MIAMI UNIVERSITY The Graduate School
Certificate for Approving the Dissertation
We hereby approve the Dissertation
of
James D. Garrity
Candidate for the Degree:
Doctor of Philosophy
______Director Dr. Michael Crowder
______Reader Dr. Gilbert Gordon
______Reader Dr. Richard Taylor
______Reader Dr. Hongcai Zhou
______Graduate School Representative Dr. Joyce Fernandes
ABSTRACT
CHARACTERIZATION OF L1, THE METALLO-β-LACTAMASE FROM Stenotrophomonas maltophilia by James D. Garrity
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 function of the metallo-β-lactamase L1 from Stenotrophomonas maltoplia, spectroscopic and mechanistic studies were performed. Mutagenesis and biochemical assays of nine L1 active site residues, predicted from computational studies to be important for tight substrate binding to the enzyme, revealed that none of the residues play a significant role in the binding of substrate to L1. Investigation of a metal-binding aspartic acid showed that, in addition to ligating a Zn(II), this residue also plays an important role in catalytic cycle of the enzyme by properly orienting a water molecule that takes part in a rate-limiting protonation event. Fluorescence studies revealed that the rate of motion of a flexible loop of amino acids that extends over the active site of L1 correlates to the formation of a non-rate-limiting intermediate in the catalytic cycle of L1. Rapid-scanning and rapid-freeze quench EPR spectrophotometry studies provided the first direct evidence that the reaction intermediate of L1 is metal bound. This dissertation offers data, which, along with previous results on L1 and other metallo-β-lactamases, can be integrated and used to guide further rational inhibitor design efforts.
CHARACTERIZARTION OF L1, THE METALLO-β-LACTAMASE FROM
Stenotrophomonas maltophilia
A DISSERTATION
Submitted to the Faculty of
Miami University in partial
fulfillment of the requirements
for the degree of
Doctor of Philosophy
Department of Chemistry and Biochemistry
by
James D. Garrity
Miami University
Oxford, Ohio
2004
Dissertation Director: Dr. Michael Crowder
TABLE OF CONTENTS
Chapter 1 Introduction 1
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 Stenotrophomonas maltophilia 18
1.7 Antibiotic Resistance in Stenotrophomonas maltophilia 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 Probing Substrate Binding to Metallo-β-Lactamase L1 34 from Stenotrophomonas maltophilia by Using Site-Directed Mutagenesis
2.1 Summary 36
2.2 Introduction 37
2.3 Materials and Methods 41
ii
2.4 Results 45
2.4.1 Wild Type L1 45
2.4.2 Ser224 Mutants 52
2.4.3 Asn233 Mutants 56
2.4.4 Tyr228 Mutants 57
2.4.5 Ile164 and Phe158 Mutants 60
2.5 Discussion 62
2.6 Conclusion 68
2.7 Acknowledgements 70
2.8 References 71
Chapter 3 Metal binding Asp120 in metallo-β-lacatamse L1 from 77 Stenotrophomonas maltophilia plays a crucial role in catalysis
3.1 Summary 79
3.2 Introduction 80
3.3 Eperimental Procedures 85
3.4 Results 90
3.5 Discussion 99
3.6 Acknowledgements 105
3.7 References 106
iii
Chapter 4 Probing the dynamics of a mobile loop above the active 112
site of L1, a metallo-β-lactamase from Stenotrophomonas
maltophilia, via site-directed mutagenesis and stopped-flow
fluorescence spectroscopy.
4.1 Summary 114
4.2 Intoduction 115
4.3 Experimental Procedures 119
4.4 Results 123
4.5 Discussion 134
4.6 Acknowledgements 141
4.7 References 142
Chapter 5 First Direct Evidence that the Reaction Intermediate of 147
Metallo-β-Lactamase L1 is Metal Bound
5.1 Summary 149
5.2 Introduction 150
5.3 Experimental Procedures 154
5.4 Results 157
5.5 Discussion 167
5.6 References 175
iv
Chaper 6 Conclusions 180
6.1 The Growing Problem of Antibiotic Resistance 180
6.2 Direct Inhibition of Metallo-β-lactamases 182
6.3 Indirect Inhibition of Metallo-β-lactamases 184
6.4 References 186
v
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. 4
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: Active site residues that were mutated in this study. 40
2-2: CD spectra of wild-type L1 and L1 mutants. 47
2-3: Intermediate formation by wild-type L1 and L1 mutants. 55
2-4 A: Stopped-flow fluorescence of wild-type L1 with nitrocefin. 59
2-4 B: Plot of observed rate constant versus concentration of nitrocefin. 59
3-1: Pictorial representation of the active site of wild-type L1 rendered 84 using Chem Draw Ultra v. 5.0 (top) and Rasmol 2.6 (bottom).
3-2: Formation of ring-opened, nitrogen anion intermediate by 94 wild-type L1 and L1 mutants.
3-3: pH dependence plots of wild-type L1 with (A) nitrocefin, 96 ∆ kcat, ◊ kcat/Km, and (B) penicillin G, Ο kcat, kcat/Km.
3-4: pH dependence plots of L1 mutants with nitrocefin. 97
3-5: Proton inventory plots of wild-type L1 and L1 mutants with nitrocefin. 98
3-6: Proposed role of Asp120 in L1. 103
4-1: Structure of L1 monomer showing the positions of key residues 118 involved in this study.
vi
4-2: Fluorescence scans of wild-type L1 and L1 mutants. 127
4-3: Fluorescence titrations of apo wild-type L1 and L1 mutants. 129
4-4: Stopped-flow fluorescence spectra of wild-type L1 and L1 mutants 130 with nitrocefin.
4-5: Stopped-flow fluorescence spectra of W39F, D160W with nitrocefin. 131
4-6: Fluorescence scans of W39F, D160W, free enzyme and 1:1 solutions 132 with nitrocefin and meropenem.
4-7: Single wavelength versus time plots of wild-type L1, W39F, and 133 W39F, D160W with nitrocefin.
5-1: Proposed metal binding site of metallo-β-lactamase L1 when bound 152 to reaction intermediate.
5-2: Rapid-scanning electronic absorption spectra of the reaction of 158 100 µM wild-type L1 with 100 µM nitrocefin at 2 oC.
5-3: Rapid-scanning electronic absorption spectra of the reaction of 158 100 µM Co(II)-substituted L1 with 100 µM nitrocefin at 2 oC.
5-4: EPR spectra and simulations of Co(II)-substituted L1 with 163 nitrocefin as the substrate.
5-5: EPR spectra and simulations of Co(II)-substituted L1 with 164 cephalothin as the substrate.
5-6: EPR spectra and simulations of Co(II)-substituted L1 with 165 meropenem as the substrate.
5-7: EPR spectra and simulations of Co(II)-substituted L1 with 166 penicillin G as the substrate.
vii
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: Oligonucleotides used in preparation of L1 mutants. 43
2-2: Metal Content of Wild-type L1 and L1 mutants. 48
2-3: Steady-state kinetic constants for hydrolysis of cephalosporins by 50 wild-type L1 and L1 mutants.
2-4: Steady-state kinetic constants for hydrolysis of penicillins by 51 wild-type L1 and L1 mutants.
