Metallo-β-Lactamase L1 form Stenotropomonas maltophilia. Antimicrob. Agents Chemother., 1998. 42(4): p. 921-926.

10. Valladares, M.H., M. Galleni, J.-M. Frere, A. Felici, M. Perilli, N. Franceschini, G.M. Rossolini, A. Oratore and G. Amicosante, Overproduction and Purification of the Aeromonas hydrophylia CphA Metallo-β-Lactamase Expressed in Escherichia coli. Microbial Drug Resistance, 1996. 2(2): p. 253-256.

11. Walsh, T.R., S. Gamblin, D.C. Emery, A.P. MacGowan and P.M. Bennett, Enzyme kinetics and biochemical analysis of ImiS, the metallo-β-lactamase from Aeromonas sobria 163a. J. Antimicrob. Chemother., 1996. 37: p. 423-431.

12. Sambrook, J., E.F. Fritsch and T. Maniatis, Molecular Cloning - A Laboratory Manual. Second ed. Vol. 1. 1989: Cold Springs Harbor Laboratory Press.

13. Crowder, M.W., Z. Wang, S.L. Franklin, E.P. Zovinka and S.J. Benkovic, Characterization of the Metal-Binding Sites of the β-Lactamase from Bacteroides fragilis. Biochemistry, 1996. 35(37): p. 12126-12132.

14. Iaconis, J.P. and C.C. Sanders, Purification and Characterization of Inducible β- lactamases in Aeromonas spp. Antimicrob. Agents Chemother., 1990. 34(1): p. 44-51.

15. Lobley, A. and B.A. Wallace, DICHROWEB: A Website for the analysis of protein secondary structure from circular dichroism spectra. Biophys. J., 2001. 80: p. 373a.

16. Lobley, A., L. Whitmore and B.A. Wallace, DICHROWEB: an interactive website for the analysis of protein secondary structure from circular dichroism spectra. Bioinformatics, 2002. 18: p. 211-212.

17. Compton, L.A. and W.C.J. Johnson, Analysis of protein circular dichroism spectra for secondary structure using a simple matrix multiplication. Anal. Biochem., 1986. 155: p. 155-167.

18. Manaualan, P. and W.C.J. Johnson, Variable selection method improves the prediction of protein secondary structure from circular dichroism. Anal. Biochem., 1987. 167: p. 76- 85.

19. Sreerama, N. and R.W. Woody, Estimation of protein secondary structure from CD spectra: Comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal. Biochem., 2000. 287: p. 252-260.

20. Sreerama, N., S.Y. Venyaminov and R.W. Woody, Estimation of protein secondary structure from CD spectra: Inclusion of denatured proteins with native protein in the analysis. Anal. Biochem., 2000. 287: p. 243-251.

21. Pace, C., F. Vajdos, L. Fee, G. Grimsley and T. Gray, How to measure and predict the molar absorption coefficient of a protein. Protein Science, 1995. 4(11): p. 2411-2423.

22. Smith, P., R. Krohn, G. Hermanson, A. Mallia, F. Gartner, M. Provenzane, E. Fujimoto, N. Goeke, B. Olson and D. Klenk, Measurement of Protein using Bicinchoninic Acid. Anal. Biochem., 1985. 150: p. 76-85.

23. Valladares, M.H., A. Felici, G. Weber, H.W. Adolph, M. Zeppezauer, G.M. Rossolini, G. Amicosante, J.-M. Frere and M. Galleni, Zn(II) Dependence of the Aeromonas

hydrophila AE036 Metallo-β-lactamase Activity and Stability. Biochemistry, 1997. 36: p. 11534-11541.

24. Valladares, M.H., M. Kiefer, U. Heinz, R.P. Soto, W. Meyer-Klaucke, H.F. Nolting, M. Zeppezauer, M. Galleni, J.-M. Frere, G.M. Rossolini and H.-W. Adolph, Kinetic and spectroscopic characterization of native and metal-substituted β-lactamase from Aeromonas hydrophila AE036. FEBS Lett., 2000. 467: p. 221-225.

25. Segatore, B., O. Massidda, G. Satta, D. Setacci and G. Amicosante, High specificity of cphA-encoded metallo-β-lactamase from Aeromonas hydrophila AE036 for carbapenems and its contribution to β-lactam resistance. Antimicrob. Agents Chemother., 1993. 37: p. 1324-1328.

26. Crowder, M.W. and T.R. Walsh, Structure and function of metallo-β-lactamases. Recent Research Developments in Antimicrobial Agents and Chemotherapy, 1999. 3: p. 105- 132.

27. Rossolini, G.M., A. Zanchi, A. Chiesurin, G. Amicosante, G. Satta and P. Guglielmetti, Distribution of cphA or related carbapenemase-encoding genes and production of carbapenemase activity in members of the genus Aeromonas. Antimicrob. Agents Chemother., 1995. 39(2): p. 346-349.

28. Rossolini, G.M., T. Walsh and G. Amincosante, The Aeromonas Metallo-β-Lactamases: Genetics, Enzymology, and Contribution to Drug Resistance. Microbial Drug Resistance, 1996. 2(2): p. 245-252.

29. Miller, R.V., Bacterial Gene Swapping in Nature. Scientific American, 1998: p. 66-71.

Chapter 3

Spectroscopic Characterization of Recombinant ImiS

3.1 Introduction

3.1.1 Zn(II)-Containing Metalloproteins

Zinc is a metal essential for growth and development in all forms of life [1]; it is the only metal that is required by enzymes in all six International Union of Biochemistry classes [2]. In biological samples, zinc always exists as a divalent cation (Zn(II)). In proteins Zn(II) can be utilized in one of four sites: (1) a structural site, such as in a zinc finger, where the Zn(II) is required to give the protein proper conformation for its functionality; (2) a protein interface site, such as in superantigens, where the Zn(II) has an influence on the quaternary structure of proteins; (3) a catalytic site, such as in alcohol dehydrogenases, where a Zn(II) is coordinated at the active site and participates in the catalytic function of the enzyme; and (4) a cocatalytic site, such as in superoxide dismutase, where two or more metals (not necessarily all Zn(II)) are in close proximity, are required for full activity, and at least one of the metals participates in the enzyme’s catalytic activity [2]. The metallo-β-lactamases appear to contain one of the latter two zinc sites.

Enzyme function is intimately related to the structure of the active site. In a metalloenzyme, where the metal cofactor is required for activity and is not simply a structural moiety, the identity of the protein ligands, their spacing and secondary interactions with supporting or “orienter” [3] amino acids, the presence of any bound solvent molecules, and the specific microenvironments created by protein folding determine the various mechanisms

through which the metal atom can be involved with the function of the enzyme [2, 3]. The previously mentioned four zinc-containing sites are all defined by characteristic protein ligands and geometries.

Zinc coordinated in structural and protein interface sites do not actively participate in the catalytic function of the enzyme. They are characterized by having four amino acid ligands, most often Cys for structural sites, and no bound solvent molecules. Catalytic sites usually coordinate Zn(II) with a coordination number of 4 or 5 in distorted tetrahedral or trigonal- bipyramidal geometries. In catalytic sites, the zinc complexes with three N/O/S donor ligands and a solvent molecule [2]. The most common amino acid ligands are His, Asp, Glu, and Cys, with His being the predominant amino acid ligand because it can disperse charge through hydrogen bonding to the non-ligating nitrogen. In addition to the amino acid ligands, a solvent molecule, usually water, is always a ligand to Zn(II) in catalytic Zn(II) sites. In a cocatalytic site, there are two or three metals (not necessarily all zinc) in close proximity, usually 3-6 Å between neighboring metals, which are ligated the same as the catalytic site zinc ions, sometimes utilizing amino acids rarely used in catalytic sites such as Asn, Gln, Ser, Thr, Tyr, and Lys. The cocatalytic sites also contain a bridging ligand; the bridging ligand can be an amino acid side chain, such as Asp, His, Glu, or Lys, or a solvent molecule, which determines the relative proximity of neighboring metals [2].

The coordination of the zinc metal ion not only determines the type of site in which

Zn(II) resides, but is also gives clues to the possible mechanism through which the enzyme proceeds. In catalytic and cocatalytic sites, a zinc bound water can be activated for ionization, polarization, or displacement by the identity and arrangement of the ligands that coordinate the

zinc [4]. An understanding of the scaffolding of a zinc binding site is important to the understanding of the function and reactivity of the Zn(II) generally and the enzyme specifically.

3.1.2 Structural Characterization of the Metallo-β-Lactamases

While the first reported metallo-β-lactamase was βLII from B. cereus in 1966 [5], it was not until 1996 that a three-dimensional (3D) structure for a metallo-β-lactamase containing two zinc ions, CcrA from B. fragilis, was reported [6]. With all of the structural information available for metallo-β-lactamases, they still are the only Zn(II) metalloenzymes that do not readily fit into either the catalytic or cocatalytic Zn(II) site families. The metallo-β-lactamases do not contain a bridging amino acid ligand; rather they have only a bridging water/hydroxide.

The second zinc is not universally important to activity. All known metallo-β-lactamases maintain some activity with one Zn(II) and some, specifically Bush group 3b β-lactamases, are inhibited by more than one equivalent of Zn(II). Even though it “takes two to tango” [7], metallo-β-lactamases are dependent upon one zinc and have the characteristics of a catalytic

Zn(II) site family member [2].

The first step in the structural elucidation of a new enzyme is comparison of its amino acid sequence with those from enzymes of known structural features. Usually, but not always, enzymes with similar amino acids sequences, in similar spatial arrangements, can be used as a guide to determine potential metal binding ligands even if the enzymes have different catalytic functions (such as metallo-β-lactamases and glyoxalase 2). This amino acid sequence comparison can also be performed for enzymes with similar functions to determine where differences may potentially exist in their structures and therefore their functions. Comparing the amino acid sequences of the metallo-β-lactamases to other enzymes with similar structure

(Chapter 1) uncovered common structural characteristics that define the zinc metallo-hydrolase family of enzymes [8], and comparisons to other metallo-β-lactamases (Chapter 2) indicated the similar structural characteristics and divergences of metallo-β-lactamases specifically [9, 10].

The coordination sphere for the two Zn(II)’s, in the majority of the crystallographically characterized metallo-β-lactamases, consists of four His, a Cys, an Asp, and at least two water/hydroxide molecules. The exception to this coordination sphere, as has been previously stated, is L1, which has an additional His in place of the Cys. The amino acid sequences of the

Bush group 3b β-lactamases reveal an Asn instead of a His in the consensus HxHxD motif, suggesting a different coordination sphere for Zn(II). These are also the two enzymes that differ the most from the general characteristics of a metallo-β-lactamase; active L1 forms a tetramer [9,

10] and the Bush group 3b β-lactamases are inhibited by more than one equivalent of zinc [11,

12].

Early 1H-NMR studies revealed the involvement of three His in the metal binding site of a Co(II)-substituted metallo-β-lactamase, with only one metal-binding site [13]. It was believed that the fourth ligand was a solvent molecule, a requirement for a catalytic site [2]. However, this model of the metal binding site conflicted with earlier UV-Vis spectra, which revealed a distinctive Cys→Co(II) LMCT (ligand-to-metal charge transfer) band and less intense features that could be assigned to ligand field transitions of a tetrahedral, high-spin Co(II) center [14]. In

1995, the first 3D structure of the same metallo-β-lactamase was determined by X-ray crystallography; however, the crystallized protein only contained one bound Zn(II) with a tetrahedral coordination sphere of three His and a solvent molecule [15]. The crystallographic data, like the previous NMR research, indicated that metallo-β-lactamase had no Cys ligand, which was clearly observable in the UV-Vis spectra; this difference in coordination sphere was

attributed to the differences between the Co(II)-β-lactamase and Zn(II)-β-lactamase [7]. It was also postulated that the arginine at position 121, which is positioned next to three of the consensus metal-binding ligands, could provide enough repulsion, destabilizing the coordination of the second Zn(II) and preventing the second equivalent of Zn(II) from binding in these early studies.

Currently, the crystal structures of four metallo-β-lactamases have been reported: CcrA from B. fragilis [6, 16], L1 from S. maltophilia [17], βLII from B. cereus [15, 18], and IMP-1 from Pseudomonas aeruginosa [19]. These crystal structures give the basic coordination sphere of the first zinc (Zn1), which binds significantly more tightly than the second equivalent of zinc, as distorted tetrahedral: three histidines, His116, His118, His196, and a solvent molecule, probably a bridging solvent molecule (a µ-aquo or µ-hydroxo) (Figure 3-1) [7, 10]. The second

Zn(II) is coordinated in a trigonal-bipyramidal geometry by the amino acids Asp120, Cys221, and His263, the bridging solvent molecule, and an additional solvent molecule (Figure 3-1) [7,

10]. The accepted 3D structure for metallo-β-lactamases containing two equivalents of Zn(II), as defined by the crystal structures, has been reaffirmed by data from other spectroscopic techniques applied to metallo-β-lactamases containing two equivalencts of Co(II), including, but not limited to, proton nuclear magnetic resonance (1H-NMR) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, extended X-ray absorption fine structure (EXAFS) spectroscopy, and electronic (UV-Vis) spectroscopy.

3.1.3 Structural Characterization of Bush Group 3b β-Lactamases

Recently, spectroscopic studies of CphA were published that gave insight into the possible metal coordination sphere [11]. UV-Vis studies on Co(II)-substituted CphA reveal a

His116

His118 Zn1 His196

µ-hydroxo- or µ-aquo- Cys221

Terminal Solvent Molecule (H2O) Asp120 Zn2

His263

Figure 3-1: Structural picture of the active site of CcrA showing the two Zn(II) ions (maroon spheres), the two solvent water/hydroxide molecules (blue spheres), and the metal binding amino acids.

strong Cys→Co(II) LMCT, and ligand field transitions were consistent with a distorted tetrahedral Co(II) metal center. EXAFS studies on Zn(II)-CphA suggested a Zn(II) binding site made up of 2 His, 1 sulfur (presumably from a Cys), and 1 N/O atom thought to be a solvent OH-

. While this coordination sphere maintains the solvent ligand required for catalytic activity, it can only be achieved through a hybridization of the Zn1 and Zn2 metal binding sites.

Currently, there is no crystal structure published on a Bush group 3b β-lactamase. ImiS was first crystallized in 1997; however, the crystals that were obtained were of insufficient quality to obtain usable diffraction patterns (Dr. James Spencer, University of Bristol, personal communication).

3.1.4 Co(II)-Substitution

3.1.4.1 Spectroscopically Silent Zn(II)

Zn(II) is a spectroscopically silent metal ion, with a diamagnetic electron configuration of

[Ar]3d10. Zn(II) has no free orbitals that would allow transitions of the appropriate energy.

Therefore, the spectroscopically-silent Zn(II) ion must be substituted with a spectroscopically- active metal ion. Co(II) substitution has repeatedly been shown to be an effective way to use spectroscopic techniques to characterize Zn(II) metal-binding sites in proteins [20]. Cobalt substitution is preferential to substitution with other metal ions because Co(II) is paramagnetic, having the [Ar]d7 electron configuration, has an ionic radius very close to Zn(II), 72 pm and 74 pm, respectively, exhibits electron relaxation times in the 10-11 to 10-12 s-1 range, and most importantly maintains all or part of the enzymatic activity, suggesting the cobalt is entering the normal metal-binding site in the proper configuration and ligation as the native Zn(II) [20, 21].