2-5: Steady-state kinetic constants for hydrolysis of carbapenems 52 by wild-type L1 and L1 mutants.
2-6: Km and KS values for Wild-type L1 and mutants with nitrocefin 60 as substrate.
3-1: Metal analysis, and Ks and kH/kD values with nitrocefin of 90 wild-type L1 and L1 mutants.
3-2: Steady-state kinetics constants for wild-type L1 and L1 mutants 92 with (2a) carbapenems, (2b) cephalosporins, and (2c) penicillins.
4-1: Analysis of CD Spectra using CDSSTR with a 43 protein reference 124 set optimized for 190-240 nm.
4-2: Steady state kinetics constants for wild-type L1 and L1 mutants. 125
4-3: Summary of fit constants for stopped-flow rapid-scanning 133 and fluorescence data of enzymes with nitrocefin at 2 °C, using IgorPro v 4.0 graphing program.
viii
Acknowledgements
I would like to sincerely thank my advisor, Dr. Michael Crowder. Leading by example, he encouraged positive interaction in the lab, and promoted the sharing of knowledge and technical expertise among his students. His guidance, support, and friendship were invaluable to the completion of this effort.
I would also like to thank my committee, Drs. Gordon, Lorigan, Taylor and Fernandes, for their support of my studies at Miami University, and Dr. Hongcai Zhou for substituting on my committee for my final defense.
Additionallly, I would like to thank Gerg Patton, Lissa Herron, and Jim Pauff for their efforts in this endeavor.
Finally, I would like to dedicate this dissertation to Lisa and my parents. To Lisa, for her day-to-day unconditional love, support, and encouragement - this made the difficult times bearable, and the good times even better. And to my parents, who, by example, have taught me what is important in life; and always given me the love, encouragement, and support necessary to succeed.
ix
Chapter 1
Introduction
Prior to 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. This work however, 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). However, few physicians held
high opinion of this work, and it was soon forgotten. It was not until 1928, when Alexander
Fleming accidentally discovered the antimicrobial effects of penicillin that a change in attitude toward the treatment of disease occurred (2). The World Wars and the proliferation of death
from sepsis, as opposed to the wound itself, likely aided this change in attitude. However,
clinical use of Fleming’s discovery was hampered by the instability of penicillin and difficulties
in obtaining large quantities of the pure substance. 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
1.1 Antibiotics
The age of antibiotics began with Fleming’s discovery of penicillin. Literally meaning
“against life”, effective antibiotics inhibit microbial growth (bacteriostatic) or kill the microbe
(bacteriocidal). The majority of antibiotics are natural products that are isolated from bacteria or fungi (4). The second largest class is the semi-synthetics. These are natural products that have been altered, by addition or subtraction of substituents, to improve efficacy or lower toxicity.
Only about 10% of the antibiotics in clinical use today are completely synthetic. Most of these compounds were designed to inhibit a specific 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, including over 50 analogs of the 3rd generation cephalosporins and quinolones that 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 of antibiotics in clinical use today, greater than 50%, 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).
2
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.
3
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.
4
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 stem 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 pentapeptide stems are then
crosslinked, by the enzyme transpeptidase, providing stability and rigidity to the cell wall. 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).
The primary bacteriocidal act of a β-lactam containing antibiotic is the interference of normal cell wall biosynthesis (5,7,12), through the mechanisim-based inhibition of the enzyme transpeptidase. The β-lactam ring 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 cannot 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, which 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 (15), resistance to carbapenems seriously limits clinical options.
5
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 A l a D A l a X D G l u
L L y s X = Gly5 D A l a
Figure 1-3: Crosslinking of building blocks of peptidoglycan layer.
6
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 containing antibiotics.
7
1.3 Antibiotic Resistance
With the emergence of penicillins, later cephalosporins, and other new semi-synthetic β- lactam containing antibiotics, the battle against bacterial infection appeared won. 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; however by 1944, over half of the Staph. strains were resistant to penicillin (5). Today, greater than 95 percent of S. aureus organisms are resistant to penicillin as well as to most cephalosporins (5). In fact there is clinical resistance to all known antibiotics, regardless of their source - natural product, semi-synthetic, or completely synthetic. A frightening example of this is linezolid (ZyvoxTM), a totally synthetic antibiotic from Pharmacia Upjohn, approved by the FDA in 2000. Linezolid is an oxazolidinone, a class of antibiotics that prevents initiation of protein synthesis in the bacterium, a novel 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 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). Misuses 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). This misuse and overuse of
8
antibiotics has provided a Darwinian bacterial selection where the susceptible (weak) organisms
are killed off and the resistant (fittest) ones survive and flourish (22).
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 (23). 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 (5,19). 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).
9
1.5 β-Lactamases
β-lactamases are enzymes that hydrolyze the carbon-nitrogen bond in the β-lactam ring
of the β-lactam containing antibiotics (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 (24). 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). 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” (6,24,25). New β-lactams lead to new β-lactamases and the spreading of resistance. Resistance is further spread through horizontal gene transfer (26).
1.5.1 Serine-β-lactamases
The majority of β-lactamases produced by bacteria, over 90 %, contain a mechanistically- significant serine at the active site (8,25). 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 (8,25). Fortunately, serine-β-lactamases do not generally hydrolyze all known,
10
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.
11
clinically-used antibiotics, such as carbapenems. In addition, β-lactamase inhibitors, such as
clavulanic acid, when used in combination with current β-lactams, prove to be a powerful
weapon against most antibiotic resistant bacteria that produce a serine-β-lactamase (27).
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
(27,28). These metallo-β-lactamases are generally broad-spectrum enzymes, able to hydrolyze
β-lactam containing antibiotics from all chemical classes with the exception of the monobactams
(27). In spite of this, 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) (27). Adding to the problem of broad-spectrum antibiotic
resistance conferred by metallo-β-lactamases is the fact that there are no clinically-useful
inhibitors against them. Metallo-β-lactamases are usually plasmid-encoded, giving them the
ability to spread resistance rapidly (5,26); however, 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 motif that is now called the β-lactamase fold (29). As more β-lactamases were identified and studied, several classification schemes evolved (Table 1-1). The first, proposed by Richmond and Sykes in 1973, divided the β-lactamases from Gram-negative bacteria into five classes (30). Sykes and
Matthews improved this scheme by including isoelectric focusing comparisons in 1976 (31).
12
Bush-Jacoby- Ambler Inhibited by Medeiros molecular Preferred substrates group (32) class (33) 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.
aCA = Clavulanic Acid bEDTA = Ethylenediaminetetraacetic acid
13
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 (33). Jaurin and Grundstrom
(34) and Medeiros (35) updated Ambler’s classification scheme by adding class C and class D and subdividing the serine-β-lactamases based upon their substrate specificity (8,25). 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 (14,32,36,37). 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) (38). Subgroup Ba 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 Ba include β-lactamase II from B. cereus and CcrA from B. fragilis. Subgroup Bb contains metallo-β-lactamases that preferentially hydrolyze carbapenems and exhibit poor activity against cephalosporins and penicillins.
Subgroup Bb 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
(39,40). 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 Bc, currently the smallest subgroup, consists of metallo-β-lactamases that target ampicillin and cephaloridine specifically while poorly hydrolyzing cephalosporins. This subgroup currently contains only the metallo-β-lactamase from Legionella gormanii and L1 from S. maltophilia (40).
14
Bush subgroup Substrate specificity Example β-Lactamase II Penicillins and to a lesser extent Ba and CcrA cephalosporins
Bb Carbapenems ImiS and CphA Metallo-β-lactamase from Bc Penicillins, cephalosporins, cephamycins Legionella gormanii and L1
Table 1-2: Bush group B metallo-β-lactamase sub-grouping scheme.
15
As metallo-β-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 metallo-β-lactamases, is growing quite rapidly (29). Members of this family of proteins participate in 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, a tandem repeat αββα pattern refered to as the β- lactamase fold, they are highly-divergent in their amino acid sequences (29). The metallo-β- lactamases, from which the family is defined, are zinc enzymes (40); however, this is not the case with every member of this family. Glyoxylase 2 has been shown to bind either two zincs or a combination of iron, zinc, and manganese; and rubredoxin oxygen:oxidoreductase (ROO) binds two irons (29). Enzymes of this family also contain 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 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 (41)), His118, His196, and a bridging water/hydroxide (40).
16
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 (29) and (40)). Sequence comparision of β-lactamase II (BLII) from B. cereus (42), CcrA from B. fragilis (43), ImiS from A. sobria (44), L1 from S. maltophilia (45), glyoxalase II (Glx2-2) from A. thaliana (46), and rubredoxin oxygen:oxidoreductase (ROO) from D. gigas (47).
17
The second metal ion-binding site, Zn2, consists of Asp120, His263, Cys221, a bridging
hydroxide/water, and a terminally-bound water (40). The exceptions to this consensus metal- binding sequence are (1) Bush group Bb 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 Stenotrophomonas maltophilia
S. maltophilia is the only member of the genus Stenotrophomonas. The genus was proposed
in 1993 by Palleroni and Bradbury (48) after many years of debate regarding the appropriate
taxonomic position of this organism. The type strain was isolated in 1958 by Hugh from an
oropharyngeal swab from a patient with an oral carcinoma (49) and named Psuedomonas
maltophilia (50). In the following years, several previously known organisms were also
reclassified as P. maltophilia (49,51). In 1981, based on DNA-rRNA hybridization techniques
and comparative enzymology, Swings et al. proposed that P. maltophilia be reclassified as
Xanthomonas maltophilia (52). Subsequent evidence (53) appeared to support this proposal;
however, it was not universally accepted, and the taxinomic status of this bacterium in the genus
Xanthamonas remained controversial. Dissatisfaction with the classification of this organism
finally gave rise to the 1993 proposal to create the new genus Stenotrophomonas with S.
maltophilia as the sole member (48). In 1995 Nesme et al. confirmed the distinction between S.
matlophilia and members of the genus Xanthomonas through the use of restriction mapping of
PCR-amplified 16S rRNA genes (54).
18
S. maltophilia is an aerobic, Gram-negative bacilli that can be found in a wide variety of
environments. The bacterium has been isolated from a variety of aquatic sources, including
lakes, rivers, wells, bottled water, and sewage (55-58). S. maltophilia has also been isolated
from a variety of soil (59-62) and plant rhizosphere environments, including grasses, sugar cane
and palms (63), from wheat (64), cabbage rape, mustard, beet (65), bananas, cotton, beans,
tobacco, citrus plants (66), orchids (67), irises (68), and stored timber (69). A partial list of nosocomial sources of S. maltophilia includes: blood sampling tubes (70,71), central venous/arterial pressure monitors (70), contact lens care systems (72,73), dionized water dispensers and disinfectant solutions (74), dialysis machines (75,76), hands of health care personnel (76,77), hydrotherapy pools, nebulizers and inhalation therapy equipement (78), oxygen analyzers (79), and shower heads, sink traps, and water faucets (80,81).
S. maltophilia is an important pathogen in nosocomial infections of immunocompromised patients suffering from cancer, cystic fibrosis, drug addition, and AIDS and in patients with organ transplants and on dialysis (80,82,83) and has been linked to bacteremia, respiratory and uniary infections, endocarditis, meningitis, conjunctivitis, and wound infections. Adherence to plastic is considered an important property of bacteria commonly implicated in line-related colonization and infection, and strains of S. maltophilia of both clinical and environmental origin have been reported to adhere to several types of plastic materials including intravenous cannulae
(84). In addition, the ability of S. maltophilia to survive and multiply in a range of intravenous infusates likely contributes to the pathogenesis of intravenous line-related infections (85,86).
Although it is regarded as being primarily a nosocomial pathogen, community-acquired infection with this bacterium has been recognized and may be more frequent than previously thought (87-
89). A range of extracellular enzymes produced by S. maltophilia, including Dnase, Rnase,
19
fibrinolysin, lipases, hyaluronidase, protease, and elastase, are proposed to play an important role
in the pathogensis of the organism (90).
1.7 Antibiotic Resistance in Stenotrophomonas maltophilia
Infections caused by S. maltophilia are particularly difficult to manage becasue clinical
isolates are frequently resistant to many antimicrobial agents. Particularly noteworthy is the
resistance to drugs of the β-lactam class. This resistance is primarily conferred by two β-
lactamases, L1 and L2 (91,92); however, the production of additional antimicrobial enzymes by
this organism has been identified (93). L1, produced by virtually all wild-type strains (94), belongs to the metallo-β-lactamase family, and is a homotetramer requiring two Zn (II) ions per monomer for maximum catalytic activity. Though most active towards penicillins, L1 also
hydrolyzes carbapenems efficiently, as well as cephalosporins to a lesser degree (95). L1 is not susceptible to classic β-lactamase inhibitors such as clavulanate, although inhibition of L1 by mercaptoacetic acid thiol ester derivatives has been reported (96). In contrast to L1, L2, which exists as a dimer, is a serine β-lactamase, and exhibits principally cephalosporinase activity (92).
Unlike L1, this enzyme is susceptible to clavulanate. Both L1 and L2 are chromosomally- encoded and are inducible, and there is evidence that they share regulatory components (97,98).
However, Bofiglio et al. have described a clinical isolate of S. maltophilia with low-level expression of L1 that retained the inducibility for the L2 enzyme (99). In addition to the production of β-lactamases, poor diffusion of penicillins and cephalosporins across the bacterial
cell membrane of S. maltophilia has also been suggested as contributing to resistance to these compounds (100,101). Instances of transferable cephalosporin resistance from nosocomial
strains of S. maltophilia to recipient strains of E. coli, Proteus mirabbilis, and Pseudomonas
aeruginosa have been reported (102), as well as transferable penicillin resistance in a clinical
20
strain of S. maltophila associated with a 5.6-kb plasmid (103). Clearly, the potential for β-
lactam resistance transfer to highly infectious bacterial strains exists.
Resistance to aminoglycosides mediated by aminoglycoside-modifying enzymes is
uncommon in S. maltophilia (104), presumably due to the low uptake of these compounds.