As has been previously discussed, Co(II)-substitution of metallo-β-lactamases has yielded useful,

accurate structural information from 1H-NMR and UV-Vis spectroscopy. Co(II)-substitution is also useful with other forms of spectroscopy, such as EPR, MCD, and even EXAFS (although in

EXAFS it is preferential to use the natural Zn(II)), which can all yield further structural information [21].

However, there are drawbacks to utilizing Co(II) as a spectroscopic probe such as air oxidation, the disadvantageous binding of the cobalt ion to other portions of the protein such as to disulfides, and tetrahedrally bound Co(II) being a borderline case for 1H-NMR. Co(II), with its slightly lower electronegativity, has a slightly higher Lewis acidity, which results in slightly shorter Co(II)-ligand bond lengths [20]. Co(II) does not have the same stereochemical preferences as Zn(II), resulting in slightly less stable Co(II)-complexes, which is why Zn(II) cannot be replaced by Co(II) through direct competitive substitution; the Zn(II) must be removed first [20].

3.1.4.2 Co(II)-Substitution

There are many different methods for introducing a metal ion into a protein, all of which can be grouped into one of three strategies: (1) biosynthetic incorporation, (2) displacement of the native metal, and (3) replacement of the native metal in an apoprotein [20]. Biosynthetic incorporation, while possible and even attempted for this work (vide infra) [22], has many limitations, not the least of which is the air-sensitive nature of aqueous solutions of Co(II) and its ability to readily oxidize to the exchange-inert Co(III) ion [23]. Direct displacement of the native metal ion is not an overly successful option when trying to replace Zn(II) with Co(II) for all of the reasons discussed above. The final, and most successful in this study (and therefore preferential), method is the replacement of the native metal in an apoprotein. This method

involves dialyzing the as-isolated enzyme versus buffer containing chelators. After further dialysis to remove Zn(II)-EDTA and excess EDTA, Co(II) can be added directly to the apoprotein. This strategy has a few problems associated with it: (1) as above, air oxidation of

Co(II) particularly in sites with a Cys can result in loss of useful protein and (2) apo-proteins can often be unstable, resulting in significant losses of protein.

Spectroscopic characterization of Co(II)-substituted enzymes is a powerful technique, yielding information about the nature of the metal ion and its interaction with the ligands [24].

The number of unique metal centers, coordination of the metal ion, the types of ligands, and the number of those ligands can all be gleaned from spectra of Co(II)-substituted enzymes. This information in conjunction with known structural information of similar enzymes can give a reliable picture of the metal coordination sphere [20, 21, 25, 26].

3.1.5 Summary of Chapter 3

Amino acid comparison between ImiS and CphA reveals that the two enzymes are 97% homologous, differing at only 7 amino acids [27]. Also, they both contain the N116H substitution in the consensus sequence of metal-binding ligands. Early anecdotal evidence, such as their carbapenemase activity and the inhibitory nature of a second equivalent of Zn(II), and their high homology lead to the basic question of whether or not ImiS and CphA are the same enzyme? While early evidence has been provided about the structure CphA, there are still many general questions about the structure of Bush group 3b β-lactamases and specifically ImiS.

What are the metal binding ligands of ImiS? What are the electronic properties of the metal(s) in

ImiS?

To address these questions, several spectroscopic techniques were utilitzed. UV-Vis spectroscopic studies were undertaken to determine the coordination number of the metal ion and whether a Cys is coordinated to the metal ion in Co(II)-substituted ImiS. 1H-NMR spectroscopic studies were undertaken to determine the number of His ligands bound to the metal ion in Co(II)- substituted ImiS. EPR spectroscopic studies were undertaken to explore any possible Co(II)-

Co(II) interactions in Co(II)2-ImiS and to probe the electronic properties of Co(II) in Co(II)- substituted ImiS. EXAFS spectroscopic studies were undertaken to probe the coordination of the metal ion center and possible amino acid ligands.

3.2 Material and Methods

3.2.1 Materials

Ethylenediaminetetraacetic acid (EDTA) from Fisher Scientific (Pittsburgh, PA), 1,10- phenanthroline (phenanthroline) from Aldrich Chemical (Milwaukee, WI), and 5,5’-dithiobis(2- nitrobenzoic acid) from Sigma Chemical (St. Louis, MO) were all used as chelators to render the protein apo. Co(II) solutions were prepared from spectroscopic grade CoCl2 or CoSO4 salts

(Fisher Scientific) and the appropriate buffer. Cacodylate buffers were prepared with cacodylic acid (Fisher Scientific) and distilled-deionized water. Buffers were made metal-free by adding approximately 1 g per L of chelex 100 resin (BioRad Laboratories, Hercules, CA), allowing the buffer to sit for 30 minutes, and then filtering the buffer through a 0.22 µm filter to remove the chelex resin. Deuterated buffers were prepared with D2O (Aldrich Chemical). Excess EDTA was removed from apo-ImiS samples using a Sephadex G-25 (Pharmacia Biotech, Uppsala,

Sweden) column prepared in sterile 3 cc syringes (Becton Dickinson, Franklin Lakes, NJ), and column fractions were collected in 15 mL conical tubes (Becton Dickinson). Quartz cuvettes for

UV/Vis spectroscopy were procured from Fisher Scientific. NMR tubes and EPR tubes, 4 mm outer-diameter, were purchased from Wilmad Glass (Buena, NJ). ImiS activity was evaluated using imipenem (Merck). N2 gas and liquid helium were purchased from Weiler Welding

(Richmond, IN).

3.2.2 Methods

3.2.2.1 Preparation of Apo-ImiS

A small concentrated sample of as-isolated, purified ImiS (0.5 mM to 1.0 mM) was dialyzed versus excess 15 mM cacodylate, pH 6.8, containing 10 mM EDTA, at 4 ºC (apo- buffer). The sample was dialyzed versus 2L of the apo-buffer for a minimum of 4 hours, and not more than 12 hours, and the buffer was changed three times. When excess precipitation was evident, the sample was removed from the dialysis tubing, centrifuged to remove the precipitate, placed in new metal-free dialysis tubing, and returned to a new reservoir of buffer. After dialysis versus the apo-buffer, the sample was dialyzed versus metal-free 15 mM cacodylate, pH 6.8, at 4

ºC (equilibrating buffer) to remove Zn(II)-EDTA and excess EDTA. The equilibrating buffer was changed a minimum of three times, but usually the sample was dialyzed versus 6-8 exchanges (2L) of the equilibrating buffer. After the final dialysis step the sample was run over an equilibrated (equilibrating buffer) G-25 column to remove any remaining EDTA. Sample concentration was determined using the UV/Vis spectrophotometer as described in Chapter 2 and concentrated, if necessary. The sample was analyzed for metal content by using an ICP-AES, as previously described. When ImiS had a Zn(II) concentration less than 10% of the protein concentration the sample was considered apo. Apo-ImiS samples could be stored at 4 ºC, for less than 24 hours without protein precipitation.

3.2.2.2 Preparation of Co(II)-Substituted ImiS

The samples of apo-ImiS were kept cold, either in the 4 ˚C cold room or on ice in the glove box, until the Co(II) was added. Co(II) addition occurred under an inert atmosphere, nitrogen (N2); either N2 gas was bubbled through the sample and all pertinent buffers or the

Co(II) was added in a glove box where the normal atmosphere was replaced with an inert N2 atmosphere and kept at a positive pressure. The appropriate volume of the Co(II) solution was added via a pasteur pipette, yielding a final Co(II):ImiS ratio of desired stoichiometry (such as

1:1 or 2:1), with vigorous mixing to ensure the homogenous distribution of Co(II) throughout the protein sample. The Co(II)/apo-ImiS mixture was allowed to incubate at room temperature

(ideally 25 ˚C) for 30 minutes to facilitate the insertion of Co(II) into the metal center of ImiS

(R. Holz, personal communication).

Upon the successful preparation of Co(II)-ImiS, a sample that appears bluish-purple in color was generated. The sample was quantitated as before, and the metal content was verified by ICP-AES. The sample was used as quickly as possible to minimize the deleterious effects of long term storage, such as sample precipitation. Co(II)-ImiS samples were stored at 4 ºC or frozen for shipping. Samples for EXAFS were made 20% in glycerol prior to shipment.

3.2.2.3 Spectroscopic Characterization of Co(II)-ImiS

3.2.2.3.1 Electronic Spectroscopy

Electronic spectra were carried out on a Hewlett Packard 5480A UV-Vis spectrophotometer with a diode array detector, using an Isotemp circulator bath to maintain the reactions at 25 °C. By using a quartz cuvette and buffer as a blank, spectra (200-700 nm) for samples that contained 0, 1, and 2 stoichiometric equivalents of Co(II) were collected.

Background spectra of apo-ImiS were used to generate difference spectra of the Co(II)- substituted samples.

3.2.2.3.2 1H-NMR Spectroscopy

Co(II)-ImiS samples for NMR spectroscopy were concentrated to greater than 1 mM. To approximately 300 µL of the concentrated Co(II)-ImiS sample, 30 µL of a 15 mM cacodylate, pH 6.8, made in 100 % D2O was added to provide a lock signal [28]. A parallel sample containing ~ 90% D2O was made by multiple dilution/concentration steps in an Amicon ultrafiltration unit equipped with YM-10 memebranes and using 15 mM cacodylate, pH 6.8 made in 100% D2O. To account for the difference in activity of a deuteron, compared to a proton, the pH of the deuterated buffer had to be adjusted according to the following equation

[29, 30]

pD = pH meter reading + 0.4 (3-1) for the buffers in ~ 100% D2O.

1H-NMR spectra were collected on a Bruker 300 MHz NMR (Nuclear Magnetic

Resonance) spectrometer. Spectra were collected at room temperature, approximately 298 K, at

300 MHz. 1H-NMR spectra were collected using a modified inversion recovery, or WEFT, pulse sequence, where a pre-saturation pulse was placed in front of an inversion recovery pulse sequence. Dr. David Tierney (University of New Mexico) supplied this pulse sequence called zgprir (D—180˚—τ—90˚—τ—AQ, where D is the presaturation pulse and the rest is the water elimination Fourier Transform (WEFT) pulse sequence) [31-34]. The value of τ was determined experimentally to minimize the 1H signal due to the solvent and to the protein resonances in the

diamagnetic region [28, 32]. Spectra were collected at room temperature, and the resonances are reported relative to the H2O or HOD proton resonance at 4.7 ppm.

3.2.2.3.3 EPR Spectroscopy

Co(II)-ImiS samples for EPR spectroscopy were prepared by pipetting Co(II)-ImiS (final concentration of a 150-300 µM) into 4 mm outer-diameter quartz EPR tubes and freezing by slow immersion into a liquid N2 bath. EPR spectra were collected on a Bruker EMX-6 X-Band

Electron Paramagnetic Resonance (EPR) Spectrometer equipped with an Oxford continuous flow cryostat and an Oxford temperature controller. The temperatures were read directly from the temperature controller, which had been calibrated with a carbon glass sensor. Typical spectra were collected at 9.47 GHz , with a modulation amplitude of 10 Gauss, a modulation frequency of 100 kHz, a sweep width of 5800 Gauss (centered at 3000 Gauss), power ranging from 1 µW to 100 mW, and at a temperature of 4.2, 5.0, 10.0, 15.0, 20.0, and 30.0 ± 0.1 K.

3.2.2.3.4 EXAFS Spectroscopy

EXAFS experiments were performed by Dr. David Tierney and Alison Costello

(University of New Mexico). ImiS samples for EXAFS were made to a concentration of 1-2 mM, so the final metal ion concentration was a minimum of 1 mM, and prepared with 20 % (v/v) glycerol. Zn(II)- and Co(II)-ImiS samples were frozen in liquid N2 for shipping to the University of New Mexico.

For spectroscopic analysis, samples were preloaded in Lucite cuvettes with 6 µm polypropylene windows and then frozen rapidly in liquid nitrogen. X-ray absorption spectra were measured at the Stanford Synchrotron Radiation Laboratory (SSRL), beamline VII-3, using

a Si(400) double crystal monochromator, and at the National Synchrotron Light Source (NSLS), beamline X9B with a Si(111) double crystal monochromator. At beamline VII-3, the monochromator was detuned 50% for harmonic rejection, while at X9B this was accomplished using a Ni focusing mirror. Fluorescence excitation spectra were measured for all samples with either a 30- (SSRL) or a 13-element (NSLS) solid-state Ge detector array. The detectors were run at a total incident count rate of <100 kHz per channel. The fluorescence count rates (Kα) were ~10 kHz per channel for 1 Zn and 1 Co imiS and ~20 kHz per channel for 2 Zn and 2 Co imiS. Samples were held at ~ 10 K in an Oxford liquid He cryostat (SSRL) or ~15 K in a

Displex cryostat (NSLS) during XAS measurements.

EXAFS spectra were measured with 10 eV steps below the edge (9459 – 9639 eV for Zn,

7509 – 7689 eV for Co), 0.5 eV steps in the edge region (9639 – 9679 eV for Zn, 7689 – 7729 eV for Co), and 0.05 Å-1 steps in the EXAFS region. Integration times varied from ~ 1 s in the pre-edge region to 13 s at k ≈ 12 Å-1 for a total integration time of approximately 45 minutes per scan. Total exposure time was approximately 9 h for the Zn samples and 4.5 h for the Co samples. X-ray energies were calibrated by reference to the absorption spectrum of the appropriate metal foil, measured at the same time as the protein spectra. The first inflection point of the foil was assigned as 9659 eV (Zn) and 7709 eV (Co).

The fluorescence and the total incident count rate scans were examined from each detector channel to confirm the absence of artifacts. Detector channels with artifacts were excluded, and the individual scans represent an average of 9 – 12 detector channels for Zn ImiS and 3 – 5 detector channels for Co ImiS. The final spectra were obtained by averaging the individual scans (12 scans for the Zn data and 5 – 6 scans for the Co data). Background subtraction of the EXAFS spectra was accomplished by fitting the data to a Gaussian equation

(centered at ca. the Kα fluorescence energy) at the pre-edge region, and fitting a 3-region spline of fourth order to the data in the EXAFS region. Data were converted from energy to k space

2 using k = 2me (E − E0 )/ h with E0 set at 9700 eV (Zn) and 7745 eV (Co). The resultant

EXAFS data were Fourier transformed over the range k = 2 – 13.8 Å-1 for Zn data and k = 1–

12.1 Å-1 for Co data. The first shell was reverse Fourier transformed [R = 0.74 – 1.93 Å (1 Zn),

1.06 – 2.35 Å (2 Zn), 0.57 – 2.31 Å (1 Co), 0.98 – 2.28 Å (2 Co)] over the same k range. The resulting EXAFS data (ca. 10 degrees of freedom) were fitted to equation (3-2) using a nonlinear least-squares algorithm.