Temperature-dependent aminoglycoside resistance was initially attributed to changes in the outer membrane (105). However, further studies from strains exhibiting both temperature-dependent and –independent resistance suggests that an altered cell surface microenvironment is secondary to changes in the O side chains of lipopolysaccharides (106-108). Resistance to quinolone agents in S. maltophilia is less well-characterized; however, resistant mutants are readily selected in vitro and are associated with qualititative and quantitative changes in outer membrane proteins. Cross-resistance to chloramphenicol and doxycycline in quinolone-resistant isolates has also been reported (109).
The development of antimicrobial resistance in vivo has received comparatively less attention. In 1997, Alonso and Martinez reported the presence of at least one multi-drug resistance system in S. maltophilia, selected for by exposure to low concentrations of tetracycline. This energy-dependent efflux mechanism is effective against quinolones and chloramphenicol, as well as tetracyclines, but not against aminoglycosides or β-lactams (6).
21
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, which is rapidly growing and bringing together researchers from many diverse disciplines. Biological systems are studied to determine the in vivo function of metals in their naturally-occurring environments, their usefulness as medical probes, and as pharmaceutical agents (110,111).
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 a variety of 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 (112). Generally, metal ions in biological systems are generally bound in a ligated form, because free metal ions are extremely toxic even at low concentrations (113).
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
22
natural sources or synthesized and purified in the lab. With sufficient compounds of high purity,
they then would be screened to determine any 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 those findings.
In an effort to help combat antibiotic resistance, rational drug design research has been
undertaken to identify and structurally-characterize critical mechanistic attributes of metallo-β-
lactamases. Of particular interest are points of contact between the enzyme and substrate during
binding and the identification of intermediate species, specifically during the rate-limiting step of
β-lactam hydrolysis. Hopefully, this information can then be used to design metallo-β-lactamase
inhibitors, which ideally would be administered in combination with existing β-lactam
antibiotics.
Research on L1 has yielded a crystal structure of the enzyme (114), computational
models that have been used to predict the manner in which L1 binds substrate, a minimal kinetic
mechanism (114), and information about the importance of several amino acid residues the
active site (115). Chapters 2 and 3 of this dissertation address the validity of the predictions
derived from computational studies. A feature common to all metallo-β-lactamases (42,115-
120) and found in other enzymes as well (121) is a flexible loop of amino acids adjacent to and
extending over the active sites of the enzymes. Prior work on metallo-β-lactamases indicated
that this loop is in motion during the binding and turnover of substrate (122,123). In Chapter 4, the motion of this loop was investigated and a link between the formation of a non-rate-limiting
23
intermediate and the motion of this loop established. To date, a sufficiently resolved crystal
structure of one of the metallo-β-lactamases with bound substrate has not been achieved. In an effort to probe substrate binding and identify reaction intermediates, Chapter 5 describes stopped-flow/rapid scanning and rapid freeze/quench EPR studies of L1 with multiple substrates.
1.8.2 Sections of the Dissertation
This dissertation describes mechanistic studies on the metallo-β-lactamase L1 from S. maltophilia. Each of the chapters comprising the main body of this dissertation is a journal publication. Chapters 2 and 3 have been published in BMC Biochemistry (124) and
J.Biol.Chem. (125), respectively; and Chapter 4 has been accepted for publication in J. Biol.
Chem. and is currently in press. Chapter 5 is a recently finished manuscript that will be
submitted for publication soon. In Chapter 2, a biochemical analysis, via site-directed
mutagensis, of protein residues predicted to be important in substrate binding to L1 is presented.
Chapter 3 describes the catalytic importance of an active site, metal-binding aspartic acid in the
hydrolysis of β-lactam containing antibiotics by L1. In Chapter 4, the motion of a flexible loop
that extends over the active site of L1, studied using tryptophan flourescence, is described.
Chapter 5 describes stopped-flow/rapid-scanning and rapid-freeze quench EPR studies of Co(II)-
substituted L1 with multiple substrates. Finally, Chapter 6 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.
24
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33
Chapter 2
Probing Substrate Binding to Metallo-β-Lactamase L1 from Stenotrophomonas maltophilia by Using Site-Directed Mutagenesis
Anne L. Carenbauer,1 James D. Garrity,1 Gopal Periyannan,1 Robert B. Yates,1 and Michael W. Crowder¶1
#Department of Chemistry and Biochemistry, Miami University, Oxford, OH USA
¶Corresponding author:
e-mail addresses: Anne L. Carenbauer: [email protected] James D. Garrity: [email protected] Gopal Periyannan: [email protected] Robert B. Yates: [email protected] Michael W. Crowder: [email protected]
34
List of abbreviations used
AES, atomic emission spectroscopy; bp, base pairs; CD, circular dichroism; ε, extinction coefficient; ICP, inductively coupled plasma; kcat, turnover number; kDa, kilodaltons; Km,
Michaelis constant; Ks, substrate binding constant; LB, Luria-Bertani media.
35
2.1 Summary
The metallo-β-lactamases are Zn(II)-containing enzymes that hydrolyze the β-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance. There are at least 20 distinct organisms that produce a metallo-β-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism. In an effort to probe substrate binding to metallo-β-lactamase L1 from
Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics. Site- directed mutations were generated of amino acids previously predicted to be important in substrate binding. Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying Km and kcat values for the different enzymes and substrates and that no direct correlation between Km and the effect of the mutation on substrate binding could be drawn. Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding. The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-β-lactamase L1. The results in this work also show that Km values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction. This work represents the first experimental testing of one of the computational models of the metallo-β-lactamases.
36
2.2 Introduction
The overuse of antibiotics in the clinic and for agricultural uses has resulted in a tremendous selective pressure for antibiotic resistant bacteria. These bacteria become resistant by a number of mechanisms, such as producing enzymes that hydrolyze or inactivate the antibiotics, producing efflux pumps that transport the antibiotic out of the cell, or modifying their cell wall components so they no longer bind effectively to the antibiotics (1-3). The most common, least expensive, and effective antibiotics are the β-lactam containing antibiotics, such as the penicillins, cephalosporins, and carbapenems (2,4,5). These antibiotics are mechanism- based inhibitors of transpeptidase, a bacterial enzyme required for the production of a strong viable cell wall (6,7). In response to the widespread use of β-lactam containing antibiotics, bacteria have acquired the ability to produce β-lactamases, which are enzymes that hydrolyze and inactivate β-lactam containing antibiotics. There are over 300 distinct β-lactamases known, and these enzymes have been grouped by a number of classification schemes (8-15). For example, Bush has developed a scheme, based on the enzymes’ molecular properties, that has four distinct β-lactamase groups (10,15). One of the more alarming groups are the Bush group 3 enzymes, which are Zn(II) dependent enzymes that hydrolyze nearly all known β-lactam containing antibiotics and for which there are no or very few known clinical inhibitors (9,14,16-
19). The metallo-β-lactamases have been further divided by Bush into subgroups based on amino acid sequence identity: the Ba enzymes share a >23% sequence identity, require 2 Zn(II) ions for full activity, prefer penicillins and cephalosporins as substrates, and are represented by metallo-β-lactamase CcrA from Bacteroides fragilis, the Bb enzymes share a 11% sequence identity with the Ba enzymes, require only 1 Zn(II) ion for full activity, prefer carbapenems as substrates, and are represented by the metallo-β-lactamase imiS from Aeromonas sobria, and the
37
Bc enzymes have only 9 conserved residues with the other metallo-β-lactamases, require 2 Zn(II) ions for activity, contain a different metal binding motif than the other metallo-β-lactamases, prefer penicillins as substrates, and are represented by the metallo-β-lactamase L1 from
Stenotrophomonas maltophilia (9). A similar grouping scheme (B1, B2, and B3) based on structural properties of the metallo-β-lactamases has recently been offered (41). The diversity of the group 3 β-lactamases is best exemplified by the enzymes’ vastly differing efficacies towards non-clinical inhibitors; these differences predict that one inhibitor may not inhibit all metallo-β- lactamases (18,20-29). To combat this problem, we are characterizing a metallo-β-lactamase from each of the subgroups in an effort to identify a common structural or mechanistic aspect of the enzymes that can be targeted for the generation of an inhibitor. It is hoped that this inhibitor, when given in combination with an existing antibiotic, will prove to be an effective therapy against bacteria that produce a metallo-β-lactamase. This work describes our efforts on metallo-
β-lactamase L1 from S. maltophilia.
S. maltophilia is an important pathogen in nosocomial infections of immunocompromised patients suffering from cancer, cystic fibrosis, drug addition, AIDS and in patients with organ transplants and on dialysis (30-32). This organism is inherently resistant to most antibiotics due to its low outer membrane permeability (33) and to β-lactam containing antibiotics due to the production of a chromosomally expressed group 2e β-lactamase (L2) and a group 3c β-lactamase
(L1) (34,35). L1 has been cloned, over-expressed, and partially characterized by kinetic and crystallographic studies (36,37). The enzyme exists as a homotetramer of ca. 118 kDa in solution and in the crystalline state. The enzyme tightly binds two Zn(II) ions per subunit and requires both Zn(II) ions for full catalytic activity. The Zn1 site has 3 histidine residues and 1 bridging hydroxide as ligands, and the Zn2 site has 2 histidines, 1 aspartic acid, 1 terminally-
38
bound water, and the bridging hydroxide as ligands. Spencer and coworkers used the crystal
structure and modeling studies to propose a substrate binding model and identified several active site residues that were involved in substrate binding (Figure 2-1) (37). However, this model has not been tested experimentally. In order to prepare tight binding inhibitors of the metallo-β- lactamases, knowledge about how substrate binds to the enzymes is needed so that all substrate- enzyme binding contacts can be maintained in any proposed inhibitor. This work describes our efforts at understanding how substrates bind to metallo-β-lactamase L1. Several site-directed mutants of L1 were generated and characterized, and the results from these studies reveal that none of the active site residues predicted from earlier computational studies (37) are essential for tight substrate binding.
39
Figure 2-1: Active site residues that were mutated in this study. Figure was rendered using Rasmol v. 2.6. The coordinates were obtained from the Protein Data Bank using the accession number 1sml.
40
2.3 Materials and Methods
E. coli strains DH5α and BL21(DE3) were obtained from Gibco BRL and Novagen,
respectively. Plasmids pET26b and pUC19 were purchased from Novagen. Primers for
sequencing and mutagenesis studies were purchased from Integrated DNA Technologies.
Deoxynucleotide triphosphates (dNTP's), MgSO4, thermopol buffer, Deep Vent DNA
polymerase, and restrictions enzymes were purchased from Promega or New England Biolabs.
Polymerase chain reaction was conducted using a Thermolyne Amplitron II unit. DNA was
purified using the Qiagen QIAQuick gel extraction kit or Plasmid Purification kit with QIAGEN-
tip 100 (Midi) columns. Wizard Plus Minipreps were acquired from Promega. Luria-Bertani
(LB) media was made following published procedures (67). Isopropyl-β-thiogalactoside (IPTG),
Biotech grade, was procured from Anatrace. Phenylmethylsulfonylfluoride (PMSF) was
purchased from Sigma. Protein solutions were concentrated with an Amicon ultrafiltration cell
equipped with YM-10 DIAFLO membranes from Amicon, Inc. Dialysis tubing was prepared
using Spectra/Por regenerated cellulose molecular porous membranes with a molecular weight
cut-off of 6-8,000 g/mol. Q-Sepharose Fast Flow was purchased from Amersham Pharmacia
Biotech. Nitrocefin was purchased from Becton Dickinson, and solutions of nitrocefin were filtered through a Fisherbrand 0.45 micron syringe filter. Cefaclor, cefoxitin, and cephalothin were purchased from Sigma; penicillin G and ampicillin were purchased from Fisher.
Imipenem, meropenem, and biapenem were generously supplied by Merck, Zeneca
Pharmaceuticals, and Lederle (Japan), respectively. All buffers and media were prepared using
Barnstead NANOpure ultrapure water.
Mutants for this study were generated by A. Carenbauer. The over-expression plasmid for L1, pUB5832, was digested with NdeI and HindIII, and the resulting ca. 900 bp piece was gel
41
purified and ligated using T4 ligase into pUC19, which was also digested with NdeI and HindIII,
to yield the cloning plasmid pL1PUC19. Mutations were introduced into the L1 gene by using
the overlap extension method of Ho et al. (60), as described previously (68). The
oligonucleotides used for the preparation of the mutants are shown in Table 2-1. The ca. 900 bp
PCR products were digested with NdeI and HindIII and ligated into pUC19. The DNA sequences were analyzed by the Biosynthesis and Sequencing Facility in the Department of
Biological Chemistry at Johns Hopkins University. After confirmation of the sequence, the mutated pL1PUC19 plasmid was digested with NdeI and HindIII, and the 900 bp, mutated L1 gene was gel purified and ligated into pET26b to create the mutant overexpression plasmids. To test for overexpression of the mutant enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated over-expression plasmids, and small scale growth cultures were used (68).
Large-scale (4 L) preparations of the L1 mutants were performed as described previously (36).
Protein purity was ascertained by SDS-PAGE.
The concentrations of L1 and the mutants were determined by measuring the proteins'
-1 -1 absorbance at 280 nm and using the published extinction coefficient of ε280nm = 54,804 M •cm
(36) or by using the method of Pace (69). Before metal analyses, the protein samples were
dialyzed versus 3 X 1 L of metal-free, 50 mM HEPES, pH 7.5 over 96 hours at 4 °C. A Varian
Inductively Coupled Plasma Spectrometer with atomic emission spectroscopy detection (ICP-
AES) was used to determine metal content of multiple preparations of wild type L1 and L1
mutants. Calibration curves were based on three standards and had correlation coefficient limits
42
Table 2-1: Oligonucleotides used in preparation of L1 mutants.