N A (k)S (k) as s c exp( 2k 2 2 )exp( 2R / )sin[2kR (k)] χ = ∑ 2 − σ as − as λ as +φas (3-2) kRas

In Equation 3-2, Ns is the number of scatterers in a shell of atoms, As(k) is the

2 backscattering amplitude of the absorber-scatterer (as) pair, σas is the mean square deviation of

the absorber-scatterer bond length Ras, φas (k) is the phase shift experienced by the photoelectron as it encounters the electron clouds of the scattering and absorbing atoms, λ is the photoelectron mean free-path, and the sum is over all shells of scatterers contributing to the EXAFS. Sc is the scale factor, a constant that is specific to each absorber-scatterer pair. The program FEFF v. 8.20

was used to calculate ab initio amplitude and phase functions, As(k)exp(-2Ras/λ) and φas (k) [35].

Calculations were performed for Zn-N and Co-N interactions at Ras = 2.05 Å and for Zn-S and

Co-S interactions at Ras = 2.30 Å. The scale factor, Sc, was calibrated by fitting a compound of known structure. The model used for calibration was cobalt (II) trispyrazolylborate, and the optimum scale factor found was Sc = 1.023. Fits to both the Zn and Co protein data were then

2 obtained for all reasonable coordination numbers by using Sc = 1.023 and varying Ras, σas , and

∆E0.

3.3 Results and Discussion

3.3.1 Co(II)-Substitution of ImiS

The first step in conducting spectroscopic studies on ImiS was to generate large quantities of Co(II)-substituted enzyme. Two strategies were attempted to prepare Co(II)-ImiS:

(1) Co(II) was added to apo-ImiS and (2) ImiS was over-expressed in minimal media in the presence of Co(II).

3.3.1.1 Generating Apo-ImiS

As-isolated ImiS contains 0.48 equivalents of Zn(II); therefore, this metal ion must be removed before the addition of Co(II). In other proteins, including other metallo-β-lactamases, the removal of Zn(II) is accomplished by dialyzing the protein exhaustively versus buffers containing 1-10 mM chelator, such as EDTA, phenanthroline (phen), or dicarboxylic acid

(DCA), followed by dialysis versus metal free buffer not containing the chelator. Initially, as- isolated ImiS was dialyzed versus 4 X 2L of 15 mM cacodylate, pH 6.8, containing 10 mM

EDTA at 4 ºC over a 4-8 hour period. During dialysis there was visible precipitation of the protein; in fact, 25-50% of the initial protein was lost during the metal removal steps. To minimize the loss of protein, the pH of the buffer was varied between 6.0 and 7.5, 20-200 mM

NaCl was added to the buffer, and 20 mM glycerol was included in the dialysis buffers; none of these changes reduced the protein loss during metal removal. In addition, different chelators (2-

10 mM phen, 5 mM DCA, and 5mM/5mM EDTA/phen) were used to reduce the amount of ImiS lost during metal removal. DCA was used because Hunt and coworkers had reported that the time required to make apo-carbonic anhydrase using this chelator was much shorter than when using EDTA [36]. It was reasoned that the shortening of dialysis times would result in reduced

protein loss. The EDTA/phen combination was used because previous studies in the lab with

GLX2-2 revealed that this was the only chelator(s) that could be used to prevent precipitation of

GLX2-2 during metal removal steps (L. Banovic and M.W. Crowder, unpublished results).

Unfortunately, none of these changes resulted in higher levels of recoverable apo-ImiS.

Therefore, EDTA was chosen as the chelator to prepare apo-ImiS samples (see Materials and

Methods for detailed protocol), and all apo-ImiS samples used to prepare Co(II)-ImiS spectroscopic samples were prepared using EDTA as the chelator.

Apo-ImiS was shown to bind less than 0.05 equivalents of Zn(II) after the dialysis steps.

Apo-ImiS exhibited steady state kinetic constants of kcat = and KM = when using imipenem as substrate, verifying how essential Zn(II) is to the activity of this enzyme. The CD spectrum of apo-ImiS showed that it maintained structural features consistent with those of the as-isolated

ImiS, 19% α-helix, 47 % β-character and 33 % unstructured (Figure 3-2).

3.3.1.2 Addition of Co(II) to apo-ImiS

As mentioned previously, the most common way to prepare Co(II)-substituted proteins is to add

Co(II) directly to an apo-protein. Initially, a small volume (1-10 µL) of a very concentrated

Co(II) solution, 10-100X the concentration of apo-ImiS sample, was added directly to a 1 mM apo-ImiS sample, and this solution was then allowed to incubate at room temperature for 30 minutes. After incubation, the mixture was purplish-blue in color, however there was a noticeable white precipitate. Over the following 30 minutes the color of the solution faded as more precipitate appeared. It was reasoned that the addition of the very concentrated CoCl2 solution, which is acidic, resulted in a pH “hot spot” [37], an area of the solution that has a significantly lower pH. These hot spots would be expected to be present no matter how quickly

) 30 g e

d 20 (m ty

i 10 tic 0 llip E

r 190 200 210 220 230 240 -10 la u c

le -20

Mo -30 Wavelength (nm)

Figure 3-2: CD spectra of approximately 5 µM as-isolated Zn(II)-ImiS (solid curve), apo-ImiS (small dashed curve), and Co(II)-ImiS (large dashed curve).

the apo-ImiS/CoCl2 solution was mixed. The areas of low pH were expected to have a denaturing effect on ImiS and cause precipitation of the protein. To minimize hot spots, two approaches were taken: (1) adding a larger volume of a more dilute aqueous CoCl2 solution to apo-ImiS and (2) adding a buffered stock CoCl2 solution to apo-ImiS.

Co(II)-substituted ImiS samples made by adding larger volumes of more dilute CoCl2 solutions were found to be too dilute for subsequent NMR and EXAFS studies; therefore, the samples were concentrated using Centricon-10’s. Significant losses of protein (as revealed by the intensity of the blue color) resulted presumably due to Co(II)-substituted ImiS being unstable to ultrafiltration or to the air-oxidation of Co(II) to Co(III). The addition of buffered CoCl2 solutions (at pH 6.8) to apo-ImiS did result in less precipitation of protein; however, the fading of the blue color over several hours continued to be a major problem. It is important to note that the buffered CoCl2 solutions had to be adjusted to pH values less than 7.0, because insoluble

Co(OH)2 readily formed and the oxidation of Co(II) to Co(III) occurs faster at alkaline pH’s [37,

38]. It was reasoned that the fading of the blue color of the Co(II)-ImiS samples was due to the air oxidation of Co(II) to Co(III). It was thought that keeping the Co(II)-ImiS solutions at slightly acidic pH’s would stabalize the protein as well as maintain Co in the +2 oxidation state.

However, previous studies have shown that the presence of a thiolate ligand to Co(II) (vida infra) favors the formation of Co(III). The presence of Co(III) bound to ImiS was very unfavorable to the subsequent kinetic and spectroscopic studies because (1) Co(III) is substitutionally-inert, (2)

Co(III) centersmost often do not absorb visible light and cannot be used to predict coordination number, and (3) Co(III) strongly prefers octahedral coordination.

In an effort to prevent air oxidation of Co(II)-ImiS, buffered CoCl2 solutions were added to apo-ImiS in a glove box maintained with a positive N2 pressure. The resulting samples were

blue and stable for days at 4 ºC. Steady state kinetics studies revealed that Co(II)1-ImiS is

-1 catalytically active with a kcat of 127 s and a KM of 129 µM, when using imipenem as the substrate. A CD spectrum of Co(II)-ImiS shows very similar spectral features as the as-isolated

Zn(II)-ImiS, and apo-ImiS, suggesting only small conformational changes in the protein between the enzymes (Figure 3-2). The CD spectra for Co(II)-ImiS were consistent with those published for Co(II)1-CphA [12]. Additionally, they were analyzed for secondary character using the program CDSSTR, as in Chapter 2, which revealed approximately 20 % a-helix, 49 % β- character, and 31 % unstructured [39-44]

3.3.2 Electronic Spectra

Electronic spectra were collected on 100 µM Co(II)-ImiS samples, with various equivalents of Co(II) added. The resulting spectra were transformed into difference spectra by subtracting the spectrum of the apo-ImiS from that of the Co(II)-substituted ImiS and analyzed to reveal the number of and possibly types of ligands. The difference spectra for all Co(II)-ImiS

-1 -1 samples contain an intense feature at 340 nm (ε340 = 605 M cm ) as well as broad features between 500 and 650 nm, with two maxima at 550 and 600 nm (Figure 3-3). Comparing the difference spectra for Co(II)1-ImiS and Co(II)2-ImiS, it is evident that the intense feature at 340 nm is at its maximum intensity with 1 equivalent of Co(II), whereas the intensity of the broad feature increases slightly upon the addition of a second equivalent of Co(II) (Figure 3-3). The

-1 -1 broad features between 500 and 650 nm have an εmax of approximately 195 and 250 M cm at

550 and 600 nm, respectively, in the Co(II)-ImiS samples with 1 equivalent of Co(II). The

-1 -1 addition of a second equivalent of Co(II) increases the εmax only slightly, ε550 = 234 M cm and

-1 -1 ε600 = 265 M cm . Not surprisingly, an electronic transition centered around 600 nm would afford a protein with a blue-to-purple color [45].

The intense feature at 340 nm is consistent with a Cys→Co(II) ligand-to-metal charge transfer

(LMCT) as has been seen in other Co(II) proteins, including Co(II)-CphA (Figure 3-3) [11, 46].

In LMCT transitions the electron moves from the molecular orbital of mainly ligand character to an orbital mainly of metal character; they are higher intensity (LaPorte allowed) than the d-d transitions and usually lie at the blue end of the visible spectrum or even into the UV region [20,

38]. Not every amino acid ligand can give rise to a LMCT transition in the visible region of the spectrum; a Co(II) center in a protein will only yield an LMCT transition if there is a cysteine or a tyrosine as a ligand [47]. Upon the addition of a second equivalent of Co(II), there was little change to the LMCT feature at 340 nm, suggesting an initial population of just one metal binding site that contains a cysteine. When taken into consideration with the fact that one equivalent of

Zn(II) is optimal for this enzyme, and a second equivalent is inhibitory (as shown in Chapter 2), this result indicates that the first equivalent of metal ion binds significantly more tightly to the metal binding site with cysteine than to the metal binding site without cysteine. This result is consistent with previous data on Co(II)-substituted CphA [11]. Additionally, the UV/Vis data suggest the molar extinction coefficient determined previously (Chapter 2) is correction since the

LMCT feature at 340 nm is completely grown in upon the addition of one equivalent of Co(II).

Therefore, ImiS does isolate with 0.54 equivalents of Zn(II).

The broad features from 500 to 650 nm are due to d-d (ligand field) transitions for a tetrahedral, high-spin Co(II) center. The intensities of absorbance maxima in the visible region of a spectrum associated with high-spin Co(II) d-d transitions, are geometry-dependent, with tetrahedral Co(II) centers being approximately 10 times more intense than octahedral centers,

800 A

600

400

200 ) -1 cm -1 M 0 300 400 500 600 700 800 cient ( i Wavelength (nm) eff o 300 B 250 Extinction C

200

150

100

0 400 500 600 700 800 Wavelength (nm)

Figure 3-3: Electronic difference spectra of Co(II)0.5-, Co(II)1- and Co(II)2-ImiS. A: Difference spectra for Co(II)0.5- (bottom curve), Co(II)1-ImiS (middle curve), and Co(II)1-ImiS (top curve), top to bottom. B: Difference spectra for Co(II)1- (bottom curve) and Co(II)2-ImiS (top curve).

and 5-coordinate Co(II) being in between [20, 31, 38]. This geometry-dependent signal intensity can be attributed to p-d orbital mixing, which allows for the relaxation of the LaPorte selection rules [48]. Symmetry character tables indicate that the p and d orbitals have different symmetries for a 6-coordinate metal ion center with Oh symmetry. Therefore, the p and d orbitals in an Oh metal ion center cannot mix. These character tables also indicate that two p orbitals and two d orbitals in centers with D3h or C4v symmetries (5-coordinate metal ion) transform as the same representation and therefore can mix. In the case of 4-coordinate, Td metal centers have all three p orbitals with the same symmetry as three d orbitals, allowing for even more pd mixing. The extent of pd orbital mixing is directly related to the degree of relaxation of

LaPorte selection rules and therefore the ligand field transition intensities.

The extinction coefficients for high-spin, tetrahedral Co(II)-complexes are often in the

200-300 M-1cm-1 range. The absorbance features of Co(II) complexes can be assigned to

4 4 specific d-d transitions by using Tanabe-Sagano diagrams: (1) the A2→ T1(P) transition is the

4 4 feature with the highest energy, (2) the A2→ T1(F) transition is in the near IR region, and (3) the

4 4 A2→ T2 is seldom observed because it is quite low in energy, in the 1000-2000 nm region of the spectrum, and is often orbitally forbidden [38]. Given the extinction coefficients for the 550 and

4 4 600 nm features in the electronic spectra of Co(II)-ImiS, we attribute the features to A2→ T1(P)

4 4 and A2→ T1(F) transitions, respectively. Our prediction assumes a pure tetrahedral geometry about the Co(II), which is rare in metalloenzymes. If there are significant distortions in the geometry about the metal ion in the Co(II)1-ImiS, it is possible that orbital degeneracy can be removed, resulting in a Jahn-Teller distortion. This distortion could possibly lead to a shoulder on the ligand field transitions (i.e. 550 nm).

Upon the addition of the second equivalent of Co(II), there was a slight increase in the intensity of the d-d transition (Figure 3-3). The magnitude of this increase, between 15 and 40

M-1cm-1, suggests that the second equivalent of Co(II) binds either very weakly (incompletely) or in 5-coordinate or octahedral site. The increase is more significant at a lower wavelength (i.e.

550 nm) suggesting that the second Co(II) site has a coordination number greater than 4, as the relative absorption maxima for higher coordination numbers shift to lower wavelength [38].

3.3.3 1H-NMR Spectra

Paramagnetic 1H-NMR spectroscopy has been used since the 1970’s to probe the active sites of metalloproteins. The paramagnetic centers, such as those containing a Co(II), Ni(II), or

Fe(II), provide fluctuating magnetic fields that can increase the relaxation rate of protons close to unpaired electrons and can often lead to isotropically shifted resonances lines [31, 32]. The altered relaxation properties and resonance positions allow 1-10 protons near the metal center to be distinguished from the other ~ 105 protons in the protein. This technique has been used very often to identify and quantitate His residues bound to metal ions, because the solvent exchangeable N—H protons yield peaks at 30-70 ppm, and these peak disappear when the H2O in the samples is replaced with D2O [31, 32].

The most important consideration in using paramagnetic 1H-NMR spectroscopy is linewidth, which is, in our case, affected greatly by the electron relaxation time (T1e) of the unpaired electron and the protein’s rotational correlation time. Because ImiS has a molecular weight of approximately 25 kDa and exists as a monomer in solution, we did not expect significant line broadening due to the size of the protein. Often the coupling of paramagnetic centers to 1H nuclei results in very broad signal that are often too broad to observe. In fact,

relatively sharp, isotropically shifted 1H signals often are not observable unless the electron

10 -1 relaxation rate (T1e) for the unpaired electron is faster than 10 s . The unpaired electron in

10 12 -1 high-spin Co(II) normally has a T1e of 10 - 10 s ; however, tetrahedral Co(II) has T1e’s of ca.

1010 s-1, making Co(II)-ImiS a borderline case for paramagnetic 1H-NMR spectroscopic studies.