Primer Sequence pUCMSZFor CTATgCggCATCAgAgCAgATT
M13Rev gATAACAATTTCACACAggA
Y228AFor CTgAgTgCACCgggCgCCCAgCTgCAgggAAAC
Y228ARev gTTTCCCTgCAgCTgggCgCCCggTgCACTCAg
Y228FFor CTgAgTgCACCgggCTTCCAgCTgCAgggAAAC
Y228FRev gTTTCCCTgCAgCTggAAgCCCggTgCACTCAg
S224DFor TACgCCgACAgCCTggACgCACCgggCTACCAg
S224DRev CAggTAgCCCggTgCgTCCAggCTgTCggCgTA
S224AFor TACgCCgACAgCCTggCCgCACCgggCTACCAg
S224ARev CAggTAgCCCggTgCggCCAggCTgTCggCgTA
S224KFor TACgCCgACAgCCTgAAggCACCgggCTACCAg
S224KRev CAggTAgCCCggTgCCTCCAggCTgTCggCgTA
F158AFor AgCgATgACCTgCACgCCggCgATggCATCACC
F158ARev ggTgATgCCATCgCCggCgTgCAggTCATCgCT
I164AFor CACTTCggCgATggCgCCACCTACCCgCCTgCC
I164ARev ggCAggCgggTAggTggCgCCATCgCCgAAgTg
N233LFor TACCAgCTgCAgggACTgCCCCgTTATCCgCAC
N233LRev gTgCggATAACggggCAgTCCCTgCAgCTggTA
N233DFor TACCAgCTgCAgggAgACCCCCgTTATCCgCAC
N233DRev gTgCggATAACgggggTCTCCCTgCAgCTggTA
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of at least 0.9950. The final dialysis buffer was used as a blank, and the Zn(II) content in the
final dialysis buffers was shown to be < 0.5 µM (detection limit of ICP) in separate ICP
measurements. The emission line of 213.856 nm is the most intense for zinc and was used to
determine the Zn content in the samples. The errors in metal content data reflect the standard
deviation (σn-1) of multiple enzyme preparations.
A. Carenbauer, G. Periyannan, and R. Yates contributed to repetitions of steady-state
kinetics. Steady-state kinetic assays were conducted at 25 oC in 50 mM cacodylate buffer, pH
o 7.0, containing 100 µM ZnCl2 on a HP 5480A diode array UV-Vis spectrophotometer at 25 C.
The changes in molar absorptivities (∆ε) used to quantitate products were (in M-1cm-1):
nitrocefin, ∆ε485 = 17,420; cephalothin, ∆ε265 = -8,790; cefoxitin, ∆ε265 = -7,000; cefaclor, ∆ε280
= -6,410; imipenem, ∆ε300 = -9,000; meropenem, ∆ε293 = -7,600; biapenem, ∆ε293 = -8,630;
ampicillin, ∆ε235 = -809; and penicillin G, ∆ε235 = -936. When possible, substrate concentrations
were varied between 0.1 to 10 times the Km value. In kinetic studies using substrates with low
Km values (cefoxitin, nitrocefin, and cephalothin) or with small ∆ε values (penicillin and
ampicillin), we typically used substrate concentrations varied between ~ Km and 10XKm and
used as much of the DA versus time data (that was linear) as possible to determine the velocity.
Steady-state kinetics constants, Km and kcat, were determined by fitting initial velocity versus
substrate concentration data directly to the Michaelis equation using CurveFit (36). The errors reported are generated by CurveFit as a result of Chi square minimization. All steady-state kinetic studies were performed in triplicate with recombinant L1 from at least three different enzyme preparations.
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Circular dichroism samples 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 diluted with
final dialysis buffer to ~75 µg/mL. A JASCO J-810 CD spectropolarimeter operating at 25 oC was used to collect CD spectra.
Rapid-scanning Vis spectra of nitrocefin hydrolysis by L1 and the L1 mutants were collected on a Applied Photophysics SX.18MV stopped-flow spectrophotometer equipped with an Applied Photophysics PD.1 photodiode array detector and a 1 cm pathlength optical cell. A typical experiment consisted of 25 µM enzyme and 5 µM nitrocefin in 50 mM cacodylate buffer,
o pH 7.0 containing 100 µM ZnCl2, the reaction temperature was thermostated at 25 C, and the
spectra were collected between 300 and 725 nm. Data from at least three experiments were
collected and averaged. Absorbance data were converted to concentration data as described
previously by McMannus and Crowder (39). Stopped-flow fluorescence studies of nitrocefin
hydrolysis by L1 were performed on an Applied Photophysics SX.18MV spectrophotometer,
using an excitation wavelength of 295 nm and a WG320 nm cut-off filter on the photomultiplier.
These experiments were conducted at 10 oC using the same buffer in the rapid-scanning Vis
studies. Fluorescence data were fitted to kobs = {(kf [S]) / KS + [S])} + kr as described previously
(40) or to kobs = kf[S] + kr by using CurveFit v. 1.0.
2.4 Results
2.4.1 Wild Type L1.
Wild-type L1 was over-expressed in Escherichia coli and purified as previously
described (36). This procedure produced an average of 50-60 mg of >90% pure, active protein
per 4L of growth culture. Circular dichroism spectra were collected on wild-type samples to
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ensure L1 expressed using the pET26b expression system had the correct secondary structure.
The CD spectrum of wild type L1 showed an intense, broad feature at 190 nm and a smaller
feature at 215 nm (Figure 2-2). These features are consistent with a sample with significant α/β
content. The Compton and Johnson algorithm (38) was used to estimate secondary structure in
the samples; wild-type L1 was estimated to have 38.3% α-helix, 26.7% β-structure (9.3%
antiparallel β-sheet, 2.1% parallel β-sheet, and 15.3% β-turn), and 34.9% other structure. These
estimates are in excellent agreement with the crystallographically determined secondary structure of ~ 40% α-helix and 30% β-structure (37). Metal analyses on multiple preparations of wild-
type L1 demonstrated that the enzyme binds 1.9 + 0.2 Zn(II) ions per monomer (Table 2-2), in
agreement with previous results (36).
Steady state kinetic studies were performed on multiple preparations of wild type L1, and
the resulting kinetic data are shown in Tables 2-3 to 2-5. When using nitrocefin as substrate and
-1 50 mM cacodylate, pH 7.0, as buffer, wild-type L1 exhibited a kcat value of 38 + 1 s and a Km
value of 12 + 1 µM. The inclusion of 100 µM ZnCl2 in the assay buffer resulted in slightly
lower values of Km and higher values for kcat (36). The inclusion of higher concentrations of
Zn(II) did not further affect the steady-state kinetic constants. Apparently, the purified,
recombinant enzyme does not bind its full complement of Zn(II); therefore, 100 µM Zn(II) was
included in all subsequent kinetic studies.
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Figure 2-2: CD spectra of wild-type L1 and L1 mutants. The data were collected at 25 oC on a JASCO J-810 CD Spectropolarimeter. The enzymes were dialyzed into 5 mM phosphate buffer, pH 7.0, and were 75 µg/mL. Figure was created using Sigmaplot v.3.5.
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Table 2-2: Metal Content of Wild-type L1 and L1 mutants.