Nonetheless, we performed 1H-NMR studies on Co(II)-ImiS to address how many His’s are bound to the metal ion(s).

1H-NMR spectra were collected on ca. 1mM samples of Co(II)-ImiS at room temperature. Early NMR experiments resulted in a noticeable precipitation that would collect in the NMR tube, presumably due to the heat generated during pulsing over the significant amount of time (hours) required to obtain the data. In an effort to prevent this precipitation, 100 mM

NaCl, 20% 2-propanol, and bovine serum albumin (BSA) were all tried as stabilizing agents.

None of these stabilizing agents significantly affected the precipitation problem. Lowering the temperature of the NMR cavity was also considered, but instrumental limitations for the 300

MHz NMR spectrometer used for these experiments and the band broadening associated with lowering the temperatures prevented this option. Finally, with help from Professor Dave Tierney

(University of New Mexico) and Dr. Ian Peat (Miami University), a modified WEFT pulse sequence, described in the Materials and Methods, was used that allowed for more rapid acquisition of the data without the concomitant precipitation of the protein.

The spectrum of Co(II)1-ImiS revealed the existence of one solvent exchangeable resonance, at 63 ppm (Figure 3-4), which can be assigned to a N—H group on a histidine bound to Co(II) [21, 31, 32]. In addition, close inspection of the spectrum revealed a resonance positioned at 174 ppm, which was not solvent exchangeable. This resonance can be attributed to a β-CH2 proton on a cysteine bound to a Co(II) (data not shown) [31]. Spectra of Co(II)2-ImiS

revealed three solvent exchangeable resonances between 45 and 55 ppm that arise from N—H protons on Co(II) bound histidines (Figure 3-4), suggesting the presence of three His in Co(II)2-

ImiS. These studies indicate that Co(II) binds only 1 His and a Cys in Co(II)1-ImiS, which correspond to amino acids from the Zn2 site in the other metallo-β-lactamases. The modified Zn1 site is populated with the second equivalent of Co(II). These 1H-NMR observations, seeing a second distinct site populated upon the addition of a second equivalent of Co(II), further supports the molar extinction coefficient determined in Chapter 2.

The first paramagnetic 1H-NMR spectra that were collected on Co(II)-ImiS also showed a significant peak at 129 ppm (Figure 3-5), which was not solvent exchangeable. Control experiments revealed that this peak was due to CH2 protons on Co(II)-EDTA. The removal of

EDTA from ImiS samples could not be accomplished using exhaustive dialysis steps alone. The use of a G-25 spin column to remove adventitious Co(II)-EDTA from subsequent spectroscopic samples resulted in a much smaller peak at 129 ppm.

3.3.4 EPR Spectra

For more than 50 years electron paramagnetic resonance (EPR) spectroscopy has been used as a tool to characterize transition ion complexes, including metalloproteins [49]. EPR has been used extensively to characterize proteins, including a few metallo-β-lactamases, which contain paramagnetic metal ion centers [50-53]. This technique has been mostly applied to di-

Co(II)-proteins because of its ability to detect intercenter spin-spin interactions between the metal ions and the ability to quantitate the zero field splitting (ZFS), which is dependent upon the symmetry and strength of the ligand field, studies have been preformed on mono-Co(II)-proteins

[21, 25, 26, 54, 55]. While the zero-field splitting parameter (∆) can be determined in principle,

100 75 50 25

ppm

B *

100 75 50 25 ppm

1 Figure 3-4: H-NMR of (A) Co(II)1-ImiS and (B) Co(II)2-ImiS showing one and three Co(II)- His resonances, respectively, indicated by an asterisk (*).

R Intensity NM

160 150 140 130 120 110 100 90 80 70 60 50 ppm

Figure 3-5: Paramagnetically shifted 1H resonances corresponding to the Co(II)-EDTA complex (129 ppm) and Co(II)-His (63 ppm).

it is difficult to obtain data for a high-spin Co(II) complex over a sufficiently wide range of temperatures and the EPR-derived value of ∆ has been shown to be unreliable [56, 57]. The majority, greater than 90 %, of the metalloprotein EPR studies has been done at X-band, where a microwave frequency of 9 – 10 GHz is used to induce spectroscopic transitions [49].

EPR studies, like the previous NMR studies, require paramagnetic complexes, such as V,

Mn, Fe, Co, Ni, and Cu-complexes, which have at least one unpaired electron. In EPR spectroscopy the paramagnetic center can be characterized by its g-tensor. The g-tensor can be defined by its principle values (gx, gy, gz) and its principle axes (x, y, z), which are determined by the unique environment surrounding the metal such as the ligand coordination sphere [25]. The g-tensor value for a free electron, ge, is 2.00 with the metal center – ligand interaction causing any deviation from this value [25]. This deviation is expected to be positive (ie. to greater g- values) if the d shell of the transition metal ion is more than half filled [25]. When there is symmetry around the metal ion center it is reflected in the g-values, with two or more of the principle g-values being identical or nearly identical.

Anisotropy occurs when the electrons of the paramagnetic center interact with the electrons of the ligands when the ligand’s electron density is not completely uniform (i.e. if the ligands are not identical or coordinated in some distorted fashion) [26]. The anisotropy of the paramagnetic metal-ligand interactions and the spin-orbit coupling removes the degeneracy of the spin states of the paramagnetic centers, giving a set of low-lying orbitals [25]. Transitions involving these orbitals give rise to the characteristic EPR resonances, which are characterized by their g-values. Often EPR investigation of paramagnetic systems must be performed on

frozen samples, near liquid helium temperatures, to significantly slow down the spin-lattice relaxation time of the unpaired electron spin to observe a signal [25, 26, 47].

X-band electron paramagnetic resonance (EPR) spectra were collected on 150-300 µM

Co(II)1-ImiS and Co(II)2-ImiS samples. The low-temperature EPR spectra (5 K) of the Co(II)1-

ImiS and Co(II)2-ImiS samples are characterized by a broad signal with geff values of 5.4 and 4.2, and a sharper signal at geff of 2.01 (Figure 3-6). These spectra are very similar in g values and shape to Co(II)-substituted CcrA and β-lactamase II, which are typical of S = 3/2 high spin, mononuclear Co(II) center [52, 53, 58]. Unfortunately, interpretation of spectra in structural terms is non-trivial when the only geff values are obtained through observation [56].

Using CoCl2 as a standard the Co(II)2-ImiS sample was integrated to 1.8 mol of Co(II) per mole of enzyme, indicating there are two Co(II)-binding sites that are not magnetically coupled [53, 55]. Further supporting the suggestion that the Co(II) are occupying two distinct, high-spin binding sites is that the signal is temperature dependent, disappearing at temperatures above 30 K (Figure 3-7) [52, 54]. Currently, attempts are being made to simulate spectra for

Co(II)1-ImiS to obtain greal and rhombic distortion of the axial zero-field (E/D) values for a Ms

=│± ½> ground state transition. These simulations will allow structural information from the spectra (Dr. Brian Bennett, National Biomedical EPR Center, Biophysics Research Institute,

Medical College of Wisconsin, personal communication)

3.3.5 EXAFS Spectra

Extended X-ray absorption fine structure (EXAFS) spectra is one of the technique of choice for biological Zn(II) protein, the other being X-ray crystallography, since Zn(II) is a spectroscopically silent metal ion [59]. X-ray crystallography characterization would be ideal

0 10002000300040005000 Gauss

Figure 3-6: EPR spectrum of Co(II)2-ImiS (top curve) and Co(II)1-ImiS (bottom curve). Both spectra were collected at 5 K, with 10 mW of power, a sweep width of 5800 Gauss, modulation amplitude of 10 Gauss, microwave frequency of 9.45 GHz, and modulation frequency of 100 kHz.

0 1000 2000 3000 4000 5000 Gauss

Figure 3-7: Temperature dependence of Co(II)2-ImiS. Spectra were recorded at 4.2, 5, 10, 15, and 30 K, top-to-bottom. All spectra were collected at 10 mW of power, a sweep width of 5800 Gauss, modulation amplitude of 10 Gauss, microwave frequency of 9.45 GHz, and modulation frequency of 100 kHz..

because it provides information both on the Zn(II)-site(s) structure and the overall structure of the enzyme; resolving these spectra are only limited by the quality of the crystals.

Unfortunately, the crystal structure of Zn(II)-ImiS has not been able to be resolved (Dr. James

Spencer, personal communication). The majority of cryrallographically characterized Zn(II) proteins, of which there are nearly 400, have exhibited four-coordinate distorted-tetrahedral geometry [59].

Where crystallography provides both metal ion binding information and tertiary structural information, EXAFS only provides information about the metal ion binding ligands. Only scatterers within 4 Å of the Zn(II) are typically detectable, with little 3-dimensional information available. The ability to study non-crystalline samples is the distinct advantage of EXAFS [59].

Unfortunately, the uncertainties and assumptions involved in Zn(II) EXAFS data analysis often lead to several valid solutions that are consistent with the EXAFS data; additionally, it has been shown to be “surprisingly easy” to obtain misleading information about a Zn(II) site [59]. Many studies have been limited to confirming a previously described structure, rather than determining a new structure for a previously uncharacterized system. Furthermore, EXAFS is limited to identifying the number of ligands to ± 1, and the atomic number of the ligands to ± 10.

Therefore, EXAFS is incapable of distinguishing between O and N ligands, and there are several examples of misidentification of a S ligand as a N or an O, and vice versa [59].

Previously, CphA had been characterized by EXAFS [11, 60]. Data analysis revealed a unique metal-binding site of one Cys, two His and an O/N atom. This active site would be unique to metallo-β-lactamases, requiring the combination of the Zn1 and Zn2 metal-binding sites. Visual analysis of the published EXAFS spectra, and their fits, revealed areas of dissimilarity between the data and the fits.

Figure 3-8 displays the Fourier transforms (FT) of the EXAFS data for Zn(II)1- and

Zn(II)2-ImiS, and Table 3-1 lists the curve fitting results as established by Dr. David Tierney and

Alison Costello (University of New Mexico). Fitting of the EXAFS data for ImiS has been very difficult (Dr. David Tierney, personal communication). Visual comparison of the EXAFS spectrum for Zn(II)-ImiS and those published for CphA reveal spectra which are not overly similar.

Two individually prepared samples of Zn(II)-ImiS were measured. Initially, data was fit with a single shell of nitrogens. However, inclusion of a sulfur leads to a four-fold improvement in the fit, justifying the inclusion of sulfur in the metal ion binding shell. This result is strong evidence that the first Zn(II) preferentially binds to the cysteine-containing side of the active site.

However, the optimum number of nitrogens/oxygens is lower than anticipated, most likely due to inaccurate calibration (a Co(II)-N interaction was used to calibrate the Zn(II)-N scale factor, above). A more realistic total coordination number is expected when better model data becomes available. The analysis that gives the optimum fit gives a Zn(II) center with a coordination number of 3. This is not a realistic coordination number, and when taken the error involved (CN ± 1), the inaccuracy of the calibration, and the uncertainties and assumptions involved in the data analysis, this Zn(II)1-ImiS could likely have the expected coordination number of 4.

Further supporting the expected coordination number of 4 with an “unseen” ligand, is a recent crystal structure of a mutant form of another metallo-β-lactamase. This mutant form of the protein is catalytically active binding just one equivalent of Zn(II) (James Garrity, personal communication). The crystal structure for this protein reveals the expected ligands in their appropriate location and no electron density in the location where the solvent molecule should be

bound (Dr. James Spencer, personal communication). It has been hypothesized that the point mutation removes the hydrogen bond that properly orients the solvent molecule. Without the hydrogen bond the solvent molecule is allowed to randomly orient around the Zn(II) spreading out its electron density over a large area and eliminating its presence in the X-ray diffraction spectra.

For Zn(II)2-ImiS, there is also a significant improvement in the fit when comparing the single shell nitrogen fit to the fit with two shells of 3 N and 0.5 S. Again, this validates the inclusion of the sulfur in the fit. For both Zn(II)1- and Zn(II)2-ImiS, the peaks in the range 2.5 –

3.5 Å are well-known multiple scattering peaks from the histidine residues [59, 61]. However, comparison of the Fourier transforms of Zn(II)1- and Zn(II)2-ImiS shows no obvious metal-metal scattering peak for the Zn(II)2-ImiS. Fits using multiple scattering techniques will be performed in the near future to help sort out imidazole multiple scattering from metal-metal scattering.

EXAFS data has also been collected for Co(II)1- and Co(II)2-ImiS samples but not yet interpreted. It should be noted that the Co(II) data is of significantly lower quality than the

Zn(II) data. Further data analysis, multiple scattering analysis, as well as collecting data from new Zn(II)- Co(II)-ImiS samples are in the process of being accomplished. However, Dr.

Tierney does not have more beam time until November.

Even, with the errors involved in the EXAFS experiments, the data strongly corroborates the previous spectroscopic experiments on Co(II)-ImiS. The EXAFS suggests that in ImiS the

Zn(II) binds to the site with the Cys, the Zn2 site, preferentially. The EXAFS data when taken in conjunction with the NMR data, which shows that the Co(II) preferentially binds to the site with one His, the Zn2 site, first, validates the hypothesis that the information gathered on Co(II)-ImiS is able to be extrapolated to the Zn(II)-ImiS, which is the active form of the enzyme in the cell.

B FT Magnitude

R + α(Å)

Figure 3-8: Fourier transform of Zn(II)1-ImiS (A) and Zn (II)2-ImiS (B).

R σ 2 as as d e CNb (Å) (Å2 x 10-3)c ∆E0 F 1 Zn ImiS January 2002 SSRL 2 N 2.01 2.56 -10.5 14.7 1 S 2.29 2.56 -10.5 (76.9) March 2003 NSLS 2 N 2.00 4.80 -14.1 15.9 1 S 2.27 3.16 -14.1 (66.9) 2 Zn ImiS March 2003 NSLS 3 N 2.03 5.77 -9.6 16.3 0.5 S 2.29 2.21 -9.6 (49.6)

Table 3-1: EXAFS data for Zn(II)-ImiS. aThe fits shown are for filtered first shell data. Fits to the unfiltered data gave similar results. The experimental b precision in Ras is 0.005 Å. However, the estimated accuracy of the bond length is ±0.02 Å. Coordination number c d giving the best fit. Mean-square deviation in absorber-scatterer bond length. ∆E0 values for N and S were linked, e 3 3 forced to be equal at all times. Goodness of fit (F) defined as k χobs – k χcalc. Fits in parentheses are the all nitrogen, single shell fits

3.4 Conclusions

To date the only structural information on a Bush group 3b β-lactamase is EXAFS and

UV/Vis data of CphA, which paint a fairly unique picture of the metal-binding site. ImiS, another Bush group 3b β-lactamase, is very similar CphA, but unique in many ways. CphA and

ImiS are highly homologous at the amino acid level, 97%, active with one equivalence of Zn(II), inhibited by greater than one equivalence of Zn(II), and they are carbapenemases. However, as shown in chapter 2, they have different steady-state kinetics. Since ImiS is a unique metallo-β- lactamase and from the same family as CphA, CphA should be a good guide/template for the structure of ImiS. Spectroscopic characterization of ImiS could serve to clarify the cloudy picture of the metal-binding structure, allowing comparisons to be drawn between ImiS and other group 3b β-lactamases, as well as between all of the metallo-β-lactamases. An understanding of the metal-binding structure is essential in the process of rational drug design.

Spectroscopic studies of the Co(II)-substituted ImiS yielded many insights into the identity of the metal-binding ligands and the nature in which they coordinate the metal ion. The purplish-blue protein has a UV/Vis spectrum consistent with a tetrahedral or distorted tetrahedral

Co(II) complex. Further analysis of the spectroscopic data reveals that the first Co(II) coordinates to a site separate and unique from the second equivalent of Co(II). The ligands for the first Co(II) site have been shown to be a single His, a Cys, and a solvent molecule (required for the catalytic site). It is hypothesized that the solvent molecule is randomly oriented. The final ligand is probably an Asp, which is consistent with the amino acid ligands associated with the Zn2 site (Figure 3-10).

Analysis of the spectra for Co(II)2-ImiS reveals the presence of another Co(II) center, which is independent of the first Co(II) ligated. This second Co(II) complex is either 5- or 6-

100

coordinate, not completely occupied by metal ion or a combination of both. 1H-NMR revealed the presence of two additional His ligands upon the coordination of a second equivalent of

Co(II), which is consistent with the ligands associated with the Zn1 site. Asn, due to the His to

Asn point difference at position 116, would most likely be the third amino acid ligand. The ligation coordination sphere would most likely be completed with solvent molecules, which is consistent with what is seen for the active site of other metallo-β-lactamases with two equivalents of metal ion bound [7, 10].

To account for the unique metal binding preference in ImiS multiple factors were given full consideration. The His to Asn point difference must have significant destabilizing effect; the loss of the imidazole ligand is associated with the switching of the preferential metal binding site. This change could possibly explain the unique hydrolytic profile, carbapenem specificity, for group 3b enzymes. The importance of this point difference becomes even more significant when the presence of Arg at position 121 is taken into consideration. Arg121, which is not present in all metallo-β-lactamases but is present in ImiS, has been hypothesized to inhibit the binding of a metal ion to the Zn2 site.

The overall purpose of the “Crowder Lab” is the development of inhibitors that would function on all metallo-β-lactamases. To accomplish this, enzymes from all groups must be characterized, both structurally and functionally and similarities determined. The metallo-β- lactamases that bind two equivalences of metal ions, which are all of them except the group 3b

β-lactamases, bind the first equivalent of Zn(II) significantly more tightly to the Zn1 site than the

Zn2 site, with His being the preferential Zn(II) binding ligand. The studies in this chapter, as well as those published on CphA, shows a preferential binding of the first metal ion to the Zn2 site. This deviation from what is known about metallo-β-lactamases presents quite an obstacle

101

because the knowledge of metallo-β-lactamases, both structural and functional, is based upon studies of enzymes that have two equivalents of metal ion bound or one equivalent bound to the

Zn1 site. The models and assumptions based on the previous, non-group 3b metallo-β- lactamases can still provide the basis for those proposed for ImiS because the same basic hydrolysis is being preformed by similar enzymes, however they must be closely scrutinized and care must be taken to ensure that false conclusions are not drawn because these are not identical systems.

102

Cys221 S O O Zn2+ H O N N O Asp120 His263

Figure 3-10: Proposed metal-binding site for ImiS.

103

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Chapter 4

Mechanistic Characterization of Recombinant ImiS

4.1 Introduction

The ultimate goal of the metallo-β-lactamase project in the Crowder Lab is the design and preparation of novel inhibitors that can be given in combination with existing β-lactams as a way to treat antibiotic resistant bacterial infections. To accomplish this goal, detailed mechanistic and structural studies are being undertaken in an effort to identify a structural or mechanistic aspect that can be targeted for inhibition. Inhibitor/drug combinations have been used successfully to treat antibiotic resistant bacterial infections. For example, Augmentin is a mixture of amoxycillin and clavulanic acid, which inhibits serine-β-lactamases [1].

Metallo-β-lactamases, from groups 3a and 3c, have been studied mechanistically, and minimal kinetic mechanisms have been proposed [2-4]. These proposed mechanisms can be divided into one of two categories: (1) a branched mechanism for metallo-β-lactamases with one

Zn(II) ion bound and (2) a linear mechanism with an intermediate for metallo-β-lactamases with two Zn(II) ion bound. However, the only mechanistic information currently available for any group 3b β-lactamases is steady-state kinetic parameters for ImiS and CphA [5, 6].

This chapter describes a mechanistic study of ImiS. The pH dependence and solvent isotope studies reveal at least one rate-significant proton transfer process. Preliminary stopped- flow kinetics suggest that ImiS utilizes a branched kinetic mechanism similar to that used by β- lactamase II [2]. Previous data and results from this work are used to offer a potential reaction mechanism for ImiS.

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4.2 Materials and Methods

4.2.1 Materials

Deuterated buffers for the solvent isotope studies were prepared with D2O purchased from Aldrich Chemical (Milwaukee, WI). Buffers were adjusted to the appropriate pH with 6 M

HCl and 10 N NaOH, or DCl and NaOD for deuterated buffers. Imipenem, a gift from Merck, was used as the substrate. MTEN buffer was prepared with 50 mM MES (2-(4- morpholino)ethane sulfonic acid), 25 mM TRIS (tris(hydroxymethyl)aminomethane), 25 mM ethanolamine, and 100mM NaCl from Fisher Scientific (Pittsburgh, PA). All other buffers were prepared as previously described (Chapter 2 or Chapter 3). Non-luer-loc syringes for stopped- flow/rapid-scanning studies were purchased from Fisher Scientific. All other materials for kinetic assays, both steady-state and pre-steady-state studies, were as previously described

(Chapter 2). Co(II)-solutions for Co(II)-substitution were prepared from CoCl2 salts purchased from Fisher Scientific. Sigma Plot version 6.1 data analysis software was purchased from SPSS

Inc.

4.2.2 Methods

4.2.2.1 Steady-State Kinetic Studies

Steady-state kinetic assays were preformed as described in Chapter 2. Inhibition studies with cefoxitin were preformed as standard steady-state assays where cefoxitin was incubated for up to 30 minutes with either substrate or enzyme before analysis.

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4.2.2.2 Solvent Isotope Effect Studies

Solvent isotope studies were performed as steady-state kinetic assays as described in

Chapter 2. The only difference was that they were performed in MTEN buffer, pH/pD = 7.0, adjusting the mole fraction of D2O (0, 0.25, 0.50, 0.75, and 1.0) contained in the buffer, as described in Chapter 3. MTEN (50 mM MES, 25 mM TRIS, 25 mM ethanolamine, and 100 mM

NaCl) was prepared metal-free, as previously described (Chapter 2), adjusting the pH/pD as necessary. Enzyme and substrate were incubated in the D2O containing buffer for 30 minutes to allow full proton-deuteron equilibration. The resulting kinetic constants were plotted versus their mole fraction D2O and fitted to the Gross-Butler equation for one (vo[1 – n + (n*(kD/kH)]),

n two (vo[1 – n + (n*(kD/kH)1][1 – n + n*(kD/kH)2]), and multiple (vo[(kD/kH) ]) protons in flight during a mechanistically important step [7].

4.2.2.3 pH Dependence Studies

pH Dependence studies were performed on ImiS samples prepared as previously described (Chapter 2). Steady-state kinetic assays, over the range of pH’s (5 – 10), were performed as previously described (Chapter 2) with the exception of using the multicomponent buffer MTEN to minimize the effects of using different buffers over the wide range of pH’s required for these studies. The logs of the resulting steady-state kinetic constants were plotted versus pH. The resulting plot was fitted with the program Leonora to the equation log(k) =

+ + + log[klim/(1 + [H ]/K1 + K2/[H ])], where, [H ] is the proton concentration at a particular pH, klim is the theoretical maximum for the corresponding kinetic constant, K1 and K2 are the Ka’s group(s) being protonated/deprotonated as pH is changed over the range [8].

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4.2.2.4 Pre-steady-state Kinetic Studies

Pre-steady-state kinetic assays were preformed on samples of ImiS in 50 mM TRIS buffer, pH 7.0, with an Applied Photophysics SX.13 stopped-flow UV-Vis/fluorescence diode array spectrophotometer at 25 ºC. Assays were run according to the instrument instructions, provided by Applied Photophysics, zeroing the instrument on the buffer. Assays were run for 48 seconds to allow the substrate, imipenem, to be hydrolyzed completely. Four hundred data points were collected over the 48 second reaction period, in 0.12 second intervals at 300 nm to monitor the disappearance of imipenem. Initially, the concentration of ImiS was held at 1.4 µM, after mixing 1:1 in the reaction cell, and the final concentration of substrate was varied between

25 µM and 135 µM in the reaction cell. Additionally, studies were performed where the imipenem concentration was held constant and the concentration of ImiS was varied between

0.46 µM and 13.75 µM in the reaction cell. Data plots were fitted to a double exponential expression (f(x) = a*e(-b*x) + c*e(-d*x)) with Sigma Plot, as well as to 3 possible reaction mechanisms with the freeware program KINSIM.

4.2.2.5 Rapid-Scanning of Co(II)-ImiS

Rapid-scanning studies of Co(II)-ImiS were performed using the same instrumentation as the pre-steady-state kinetic studies described above. Rapid-scanning assays were performed on samples of Co(II)1-ImiS in 50 mM TRIS buffer, pH 7, with imipenem held at approximately 40

µM. Co(II)1-ImiS was prepared as described in Chapter 3. The spectrophotometer was blanked on buffer. Data scans were collected from 295 nm to 700nm for the first 1 second of the reaction. Again, 400 scans were collected for the time period of the reaction. Data were analyzed both as whole spectrum and single wavelength sets.

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4.3 Results and Discussion

4.3.1 Steady-State Kinetics

Metallo-β-lactamases are characterized by their broad substrate specificity, hydrolyzing members of all classes of β-lactams [4]; however, ImiS, as well as other Bush Group 3b β- lactamases, have shown a fairly narrow specificity for carbapenems (Chapter 2). This unique characteristic mirrors the unique metal coordination profile of these enzymes (Chapter 3).

Reported mechanistic studies on non-Bush Group 3b β-lactamases suggest that metallo-β- lactamases utilize at least two different reaction mechanisms, one for mononuclear Zn(II)-β- lactamases, such as β-lactamase II, and one for dinuclear Zn(II)-β-lactamases, such as CcrA and

L1 [3]. Since Bush Group 3b β-lactamases are mononuclear, with the dinuclear form of the enzyme being less active, we hypothesized that ImiS utilizes the mononuclear β-lactam hydrolysis mechanism.

In an effort to understand better the function of metallo-β-lactamases and potentially determine a method by which to inhibit them, studies have been undertaken to explore the mechanistic characteristics of Bush Group 3a and 3c β-lactamases, and several features of the reaction mechanism were uncovered/predicted (general-base, nucleophilic attack, electrophilic stabalization, etc.) [4, 9, 10]. Studies have even been undertaken to explore the effect of the key amino side-chain (R1) on the effectiveness of the β-lactam, but these studies have only been performed on hydrolysis of penicillins and cephalosporins by non-group 3b metallo-β-lactamases

[11]. While many have undertaken studies to understand the unique metal coordination of group

3b β-lactamases, little has been done to understand the mechanistic implications of this unique structure.

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Steady-state kinetic studies have previously been performed (Chapter 2) to establish basic mechanistic preferences. In these studies it was shown that ImiS experiences substrate inhibition at substrate concentrations as low as 2X KM (Figure 4-1). Additionally, cefoxitin has been shown to be a slow binding inhibitor of ImiS, at pH 7.0, which means the amount of inhibition is dependent upon how long the enzyme is incubated with the inhibitor (data not shown) [4, 12].

Incubation of the inhibitor with the substrate, and not ImiS, prior to steady-state assays lead to minimal inhibition. Currently, there are no published reports of other inhibitors for group 3b β- lactamases.

4.3.2 Solvent Isotope Effects

Solvent isotope effects and proton inventories are experiments that can reveal the number of protons involved in rate significant steps of the catalytic mechanism [7]. By comparing enzymatic activity in the presence of protons, deuterons, and proton/deuteron mixtures, solvent isotope studies can be performed as simple kinetic assays. Comparison of kinetic constants in buffers containing varying amounts of H2O and D2O can give insight into the number of protons being transferred during the rate-limiting step and potentially to the nature of the proton donor/acceptor.

Unfortunately, very little is known about the proton inventories of most metallo-β- lactamases; however, existing proton inventories have been performed on a metallo-β-lactamase in the mononuclear and dinuclear Zn(II) forms. β-lactamase II, with cephaloridine and benzylpenicillin as substrates and excess Zn(II) present, exhibited a solvent isotope effect of

H D H D kcat /kcat of 1.4-1.6 and (kcat/KM) /(kcat/KM) of 0.85-1.82 [13]. These isotope effects have been assigned to a combination of an inverse isotope effect in a non-rate-limiting step and a large

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-7 8x10

6 ) -1 e t

ra 4 rate (s 2

0 50 100 150 200 Imipenem (µM)

Figure 4-1: Michaelis-Menton plot of the velocity of reaction versus substrate (imipenem) concentration exhibiting substrate inhibition.

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isotope effect in a rate-limiting step. However, the conclusions drawn from this study are questionable because the experimental conditions used in this study resulted in mixtures of mono- and dinuclear Zn(II)-containing β-lactamase II. Additionally, a dinuclear Zn(II) metallo-

H D β-lactamase CcrA, with nitrocefin as a substrate, exhibited a solvent isotope effect of a kcat /kcat

H D of approximately 2.5 and a (kcat/KM) /(kcat/KM) of approximately 1.6, indicating that the proton transfer occurs in a kinetically important step [14]. Since these two metallo-β-lactamases exhibited different solvent isotope effects, it was unclear what behavior ImiS would exhibit.

Proton inventory studies were performed with recombinant ImiS in MTEN buffer at pH/pD = 7.0 using imipenem as the substrate. Data were collected for samples that were 0, 25,

50, 75, and 100 % in D2O buffer (mole fraction of D2O (n) = 0, 0.25, 0.50, 0.75, and 1.0, respectively). The hydrolysis of imipenem by recombinant ImiS yielded a solvent isotope effect

H D H D of 1.8 for kcat /kcat and 1.4 for (kcat/KM) /(kcat/KM) , which is consistent with a proton transfer occurring during a kinetically important step. A single proton transfer with a solvent isotope effect in the above range is most commonly observed for a proton transfer among O, N, and S atoms [3, 7]. Further analysis of the data was accomplished by fitting the kinetic constants versus the mole fraction D2O (or percent D2O) data to the Gross-Butler equation [7]. When using the Gross-Butler equation, one would expect a linear plot for one proton transfer, a quadratic curve for two proton transfers, a cubic curve for three proton transfers, and an exponential curve for infinite proton transfers. However, in practice it is only possible to distinguish between mechanisms with zero, one, two, and multiple (three through infinite) proton transfers. Unfortunately, studies with ImiS yielded plots of kcat and kcat/KM versus mole fraction of D2O that could not unambiquously determine whether there was 1, 2, or multiple protons in flight during catalysis, given the errors associated with the steady-state kinetic constants (Figure

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265

240

) t 215 ca k 190 og( l

165

140 0 0.2 0.4 0.6 0.8 1 n

4.6 4.4

) 4.2 M /K 4 t ca

k 3.8 og(

l 3.6

3.4

3.2 0 0.2 0.4 0.6 0.8 1

Figue 4-2: Proton inventory for ImiS at pH 7.0 using imipenem as a reporter substrate. The experimental data are represented with black diamonds, and there is ~ 5 % error in the steady- state kinetic constants. Theoretical Gross-Butler curves for 0, 1, 2, and multiple proton transfers (top to bottom) are shown in each plot.