Sample Zn(II) Content (moles Zn(II)/mole of enzyme)a
Wild-type 1.9 + 0.2
S224A 1.8 + 0.2
S224D 1.7 + 0.3
S224K 1.0 + 0.1
Y228A 1.8 + 0.1
Y228F 1.7 + 0.3
F158A 1.5 + 0.2
I164A 1.6 + 0.2
N233L 1.8 + 0.2
N233D 1.5 + 0.1
a The final dialysis buffer was used as a blank, and the Zn(II) content in the final dialysis buffers was shown to be < 0.5 µM in separate ICP measurements.
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-1 -1 -1 -1 Wild-type L1 exhibited kcat values of 41 + 1 s , 1.9 + 0.1 s , 42 + 1 s , and 82 + 5 s for the cephalosporins, nitrocefin, cefoxitin, cefaclor, and cephalothin (Table 2-3). For these same substrates, the Km values were 4 + 1 µM, 1.1 + 0.1 µM, 13 + 1 µM, and 8.9 + 1.5 µM,
respectively. Two penicillins were tested as substrates, and penicillin G and ampicillin exhibited
-1 -1 Km values of 38 + 12 µM and 55 + 5 µM and kcat values of 600 + 100 s and 520 + 10 s , respectively (Table 2-4). Three carbapenems were also used as substrates for L1, and biapenem, imipenem, and meropenem exhibited Km values of 32 + 1 µM, 57 + 7 µM, and 15 + 4 µM and
-1 -1 -1 kcat values of 134 + 4 s , 370 + 5 s , and 157 + 9 s , respectively (Table 2-5). L1’s preference for penicillins and carbapenems over cephalosporins, as exemplified by the kcat values, is in agreement with previous studies and supports L1’s placement in the β-lactamase 3c family (9).
Rapid-scanning visible spectra of 25 µM wild-type L1 with 5 µM nitrocefin demonstrated a decrease in absorbance at 390 nm, an increase at 485 nm, and a rapid increase and slower decrease in absorbance at 665 nm. These spectra are similar to those previously reported for wild-type L1 and nitrocefin (39), and the features can be attributed to substrate decay, product formation, and intermediate formation and decay, respectively. Under these conditions, 2.2 µM intermediate was formed during the first 10 milliseconds of the reaction
(Figure 2-3), and the rate of decay of this intermediate corresponds to the steady-state kcat (Table
2-3). To probe further the binding of nitrocefin to wild-type L1, stopped-flow fluorescence studies were conducted as previously described (40)(Figure 2-4). The reaction of wild-type L1 with nitrocefin under steady-state conditions at 10 oC resulted in a rapid decrease in fluorescence
followed by a rate-limiting return of fluorescence (Figure 2-4A). Fitting of the data, as described
by Spencer et al. (40), resulted in a KS value for wild-type L1 of 38 + 5 µM (Figure 2-4B).
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Table 2-3: Steady-state kinetic constants for hydrolysis of cephalosporins by wild-type L1 and L1 mutants.
nitrocefin cefoxitin cefaclor cephalothin Enzyme Km kcat kcat/Km Km kcat kcat/Km Km kcat kcat/Km Km kcat kcat/Km (µM) (s-1) X107 (µM) (s-1) X107 (µM) (s-1) X107 (µM) (s-1) X107 M-1s-1 M-1s-1 M-1s-1 M-1s-1 w.t. 4 + 1 41 + 1 1.0 1.1 + 1.9 + 0.1 0.17 13 + 1 42 + 1 0.32 8.9 + 82 + 5 0.92 0.1 1.5 S224A 7 + 1 48 + 5 0.69 3.0 + 1.0 + 0.1 0.033 14 + 2 14 + 1 0.10 3.6 + 19.8 + 0.2 0.55 0.5 0.1 S224K 14 + 2 48 + 10 0.34 2.0 + 0.60+ 0.030 13 + 2 6.5 + 0.3 0.050 6.2 + 26 + 1 0.42 0.3 0.06 0.6 S224D 11 + 5 2.3 + 0.4 0.021 50 + 6 1.4 + 0.2 0.0028 215 + 3 + 1 0.0014 75 + 7 32 + 2 0.043 17 I164A 8 + 2 130 + 30 1.6 11 + 1 8 + 1 0.073 3.3 + 30 + 3 0.91 16 + 1 146 + 2 0.91 0.5 F158A 123 + 1290 + 20 1.0 11 + 2 16 + 2 0.15 135 + 99 + 9 0.073 63 + 17 353 + 35 0.56 20 23 Y228A 23 + 3 72 + 2 0.31 10 + 2 5.8 + 0.3 0.058 550 + 40 + 3 0.0073 290 + 320 + 40 0.11 100 60 Y228F 36 + 16 81 + 18 0.23 8.2 + 5.5 + 0.5 0.067 240 + 74 + 4 0.031 58 + 7 190 + 50 0.33 1.1 60 N233L 7 + 2 62 + 12 0.89 4.4 + 0.90 + 0.020 14 + 2 32 + 1 0.23 8.0 + 51 + 1 0.64 1.6 0.17 0.7 N233D 9 + 2 21 + 2 0.23 1.1 + 1.1 + 0.1 0.10 25 + 5 34 + 3 0.14 18 + 4 65 + 2 0.36 0.2
Table 2-4: Steady-state kinetic constants for hydrolysis of penicillins by wild-type L1 and L1 mutants.
penicillin G ampicillin Enzyme Km kcat kcat/Km Km kcat kcat/Km (µM) (s-1) X107 (µM) (s-1) X107 w.t. 38 + 12 600 + 100 1.6 55 + 5 520 + 10 0.95 S224A 70 + 20 580 + 100 0.83 125 + 13 339 + 1 0.27 S224K 44 + 8 124 + 12 0.28 25 + 3 152 + 2 0.61 S224D 1600 + 200 42 + 9 0.0026 1100 + 240 10 + 1 0.00091 I164A 60 + 5 698 + 100 1.2 43 + 3 524 + 100 1.2 F158A 50 + 5 138 + 10 0.28 165 + 20 270 + 30 0.16 Y228A 410 + 60 609 + 64 0.15 710 + 74 443 + 10 0.062 Y228F 140 + 14 630 + 30 0.45 271 + 40 243 + 30 0.090 N233L 33 + 9 184 + 35 0.56 90 + 20 508 + 40 0.56 N233D 60 + 2 440 + 86 0.73 117 + 18 621 + 31 0.53
Table 2-5: Steady-state kinetic constants for hydrolysis of carbapenems by wild-type L1 and L1 mutants.
biapenem imipenem meropenem Enzyme Km kcat kcat/Km Km kcat kcat/Km Km kcat kcat/Km (µM) (s-1) X107 (µM) (s-1) X107 (µM) (s-1) X107 w.t. 32 + 1 134 + 4 0.42 57 + 7 370 + 5 0.65 15 + 4 157 + 9 1.0 S224A 34 + 5 56 + 2 0.16 29 + 4 100 + 3 0.34 50 + 10 244 + 1 0.49 S224K 100 + 22 43 + 2 0.043 60 + 4 14 + 1 0.023 12 + 1 212 + 1 1.8 S224D 64 + 7 22 + 1 0.034 42 + 4 17 + 1 0.040 132 + 23 69 + 7 0.052 I164A 55 + 3 112 + 1 0.20 92 + 11 570 + 43 0.62 14 + 1 96 + 3 0.69 F158A 50 + 11 70 + 5 0.14 100 + 20 370 + 50 0.37 7 + 2 36 + 2 0.51 Y228A 175 + 25 21 + 2 0.012 350 + 94 134 + 36 0.038 4.5 + 0.2 26 + 1 0.58 Y228F 150 + 23 51 + 4 0.034 107 + 13 83 + 10 0.078 13 + 1 70 + 1 0.54 N233L 29 + 3 105 + 6 0.36 36 + 5 250 + 20 0.69 12 + 1 67 + 3 0.56 N233D 28 + 8 7 + 1 0.025 71 + 16 158 + 13 0.22 16 + 4 3.5 + 0.1 0.022
2.4.2 Ser224 Mutants (the BBL numbering scheme proposed in reference 41 was used
throughout this manuscript).