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4-2). Nonetheless, the data did unequivocally demonstrate that there was at least one proton transfer during a rate significant step. By using literature precedents of proton inventory data from similar hydrolytic enzymes [3, 9, 15], it is most likely that ImiS utilizes a reaction mechanism in which there are 1 or 2 proton transfer processes during a rate-limiting step.

4.3.3 pH Dependence

pH-Dependence studies, performing steady-state kinetic studies at multiple pH’s, can yield many interesting insights into the mechanism of an enzyme due to the forced protonation or deprotonation of essential enzyme and substrate groups as the pH is varied. Most simply, pH dependence studies can reveal the number of protons, over a defined range, involved in a rate- limiting step. Additionally, pH dependence studies can be used to reveal the pKa values of groups with ionizable protons as well as determine whether the ionizable proton is associated with the free enzyme, free substrate, or the enzyme-substrate complex (ES) [3].

In typical studies, steady-state kinetic constants, kcat and KM, are determined at different pH’s, and plots of the log(kcat) and log(kcat/KM) versus the pH are generated. Any inflection points in the log(kcat) versus pH plots can be attributed to groups on the ES complex, while inflections points in the log(kcat/KM) versus pH plots can be assigned to groups on the free enzyme or free substrate [4, 16].

There have been many published pH dependence studies on metallo-β-lactamases; however, the majority of these studies have involved β-lactamase II. Studies on Zn(II)- containing β-lactamase II yielded log kcat versus pH plots with an asymmetric bell-shaped curve, when using benzylpenicillin as the substrate [3, 4]. These plots have been analyzed, and the researchers report two inflection points. The basic pKa of 9.5 was assigned to one proton

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transfer, and the acidic pKa of 5.6 was attributed to two protons being transferred. It has been suggested that one of the groups at 5.6 and the group at 9.5 are involved in inhibitor/substrate binding, and that the other group at 5.6 has to be a Zn(II)-bound water molecule or the protonation of Asp120, an amino acid suggested to be mechanistically significant in the mono-

Zn(II) enzyme mechanism [4, 17, 18]. A pKa of 5.6 for a Zn(II)-bound water is lower than expected for a free water molecule, but it is not that far removed for the pKa of a Zn(II)-bound water molecule in carboxypeptidase A (pKa = 6.2) [4, 17, 18]. These pH dependence studies were performed in the presence of excess Zn(II); however, it is still likely that the β-lactamase II existed as a mixture of mono- and dinuclear Zn(II) forms [3, 4].

pH dependence studies have also been performed on β-lactamase II, as well as CcrA and

L1, using nitrocefin as a substrate [3, 4, 14]. Interestingly, these studies yielded log kcat versus pH plots with no inflection points over the range of pH 5.25 to 10. Previously reported proton inventories for CcrA revealed the presence of a proton transfer at a kinetically significant step [3,

4]. These seemingly mutually exclusive results can be reconciled if one assumes that the proton in flight is associated with a Zn(II)-OH2-Zn(II) and that the pKa of this proton should be well below 5.25 [3, 14]. Surprisingly, these different pH dependence profiles suggest different reaction mechanisms utilized by the tested metallo-β-lactamases. The existence of different reaction mechanisms can be explained by the different properties of the enzymes (mono- or di-

Zn(II) form), as well as by any differences related to the substrate (penicillin or cephaloridine)

[3, 11].

pH Dependence studies were performed on recombinant ImiS over the pH range of 5 - 10 using a multicomponent buffer MTEN. MTEN (50 mM MES, 25 mM TRIS, 25 mM

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ethanolamine, and 100 mM NaCl) was used to minimized the effects of using different buffers over wide pH ranges; the NaCl was included to normalize the ionic strength effects in the assays.

The log(kcat) versus pH plot revealed an apparent inflection point in the acidic region of the plot and a slow decrease in activity in the basic region of the plot, with a maximum activity at a pH of 6.5 (Figure 4-3A). When the experimental data were fitted to the equation from

Leonora, a pKa of 5.6 ± 0.3 was determined. In the proposed active site for ImiS (Chapter 3), there are two groups that could have a pKa of 5.6: (1) a Zn(II)-bound water or (2) Asp120.

Since Asp120 is predicted to be bound to Zn(II), the pKa for Asp120 should be significantly lower than 4. A direct test of this idea was not possible since ImiS was not stable at pH’s under

5.0. Therefore, the observed pKa of 5.6 is assigned to the dissociation of the Zn(II)-OH2 group.

In the basic region (Figure 4-3A), there is a gradual decrease in the activity, which corresponds to a change in the log(kcat) of 0.73 over 3.5 pH units, with a third of the decrease occurring between pH of 6.5 and 7.0. By definition, this decrease in activity does not constitute an inflection point [16]. In an effort to determine the origin of this decrease in activity, the stability of imipenem was tested at all of the pH’s utilized in these studies. It was determined that there was no apparent background hydrolysis of imipenem in the absence of ImiS. pH

Dependence studies of other metallo-β-lactamases have yielded pKa’s of 9.5; however, the testing for pKa’s above 10 was not possible due to enzyme instability at pH’s greater than 10 [3].

The log(kcat/KM) versus pH plots revealed no inflection points, indicating that the protonation/deprotonation events involved in the rate-limiting step are in the ES complex and not in the free E or free S (Figure 4-3B). A plot of log(KM) versus pH, which includes all the possible pKa’s revealed only the pKa of 5.6 from the ES complex (data not shown).

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3 A 2.5 2 ) cat

k 1.5

log( 1 0.5 0 5678910 pH

1.5 B

) 1 M 0.5 /K t

ca 0 k -0.5 og( l -1 -1.5 -2

5678910 pH

Figure 4-3: The pH-dependence of kcat and kcat/KM for recombinant ImiS hydrolyzing imipenem in MTEN buffer. (A) Plot of log(kcat) versus pH. The data points represent average values from 3 runs. The solid line was determined using the program Leonora. (B) Plot of the log(kcat/KM) versus pH.

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4.3.4 Pre-steady State Kinetics

The use of stopped-flow and/or rapid-scanning spectrophotometers allows for the monitoring of enzyme reactions a few milliseconds after mixing of the enzyme and substrate and before the reactions reach steady-state. Therefore, this technique has been used extensively to detect short-lived reaction intermediates and employed to propose kinetic mechanisms for many enzymes. In an effort to study the reaction mechanism of ImiS in more detail, pre-steady state kinetic studies were undertaken. In previous pre-steady state kinetic studies on metallo-β- lactamases, nitrocefin was used as the substrate because this substrate and its corresponding hydrolyzed product absorb visible light, thereby allowing for simultaneous monitoring of substrate disappearance and product formation [3]. Due to the narrow substrate specificity profile of ImiS (this study), nitrocefin could not be used in these pre-steady state kinetic studies, and imipenem was used as the substrate. Unlike nitrocefin, hydrolyzed imipenem does not absorb in the visible region of the electromagnetic spectrum; therefore, the amount of information that could be uncovered using pre-steady state kinetics was limited. Nonetheless, these studies were expected to reveal whether ImiS utilizes a mechanism either like L1 and CcrA or like β- lactamase II.

Stopped-flow kinetic experiments with 1.4 µM ImiS and various concentrations of imipenem ranging from 25 µM to 135 µM were conducted (Figure 4-4). Higher concentrations of substrate were not used because of substrate inhibition of ImiS at high substrate concentrations, which was observed in previous steady-state kinetic studies. Lower concentrations of substrate were not used due to the low absorbance of imipenem at concentrations less than 10 µM. These stopped-flow kinetic experiments yielded biphasic kinetic traces (data points in Figure 4-4), and the same traces were observed in studies

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1.6e-4 1.4e-4 1.2e-4 (M) 1.0e-4 8.0e-5 6.0e-5 4.0e-5 Concentration 2.0e-5 0.0 0 10203040 Time (s)

Figure 4-4: Stopped-flow kinetic experiments with 1.4 µM ImiS and various concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to a double exponential equation (solid line).

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at fixed substrate concentrations and varied enzyme concentrations (data not shown). These stopped-flow traces were fitted well with a double exponential equation (Figure 4-4), and the data from these fits are given in Table 4-1. There are two common enzymatic reaction mechanisms that give rise to biphasic progress curves: (1) branched kinetic pathways and (2) pathways that allow for the buildup of a reaction intermediate whose breakdown is rate-limiting.

The kinetic mechanisms for these pathways are shown below.

Branched kinetic pathway:

k 1 k2 k3 E + S ES ES1 E + P k-1 k-2 k-3 k4 k-4 ES2

Pathway with buildup of intermediate: k 3 k1 k2 slow k4 E + S ES EI EP E + P k-1 k-2 k-3 k-4

Interestingly, previous studies have shown that both of these pathways are utilized by metallo-β-lactamases: L1 and CcrA have been reported to use the pathway with a buildup of an intermediate whose breakdown is rate-limiting, and β-lactamase II has been reported to use the branched kinetic pathway. The previous studies with L1 and CcrA did not yield biphasic substrate breakdown progress curves because the breakdown of substrate occurred so quickly as to preclude detection by stopped-flow techniques. Analysis of the progress curves in Figure 4-4 does offer some information about which of the two mechanisms may be being utilized by ImiS.

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Imipenem (µM) a b (s-1) c d (s-1) R2

25 1.138 x 10-5 6.00 1.44 x 10-5 7.99 x 10-2 0.9985

65 3.01 x 10-5 3.24 4.01 x 10-5 1.11 x 10-1 0.9996

135 7.19 x 10-5 4.51 8.61 x 10-5 9.95 x 10-2 0.9997

Table 4-1: Data of stopped-flow traces fitted to the double exponential equation with the Sigma Plot data analysis program. Double exponential equation used f(x) = a*e(-b*x) + c*e(-d*x). Plots of these data are shown in Figure 4-4.

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The amplitudes of the first phase of the biphasic plots are much higher than the concentration of the enzyme in the reactions. For example, the amplitude of the first phase of the trace with 135

µM imipenem is ca. 55 µM, which is 39 times higher than the concentration of the enzyme in this reaction. Similarly, the amplitudes of the reactions with 65 µM and 25 µM imipenem are ca.

15 and 5 times greater than the enzyme concentration, respectively. This characteristic rules out the pathway with a buildup of an intermediate whose breakdown is rate-limiting because the amplitude of progress curve for such a mechanism would be equal to the concentration of the enzyme [15].

To probe further the kinetic mechanism of ImiS and its reaction with imipenem, kinetic simulations of the data were conducted. KINSIM, freeware initially developed by Carl Frieden, is software that generates predicted progress curves based on user-inputed kinetic mechanisms, rate constants, and initial enzyme and substrate concentrations. The successful simulation of progress curves cannot unambiguously prove that an enzyme is using a specific kinetic mechanism; however, simulations can be used to rule out other kinetic mechanisms. To simulate the progress curves in Figure 4-4, three kinetic mechanisms were tested: (1) a simple Michaelis mechanism (shown below), (2) a modified branched mechanism similar to that previously reported by Waley and coworkers [2], and (3) the mechanism that has been proposed for L1 and

CcrA [4, 15].

Michaelis mechanism:

k 1 k2 k3 E + S ES EP E + P k k-1 k-2 -3

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1.6e-4

1.4e-4 [E] = 1.375 µM

1.2e-4 135 µM 1.0e-4 8 -1 -1 k1 = 10 M s -1 8.0e-5 k-1 = 15,400 s tration (M) -1 k2 = 23 s 6.0e-5 -1 k-2 = 0.0005 s k = 30,000 s-1 4.0e-5 3 Concen 65 µM 8 -1 -1 k-3 = 10 M s 2.0e-5 0.0 25 µM 0 10203040 time (sec)

Figure 4-5: Stopped-flow kinetic experiments with 1.4 µM ImiS and various 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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Since none of the kinetic constants could be measured independently, several assumptions had to be made in order to simulate the enzymatic progress curves. It was assumed that substrate and product bind to the enzyme at the diffusionally-controlled limit of 108 M-1s-1 [15]. The value of k-1 was calculated using the previously reported Km value for imipenem of 154 µM and the assumed value for k1. Secondly, it was assumed that the chemistry, or rate-limiting, step was essentially irreversible. In the first simulations, it was assumed that product binding to ImiS was

-1 very weak with a KD of >300 µM [15], and k3 was set to 30,000 s . In the simulations, k2 was

-1 varied over several orders of magnitude, and the “best” fit was achieved with k2 equal to 23 s .

As can be seen in Figure 4-5, the simulated curves did not fit well the data. In a second set of simulations with the Michaelis mechanism, k2, the chemistry step, was set to the steady-state kcat

-1 value of 233 s , and k3 was varied over several orders of magnitude. The best fits were achieved

-1 -1 with k3 equal to 500 s for the reaction with 135 µM imipenem, 260 s for the reaction with 65

µM imipenem, and 80 s-1 for the reaction with 25 µM imipenem. Since the rate constants had to be varied from reaction to reaction and the observed fits do not fit the data well (Figure 4-6), it is clear that the Michaelis mechanism is not being utilized in the reaction of ImiS with imipenem.

Despite the fact that the amplitudes of the progress curves suggested that the L1/CcrA mechanism was not being used by ImiS, kinetic simulations were used to be at the diffusionally- controlled limits, k-1 was assumed to be the same value as in the previous simulations, and the chemistry steps (k2 and k3) were assumed to be essentially irreversible. In the first simulations using this mechanism, it was assumed that product binding to enzyme was very weak, and as

-1 before, k4 was set to 30,000 s . The rate constants k2 and k3 were varied over further rule the mechanism out. As with the simulations with the Michaelis mechanism, none of the microscopic rate constants could be independently measured; therefore, a number of assumptions were made.

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1.6e-4

1.4e-4 [E] = 1.375 µM 1.2e-4 135 µM k = 500 s-1 1.0e-4 3 8 -1 -1 k1 = 10 M s -1 8.0e-5 k-1 = 15,400 s tration (M) -1 -1 k = 233 s 65 µM k = 260 s 2 6.0e-5 3 -1 k-2 = 0.0005 s k = varied 4.0e-5 -1 3 Concen 8 -1 -1 25 µM k3 = 80 s k-3 = 10 M s 2.0e-5 0.0 0 10203040 time (sec)

Figure 4-6: Stopped-flow kinetic experiments with 1.4 µM ImiS and various concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to the Michaelis-Menton mechanism with KINSIM varying k3.

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As before, product and substrate binding to enzyme were assumed to several orders of magnitude, and in most simulations, k2 was set to be at least 10 times greater than k3. The fits were terrible and were nearly identical to those in Figure 4-5 (data not shown). In the subsequent

-1 -1 simulations, k2 was set at 2,330 s , and k3 was set to the steady-state kcat value of 233 s . The value of k4 was varied over several orders of magnitude, and the best fits yielded k4 ranging from

10-50 s-1 (Figure 4-7). These simulated curves clearly fit the second phase of the ImiS progress curves, but they do not account for the fast initial phase. In addition, the values of k4 would suggest very tight binding of product to the enzyme (Ki values in the low micromolar range).

Yet, steady-state kinetic studies do not reveal any evidence for product inhibition. These lines of evidence indicate that the reaction of ImiS and imipenem does not utilize the mechanism used by

L1 and CcrA to hydrolyze nitrocefin.

The final mechanism tested was a branched mechanism (see above) that was modified from that reported by Waley and coworkers [2]. Previously, Waley and coworkers performed stopped-flow and rapid-scanning UV-Vis studies on Co(II)-substituted β-lactamase II and used penicillin G and nitrocefin as substrates [2]. These studies yielded biphasic progress curves with burst amplitudes that were much larger than the concentration of the enzyme. Waley and coworkers predicted a branched mechanism similar to that above, except that ES2 was predicted to proceed to another intermediate that then produced free enzyme and product. Since we were unable to provide evidence of other intermediates in the reaction of ImiS and imipenem and since we could not independently measure any microscopic rate constants, the inclusion of these extra steps would unnecessarily complicate the kinetic simulations. Simulations with the branched mechanism (shown above) were conducted, and as before, several assumptions were made. Substrate and product binding were assumed to be at the diffusionally-controlled limits.

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[imipenem] dependence

1.6e-4 1.4e-4 1.2e-4 1.0e-4 8.0e-5 tration (M) 6.0e-5 4.0e-5 Concen 2.0e-5 0.0 0 10203040 time (sec)

Figure 4-7: Stopped-flow kinetic experiments with 1.4 µM ImiS and various concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to the L1/CcrA, buildup of intermediate mechanism with KINSIM.

131

-1 The steady-state KM value was used to set k-1 to 15,400 s . The chemistry step (k-2) was assumed to be essentially irreversible, and k3 was set to be equal to the steady-state kcat value.

The value of k2 was set to be roughly 10 times that of k3. It should be noted that a separate product dissociation step was not included in this mechanism in an effort to more closely follow

Waley’s mechanism [2]. The values of k4 and k-4 were varied until the best fits to the data were achieved (Figure 4-8); these fits could not be achieved using only one single value of k4 as this had to be varied between 2.5 and 20 s-1. Although the requirement to vary a rate constant suggests that this mechanism is not correct/complete for ImiS, it should be noted that this mechanism does reasonably approximate the biphasic nature of the ImiS progress curves.

Additional experiments in the future will be necessary to probe for other intermediates and to independently determine microscopic rate constants.

4.3.5 Rapid-scanning Studies of Co(II)-Substituted ImiS

Low temperature pre-steady state studies on Co(II)-substituted β-lactamase II, with benzylpenicillin as substrate, showed the existence of three intermediates; the first two have spectra consistent with a 4-coordinate metal center that completely disintegrates after 8 s at 3 ºC and the final intermediate appearing to be 5-coordinate [2]. The differences between 4- and 5- coordinate high-spin Co(II) complexes can be quantitated due to the relative intensities of the d-d transitions, with 4-coordinate being up to an order of magnitude more intense [19, 20]. These data were used to support a branched pathway and a change in coordination of the metal ion during the hydrolysis of benzylpenicillin by β-lactamase II [2].

Rapid-scanning studies of Co(II)1-ImiS, using imipenem as the substrate, were performed at 25 ºC in an effort to understand more fully the reaction mechanism, especially the role of the

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1.6e-4 [E] = 1.375 µM 1.4e-4

8 -1 -1 1.2e-4 k1 = 10 M s k = 15,400 s-1 (M) -1 1.0e-4 -1 k2 = 3000 s -1 k-2 = 0.0005 s 8.0e-5 -1 k3 = 250 s 8 -1 -1 6.0e-5 k-3 = 10 M s -1 k4 = varied between 2.5 and 20 s 4.0e-5 -1 Concentration k-4 = 0.5 s 2.0e-5 0.0 0 10203040 time (sec)

Figure 4-8: Stopped-flow kinetic experiments with 1.4 µM ImiS and various concentrations of imipenem ranging from 25 µM to 135 µM were conducted and fitted to the β- lactamase II branched mechanism with KINSIM.

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e c

n a b r o

s Ab

400 450 500 550 600 650 700 Wavelength (nm)

0.14 B 0.12

nm 0.10 00 6 t 0.08 ce a

rban 0.06 o s

Ab 0.04

0.02

0.00 0.00.20.40.60.81.0 Time (s)

Figure 4-9: Rapid-scanning of Co(II)-substituted ImiS with imipenem as substrate. (A) Full spectrum absorbance showing the d-d transition bands (500 – 650 nm). (B) Absorption at 600 nm for the first 1 s of the reaction.

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metal ion and any potential change to its coordination sphere during hydrolysis. Rapid-scanning

UV/Vis studies across a wide spectrum (300-700 nm), which includes the Co(II) ligand field absorption bands (500-650 nm), were used to probe the reaction of Co(II)1-ImiS with imipenem

(Figure 4-9A). Similar to previous pre-steady state studies these rapid-scanning studies with

ImiS provided only a limited amount of information. There was no evidence for any reaction intermediates nor for any change to the coordination of the active-site bound Co(II) during the reaction (Figure 4-9B). Therefore, ImiS is does not apparently proceed through the same 5- coordinate intermediate that β-lactamase II does. It should be noted though that the studies on β- lactamase II were conducted at low temperatures [4, 13]. It is possible that the formation of

ImiS intermediates or changes in coordination number occurred too fast for detection with our stopped-flow instrument.

4.4 Conclusions: ImiS’ Mechanism of β-Lactam Hydrolysis

By using previously published results and the data generated in this dissertation, a minimal reaction mechanism for the hydrolysis of imipenem by ImiS can now be proposed. For this proposed mechanism, the branched mechanism, proposed by Waley et al. for the mononuclear Zn(II) β-lactamase II, was used as a basis [2, 3]. This mechanism was adjusted to account for the structural differences between ImiS and β-lactamase II as well as the differences in the mechanism (i.e. one proton transfer for ImiS rather than three for β-lactamase II).

The proposed mechanism of imipenem hydrolysis by Zn(II)1-ImiS is shown in Figure 4-

10. As established in Chapter 3, the Zn(II) is bound tightly to the Zn2-site, with Asp120,

Cys221, His263, and a randomly oriented, terminally bound hydroxide shown. Additionally,

Arg121 is shown in the active site. All other amino acids and waters of solvation are omitted for

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clarity. It should be noted that some of these amino acid residues may be important for substrate binding or catalysis, many of which are currently being explored (Dr. Michael W. Crowder and

Sowmya Chandrasekar, unpublished data).

In the initial step substrate enters the active site and is oriented by Arg121, forming the

ES complex. For β-lactamase II, Arg121 is believed to interact with the β-lactam ring nitrogen while occupying the position normally occupied by Zn(II)2 [3, 4, 13]. This orientation is not possible for ImiS, since the metal ion occupies the Zn2 site, but the Arg121 is predicted to interact with the substrate, polarizing the β-lactam carbonyl for nucleophilic attack.

After the substrate enters the active site, the Zn(II)-bound hydroxide is properly oriented by Asp120, forming ES1. Asp 120 is predicted to be in position to hydrogen bond with the

Zn(II)-bound hydroxide [2-4, 21]. Once the hydroxide is properly oriented there are two possible paths that could be followed. If ImiS were to follow the non-catalytic branch the hydroxide could become unoriented againg (ES2). However, if ImiS were to follow the catalytic branch the properly oriented Zn(II)-bound hydroxide would attack the β-lactam carbonyl carbon yielding a mono-anionic tetrahedral intermediate. The anionic charge on this species is stabilized by the presence of Arg121. While there is not direct evidence this intermediate exists, a similar intermediate has been seen or proposed in every metallo-β-lactamase mechanism [3, 4].

The breakdown of the tetrahedral intermediate, which can be achieved by simply pushing electrons, leads to ring opening and the generation of a nitrogen anion intermediate. Although there is no direct evidence that this intermediate forms in the reaction of ImiS and imipenem, previous studied on L1 and CcrA indicate the formation of such an intermediate [3, 15, 22]. The protonation of the nitrogen, the rate-limiting step of this proposed mechanism, results in product

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release leaving an open site for solvent binding. This proton transfer event can be associated with the pKa of 5.6 seen in the pH dependence studies.

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R' S

Cys221 CO - R P 2 H - O Cys221 OHN 2+ Zn H O O His263 NH2 Zn2+ O H N + H O 2 His263 NH2 O H N + Arg121 O 2 Asp120 Arg121 Asp120 S

H O 2 R' S R' S - CO2 R CO - R Cys221 N 2 - Cys221 N O O O Zn2+ H 2+ O Zn H His263 NH2 H N + His263 NH2 O O 2 O H N + O 2 Arg121 Arg121 Asp120 Asp120 ES

R' S R' S

- - CO R CO2 R 2 N Cys221 N Cys221 O - O O Zn2+ O Zn2+ H H His263 NH2 His263 NH2 + O H N + H2N O 2 O O Arg121 Arg121 Asp120 Asp120 ES1

R' S

- CO2 R Cys221 H N O Zn2+ O His263 NH2 H N + O O 2 Arg121 Asp120 ES2

Figure 4-10: Proposed mechanism for the hydrolysis of imipenem by ImiS.

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4.5 References

1. Swanson-Biearman, B., B.S. Dean, G. Lopez and E.P. Krenzelok, The effects of penicillin and cephalosporin ingestions in children less than six years of age. Vet Hum Toxicol, 1988. 30: p. 66-67.

2. Bicknell, R., A. Schaffer, S.G. Waley and D.S. Auld, Changes in the Coordination Geometry of the Active-Site Metal during Catalysis of Benzylpenicillin Hydrolysis by Bacillus cereus β-Lactamase II. Biochemistry, 1986. 25: p. 7208-7215.

3. Crowder, M.W. and T.R. Walsh, Structure and function of metallo-β-lactamases. Recent Research Developments in Antimicrobial Agents and Chemotherapy, 1999. 3: p. 105- 132.

4. Page, M.I. and A.P. Laws, The mechanism of catalysis and the inhibition of β- lactamases. Chem. Comm., 1998: p. 1609-1617.

5. Valladares, M.H., M. Kiefer, U. Heinz, R.P. Soto, W. Meyer-Klaucke, H.F. Nolting, M. Zeppezauer, M. Galleni, J.-M. Frere, G.M. Rossolini and H.-W. Adolph, Kinetic and spectroscopic characterization of native and metal-substituted β-lactamase from Aeromonas hydrophila AE036. FEBS Lett., 2000. 467: p. 221-225.

6. Walsh, T.R., S. Gamblin, D.C. Emery, A.P. MacGowan and P.M. Bennett, Enzyme kinetics and biochemical analysis of ImiS, the metallo-β-lactamase from Aeromonas sobria 163a. J. Antimicrob. Chemother., 1996. 37: p. 423-431.

7. Venkatasubban, K. and R.L. Schowen, The Proton Inventory Technique. CRC Chem. Rev. Biochemistry. 17(1): p. 1-44.

8. Cornish-Bowden, A., Analysis of Enzyme Kinetic Data. 1995, Oxford: Oxford University Press.

9. Yanchak, M.P., R.A. Taylor and M.W. Crowder, Mutational Analysis of Metallo-β- lactamase CcrA from Bacteroides fragilis. Biochemistry, 2000. 39: p. 11330-11339.

10. Crowder, M.W., T.R. Walsh, L. Banovic, M. Pettit and J. Spencer, Overexpression, Purification, and Characterization of the Cloned Metallo-β-Lactamase L1 form Stenotropomonas maltophilia. Antimicrob. Agents Chemother., 1998. 42(4): p. 921-926.

11. Caselli, E., R.A. Powers, L.C. Blasczcak, C.Y.E. Wu, P. Fabio and B.K. Shoichet, Energetic, structural, and antimicrobial analyses of β-lactam side chain recognition by β-lactamases. Chem. Biol., 2001. 8: p. 17-31.

12. Ustynyuk, L., B. Bennett, T. Edwards and R.C. Holz, Inhibition of the Aminopeptidase from Aeromonas proteolytica by Aliphatic Alcohols. Characterization of the Hydrophobic Substrate Recognition Site. Biochemistry, 1999. 38: p. 11433-11439.

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13. Bounaga, S., A.P. Laws, M. Galleni and M.I. Page, The mechanism of catalysis and the inhibition of the Bacillus cereus zinc-dependent β-lactamase. Biochem. J., 1998. 331: p. 703-711.

14. Wang, Z. and S.J. Benkovic, Purification, Characterization, and Kinetic Studies of a Soluble Bacteroides fragilis Metallo-β-lactamase that Provides Multiple Antibiotic Resistance. J. Biol. Chem., 1998. 273(35): p. 22402-22408.

15. McManus-Munoz, S. and M.W. Crowder, Kinetic Mechanism of Metallo-β-Lactamase L1 from Stenotrophomonas maltophila. Biochemistry, 1999. 38: p. 1547-1553.

16. Fersht, A., Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. 1999, New York: W. H. Freeman and Company.

17. Mock, W. and J.-T. Tsay, pK values for active site residues of Carboxypeptidase A. J. Biol. Chem., 1988. 263: p. 8635.

18. Mock, W. and J.-T. Tsay, A probe of the active site acidity of Carboxypeptidase A. Biochemistry, 1986. 25: p. 2920.

19. Maret, W. and B.L. Vallee, Cobalt as Probe and Label of Proteins. Meth. Enzymol., 1993. 226: p. 52-71.

20. Bertini, I. and C. Luchinat, High Spin Cobalt(II) as a Probe for the Investigation of Metalloproteins, in Advances in Inorganic Biochemistry, G.L. Eichhorn and L.G. Marzilli, Editors. 1984, Elsevier: New York. p. 71-111.

21. Wang, Z., W. Fast and S.J. Benkovic, On the Mechanism of hte Metallo-β-lactamase from Bacteroides fragilis. Biochemistry, 1999. 38: p. 10013-10023.

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Chapter 5

Conclusions: ImiS in Context

5.1 Growing Problem of Antibiotic Resistance

Antibiotic resistance is currently a major clinical and public health concern, presenting dilemmas to clinicians, and an escalating problem in nearly every infectious disease [1-3].

Antibacterial products were developed to prevent transmission of disease-causing microorganisms among those with compromised immune systems, especially for patients in hospitals [4]. Eventually these antibacterial products made it to consumers, and today there are more than 700 household products containing antibacterial agents, up from a few dozen in the mid-1990s [4]. Initially, these products were designed for use in households containing immuno compromised patients, such as cancer patients; however, these products are now making it into healthy households. Currently, it is nearly impossible to purchase hand soap that does not claim to be antibacterial. Interestingly, added health benefits have not been demonstrated in consumers who use these antibacterial household products. On the other hand, these products more likely have the negative side effects of selecting bacteria resistant to these antibacterial agents and altering a person’s microflora [4].

Currently, more penicillin is administered in one single dose than was used during WWII.

This is due primarily to the development of bacterial resistance to the antibiotics. The use of antibiotics and surface antibacterials for agricultural and other non-clinical purposes, such as in household products, has allowed these antibacterial agents to enter the environment in an uncontrolled manner [3]. Residues of these antibiotics can be found in the environment for long

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periods of time after they are administered, allowing the bacteria to develop resistance during or after antibiotic treatment [3]. The efficacy of all of the most useful and powerful antibiotics has been challenged by bacterial resistance [5]. There are already bacteria that are resistant to multiple antibiotics, and the resulting infections are difficult, if not impossible, to treat. Without addressing the proliferation of resistance genes, the inability to effectively treat bacterial infections will only become worse [3].

In the past, bacterial resistance to an antibiotic led to the development of new and varied antibiotics [6]. This was acceptable due to the “stunning success of the pharmaceutical industry”

[7]; but recently, the development of new antibiotics has been slow [8] and history shows that resistant phenotypes are quick to follow [7, 9]. Penicillin resistance emerged before the end of

WWII, only a couple of years after the introduction of penicillin into the clinical realm [7, 9].

Improvements in antibiotic use and decreasing the proliferation of resistance genes could reverse the problem of resistance [2].

There is a strong correlation between antibiotic use and the presence of antibiotic resistance [6, 10-12]. Exposure of a population to antibiotics promotes the acquired antimicrobial resistance of the pathogens in that community. This is especially evident in the increased frequency of antibiotic resistance associated with the increased use of an antibiotic in hospitals [12]. This problem is exacerbated by the increased use of antimicrobial agents in the developed world as well as in developing countries [12]. The use (misuse or overuse) of antibiotics is evident; within 200 days of birth, 70% of all newborns are exposed to at least one antibiotic [12]. Antibiotics are prescribed for many reasons including for approximately 40% of viral respiratory tract infections, for which they have little or no benefit, and for about one-third of all hospital patients, with half of those prescriptions being unnecessary [6, 12].

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Interestingly, there is more than casual evidence that altering this use of antibiotics could help impede, if not reduce, the spread of antibiotic resistance. The reduced consumption of antibiotics is often followed by the reduction of resistance to that specific drug [6, 12].

Apparently it is too biologically expensive for the bacteria to maintain resistance in the absence of antibiotics [11]. A reduction in the consumption of antibiotics does not mean that one would be unable to obtain antibiotics if he/she had a bacterial infection. Rather, the reduction in antibiotic consumption could be achieved through several methods: (1) elimination of antibiotic misuse, (2) consumption management of antibiotics, whether it be by limiting those available for use for specific infections or cycling the prominent general antibiotics, and (3) educational programs, so people no longer believe that antibiotics are cure-all drugs independent of the cause of disease [6].

The financial costs of antibiotic resistance is staggering. Livermore and Dudley have estimated that it costs $500,000,000 and takes 7-10 years to develop a new drug [8], with resistance to the new antibiotic occurring in 1-2 years after introduction into the clinic [12]. This estimate only accounts for the obvious capital costs of developing a new antibiotic, ignoring the many biological and hospital costs associated with antibiotic resistance [11]. Rather than depend solely on the development of new antibiotics, other methods to control antibiotic resistance should be considered. The development of inhibitors to the bacterial resistance mechanisms, such as to β-lactamases, would allow the continued use of current antibiotics for which resistance has already developed. Simply modifying the use of antibiotics could potentially limit the proliferation of resistance genes and possibly lead to a decline in antibiotic resistance. The pursuit of all three paths together could have a strongly positive synergistic effect on the problem of antibiotic resistance.

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5.2 ImiS Conclusions

ImiS is a Bush Group 3b β-lactamase that has been shown to have optimal activity coordinating one equivalent of Zn(II), where all other groups of metallo-β-lactamases require two Zn(II) ions for optimal activity. In this work it was shown that the metal ion preferentially binds to the Zn2 site, most likely in a distorted tetrahedral geometry. The Zn(II) ion ligated is by an aspartic acid, a histidine, a cysteine, and a solvent water molecule. In other non-3b metallo-β- lactamases, the Zn2 site is not the preferential metal-binding site; in fact, the other metallo-β- lactamases that require two Zn(II) for maximal activity tend to bind Zn(II) to the Zn1 site over

1000 times more tightly than to the Zn2 site [13]. Recombinant ImiS is purified with only about half of Zn2 sites populated, while most other metallo-β-lactamases are isolated with their full, or nearly full, complement of metal ions.

Even though ImiS utilizes a unique metal binding geometry for metallo-β-lactamases, the mechanism of its carbapenemase activity is similar to the mechanism of β-lactam hydrolysis utilized by other metallo-β-lactamases. The mechanistic similarities between members of all of the different groups of metallo-β-lactamases means that there is a possibility that one inhibitor may work across the spectrum of these enzymes. A clinically significant inhibitor, which is able to inhibit all metallo-β-lactamases, would be a significant advancement; the ability to administer existing antibiotics in combination with this inhibitor would be beneficial to society as a whole.

5.3 ImiS in Context

5.3.1 Inhibitor Design

The use of agents that inhibit metallo-β-lactamases, when used in conjunction with existing antibiotics, is one strategy for treating resistant bacterial infections. These agents have

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to inhibit the enzyme at extremely low concentrations to be clinically useful. The use of inhibitor/drug combinations has been successfully used to treat bacteria that produce a serine-β- lactamase [14]. However, all of the inhibitors for serine-β-lactamases have been shown to be ineffective inhibitors of metallo-β-lactamases.

To date, several metallo-β-lactamase inhibitors that have been designed, tested, and shown to be nonclinically-useful [15, 16]. However, the structural and mechanistic information gained from the study of these inhibitors, as well as using the structural and mechanistic information for ImiS and the other metallo-β-lactamases will be useful aids in the rational design of a clinically-useful inhibitor. Many of the previously reported inhibitors, such as thiol- containing compounds and 2-(4-morpholino)ethane sulfonic acid (MES), have been shown to be effective against some metallo-β-lactamases but unable to inhibit all metallo-β-lactamases [15,

17]. In the worst cases, inhibitors, such as cefoxitin, have been shown to be effective against one enzyme but hydrolyzed by other metallo-β-lactamases [18, 19]. Based on the data collected to date on the metallo-β-lactamases, a potentially good inhibitor for all enzymes may be a β-lactam with a methoxy group at the 3’ position and an electron donating group near the β-lactam nitrogen. Such a molecule would be slowly hydrolyzed (like cefoxitin) and be slowly protonated after ring opening. Efforts to prepare such a compound are underway (Dr. Michael W. Crowder, personal communication).

5.3.2 Regulation of β-Lactamases

β-Lactamase production is the most common methods of bacterial antibiotic resistance.

While metallo-β-lactamases make up only about 7 % of the known β-lactamases, the diversity of the members of the metallo-β-lactamase containing bacteria family are an evolving clinical threat

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[20, 21]. Currently, the efforts to overcome this threat involve the design of new antibacterial agents and/or metallo-β-lactamase inhibitors. Very little effort has been made to understand the control and regulation of β-lactamases. Some believe that metallo-β-lactamases exist as apo- or mono-Zn(II)-enzymes and only in the presence of antibiotics do they coordinate a second equivalent of Zn(II) [20]. Many metallo-β-lactamases are active as mono-Zn(II)-enzymes, with

Bush Group 3b β-lactamases being most active with in the mono-Zn(II) form, but many studies have shown the majority of the metallo-β-lactamases require two Zn(II) for full activity. Since it has been shown that there is only 1 “free” Zn(II) per every second bacterial cell, how does the cell regulate the Zn(II) loading of the metallo-β-lactamases [22]? The homeostasis of Zn(II) in a cell is not a well understood process, due mostly to the difficulty of studying Zn(II) because of its spectroscopic silence. The understanding of how Zn(II) is loaded into enzymes may reveal other possible ways to inhibit the activity of metallo-β-lactamases, which have been shown to be inactive without the Zn(II) [13]. Furthermore, little is known about the cellular regulation of metallo-β-lactamases. Limiting β-lactamase production, if the regulation controls were unique to bacteria, could allow existing antibacterial agents to be effectively administered. Inhibiting β- lactamases directly is not the only method capable of allowing existing antibiotics to be effective again, however, it is currently the most promising method.

5.3.3 Metal Requirements

Carbapenems are a relatively new class of β-lactams, and many of the carbapenemases are mono-Zn(II) β-lactamases. This information leads one to wonder how these enzymes evolved from the other metallo-β-lactamases. This question leads to an additional corollary:

146

have the metallo-β-lactamases evolved from a metalloenzyme that requires two zinc ions and are they currently evolving into mono-Zn(II)-enzymes?

The mononuclear forms of metallo-β-lactamases tend to exhibit greater substrate specificity, while the dinuclear forms of metallo-β-lactamases tend to hydrolyze a rather broad spectrum of β-lactam antibiotics [23]. This substrate specificity-metal ion relationship is true even for mononuclear and dinuclear forms of the same enzymes [23]. This zinc requirement of metallo-β-lactamases has led to the testing of many metal ion chelators as inhibitors. Removing the Zn(II) with a chelator, such as EDTA or 1,10-phenanthroline, can inactivate the enzymes; however, the selectivity of a metal chelator is rather poor and therefore their use in a clinical setting is undesirable.

An additional corollary to the above question, when this idea is considered in the context of the aforementioned Zn(II) concentrations and regulating metallo-β-lactamases, is whether metallo-β-lactamases are functional as mononuclear Zn(II) enzymes in the bacterial cell, and the dinuclear form is a purification artifact?

Metallo-β-lactamases are members of the larger zinc metallo-hydrolase family of β- lactamase fold enzymes [24]. In recent years, this family has expanded quite rapidly due to accumulation of sequence and structural data. The diverse nature of the members of this family belies the highly divergent functions of these enzymes [24]. However, these enzymes are characterized by the same folding patterns and sequence motifs. Members of this family are distributed over three domains of living organisms (Eukarya, Archaea, and Bacteria) suggesting an ancient origin and functional importance of this protein family and the β-lactamase fold.

Interestingly, not every member of this family requires Zn(II) as the metal ion, rubredoxin

147

oxygen:oxidoreductase (ROO) requires two iron ions per monomer and glyoxalase II, specifically 2-2, utilizes a mixture of ions in vivo including iron, manganese, and zinc.

The β-lactamase fold that defines this family of enzymes is an αββα fold. This fold is found in all of the members of this family thus far characterized; however, the origin of this fold is unknown. Many believe that the fold originated in from the splicing together of two proteins

[24].

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5.4 References

1. Lipsitch, M., The rise and fall of antimicrobial resistance. Trends Microbiol., 2001. 9(9): p. 438-444.

2. Levy, S., Antibiotic Resistance: Consequences of Inaction. Clinical Infectious Diseases, 2001. 33: p. S124-S129.

3. Levy, S., Factors Impacting on the Problem of Antibiotic Resistance. J. Antimicrob. Chemother., 2002. 49(1): p. 25-30.

4. Levy, S., Antibacterial Household Products: Cause for Concern. Emerging Infectious Diseases, 2001. 3: p. 512-515.

5. Lakaye, B., A. Dubus, S. Lepage, S. Groslambert and J.-M. Frere, When drug inactivation renders the target irrelevant to antibiotic resistance: a case story with β- lactams. Mol. Microbiol., 1999. 31(1): p. 89-101.

6. Monroe, S. and R. Polk, Antimicrobial use and bacterial resistances. Curr. Opin. Microbiol., 2000. 3: p. 496-501.

7. Neu, H.C., The crisis of antibiotic resistance. Science, 1992. 257: p. 1064-1073.

8. Livermore, D.M. and M.N. Dudley, Antibiotics: better use, better drugs, or both? Curr. Opin. Microbiol., 2000. 3: p. 487-488.

9. Knowles, J.R., Penicillin Resistance: The chemistry of β-lactamase inhibition. Acc. Chem. Res., 1985. 18: p. 97-104.

10. Austin, D., K. Kristinsson and R. Anderson, The relationship between the volume of antimicrobial consumption in human communities and the frequency of resistance. Proc. Natl. Acad. Sci. U. S. A., 1999. 96: p. 1152-1156.

11. Andersson, D.I. and B.R. Levin, The biological cost of antibiotic resistance. Curr. Opin. Microbiol., 1999. 2: p. 489-493.

12. Guillemot, D., Antibiotic use in humans and bacterial resistance. Curr. Opin. Microbiol., 1999. 2: p. 494-498.

13. Crowder, M.W. and T.R. Walsh, Structure and function of metallo-β-lactamases. Recent Research Developments in Antimicrobial Agents and Chemotherapy, 1999. 3: p. 105- 132.

14. Knowles, J.R., The Inhibition and Inactivation of β-Lactamase, in Enzyme Inhibitors, U. Brodbeck, Editor. 1980, Verlag Chemie: Meinheim. p. 163-167.

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15. Payne, D.J., J.H. Bateson, B.C. Gasson, T. Khushi, D. Proctor, S.C. Pearson and R. Reid, Inhibition of metallo-β-lactamases by a series of thiol ester derivatives of mercaptophenylacetic acid. FEMS Microbiol. Lett., 1997. 157: p. 171-175.

16. Yang, K.W. and M.W. Crowder, Inhibition Studies on the Metallo-β-lactamase L1 from Stenotrophomonas maltophilia. Arch. Biochem. Biophys., 1999. 368(1): p. 1-6.

17. Fitzgerald, P.M., J.K. Wu and J.H. Toney, Unanticipated Inhibition of the Metallo-β- lactamase from Bacteroides fragilis by 4-Morpholineethanesulfonic Acid (MES): A Crystallographic Study at 1.85-A Resolution. Biochemistry, 1998. 37: p. 6791-6800.

18. Felici, A., G. Amicosante, A. Oratore, R. Strom, P. Ledent, B. Joris, L. Fanuel and J.-M. Frere, An overview of the kinetic parameters of class B β-lactamases. Biochem. J., 1993. 291: p. 151-155.

19. Crowder, M.W., T.R. Walsh, L. Banovic, M. Pettit and J. Spencer, Overexpression, Purification, and Characterization of the Cloned Metallo-β-Lactamase L1 form Stenotropomonas maltophilia. Antimicrob. Agents Chemother., 1998. 42(4): p. 921-926.

20. Cricco, J.A., E.G. Orellano, R.M. Rasia, E.A. Ceccarelli and A.J. Vila, Metallo-β- lactamases: does it take two to tango? Coord. Chem. Rev., 1999. 190-192: p. 519-535.

21. Fast, W., Z. Wang and S.J. Benkovic, Familial Mutations and Zinc Stoichiometry Determine the Rate-Limiting Step of Nitrocefin Hyrdolysis by Metallo-β-Lactamase from Bacteroides fragilis. Biochemistry, 2000.

22. Outten, C.E. and T.V. O'Halloran, Femtomolar Sensitivity of Metalloregulatory Proteins Controlling Zinc Homeostasis. Science, 2001. 292: p. 2488-2492.

23. Paul-Soto, R., M. Hernandez-Valladares, M. Galleni, R. Bauer, M. Zeppezauer, J.-M. Frere and H.-W. Adolph, Mono- and binuclear Zn-β-lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions. FEBS Lett., 1998. 438: p. 137-140.

24. Daiyasu, H., K. Osaka, Y. Ishino and H. Toh, Expansion of the zinc metallo-hydrolase family of the β-lactamase fold. FEBS Lett., 2001. 503: p. 1-6.

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