All sequenced subclass Ba and Bb metallo-β-lactamases (except VIM-1) have a lysine
residue at position 224 (41), and all computational models for substrate binding to the metallo-β- lactamases assume that the invariant carboxylate on substrates forms an electrostatic interaction with this lysine. In L1, the residue at position 224 is a serine (35), and the substrate-binding model for L1 predicts that this serine residue interacts with the carboxylate on substrate via a water molecule (37). To test the proposed role of Ser224 in L1, serine was changed to an alanine
(S224A), aspartic acid (S224D), and lysine (S224K), and these mutants were characterized using
metal analyses, CD spectroscopy, steady-state kinetics, and pre-steady state kinetic studies.
Small-scale growth cultures showed that all three mutants were over-expressed at levels comparable to those of wild-type L1. Large-scale over-expression and purification of the mutants showed that all three mutants were isolatable at levels comparable to those of wild-type
L1. Metal analyses of the S224A and S224D mutants showed that both mutants bind nearly two
Zn(II) ions (Table 2-2), like wild-type L1 (36); however, the S224K mutant binds only 1.0 Zn(II) per protein. CD spectra of the mutants were similar to those of wild-type L1 (Figure 2-2).
Steady-state kinetic studies were conducted with all three mutants in buffer containing 100 µM
ZnCl2 to ensure that both Zn(II) binding sites were saturated in these studies. Addition of higher
concentrations of Zn(II) did not result in different values for the steady-state kinetic constants in
Tables 2-3 to 2-5.
When the cephalosporins were used as substrates, the S224A and S224K mutants
exhibited 2- to 4-fold changes in Km values (Table 2-3). In studies with cefoxitin, cefaclor, and
cephalothin as substrate, the observed kcat values for the S224A and S224K mutants were 2- to 7-
53
fold lower; however, the kcat values when using nitrocefin as substrate were slightly higher (< 2- fold). On the other hand, the S224D mutant exhibited 3- to 50-fold higher Km values and 2- to
20-fold lower kcat values for the cephalosporins tested. A similar trend was observed in kinetic studies when using penicillins as substrates (Table 2-4). Generally, the S224A and S224K mutants exhibited small changes in Km and kcat, while the S224D mutant yielded 20- to 40-fold increased values for Km and >10-fold decreases in kcat when using the penicillins as substrates.
When the carbapenems were used as substrates however, the changes in Km values were relatively smaller than with the other substrates, and 2- to 37-fold changes in kcat were observed
(Table 2-5).
Rapid-scanning Vis studies of the S224X mutants were conducted to probe whether the mutations caused changes in the amount of intermediate that accumulates during catalysis.
When 50 µM S224A was reacted with 5 µM nitrocefin, 1.7 µM intermediate formed during the first 10 milliseconds of the reaction (Figure 2-3), and rate of decay of this intermediate was equal to the steady-state kcat (Table 2-3). In spite of utilizing a number of reaction conditions, the
S224K and S224D mutants yielded rapid-scan spectra with no detectable absorbances at 665 nm
(Figure 2-3), indicating that the intermediate is not stabilized as well in these mutants as in wild- type L1. Stopped-flow fluorescence studies at 10 oC with the S224A, S224D, and S224K mutants and nitrocefin as the substrate resulted in KS values of 39 + 10, 213 + 63, and 33 + 5
µM, respectively.
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Figure 2-3: Intermediate formation by wild-type L1 and L1 mutants. The spectra were collected using rapid scanning Vis studies, and the absorbance values at 668 nm were converted to concentration values as described in Materials and Methods. Typical reactions were conducted with 25 µM L1 (or mutant) and 5 µM nitrocefin in 50 mM cacodylate, pH 7.0, containing 100 o µM ZnCl2 at 25 C.
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2.4.3 Asn233 Mutants.
Two-thirds of all sequenced metallo-β-lactamases have an Asn at position 233 (41), and this residue was predicted (42) and shown (43) to be involved with substrate binding and activation by interacting electrostatically with the substrate β-lactam carbonyl. However, in L1,
Asn233 is 14 Å away from the modeled position of the substrate β-lactam carbonyl (37). To test the role of Asn233 in substrate binding, the Asn was changed to a leucine (N233L) and to an aspartic acid (N233D), and these mutants were characterized by using metal analyses, CD spectroscopy, steady-state kinetics, and pre-steady state kinetic studies.
Small-scale growth cultures showed that both mutants were over-expressed at levels comparable to that of wild-type L1. Large-scale over-expression and purification of the mutants showed that both mutants were isolatable at levels comparable to that of wild-type L1. Metal analyses of the N233L and N233D mutants showed that both bind nearly two Zn(II) ions (Table
2-2), like wild-type L1 (36). CD spectra of the mutants were similar to those of wild-type L1.
Steady-state kinetic studies were conducted with both mutants in buffer containing 100 µM
ZnCl2 to ensure that both Zn(II) binding sites were saturated in these studies. Addition of higher
concentrations of Zn(II) did not result in different values for the steady-state kinetic constants in
Tables 2-3 to 2-5.
With all substrates tested, the N233L and N233D mutants exhibited Km values that
differed less than a factor of 4 than that observed for wild-type L1 (Tables 2-3 to 2-5). The kcat values exhibited by these mutants for all substrates also differed by less than a factor of 4, except when biapenem and meropenem were used as substrates for the N233D mutant. With these two substrates, there was a 19-fold and 45-fold decrease in the kcat values when using biapenem and
56
meropenem, respectively (Table 2-5). The steady-state kinetic data generally support the
prediction that Asn233 does not play a large role in binding or catalysis.
However, rapid-scanning Vis studies of N233L and N233D with nitrocefin demonstrate
that no detectable amounts of intermediate are formed during the reaction, even when using a
wide number of reaction conditions (Figure 2-3). Stopped-flow fluorescence studies at 10 oC
with the N233L and N233D mutants and nitrocefin as substrate resulted in KS values of 26 + 9
and 25 + 8 µM, respectively.
2.4.4 Tyr228 Mutants.
The substrate-binding model showed that Tyr228 in L1 was position-conserved with
Asn233 in the other crystallographically characterized metallo-β-lactamases (37,42,44-46).
Spencer and coworkers postulated that Tyr228 is part of an oxyanion hole that interacts with the
β-lactam carbonyl on substrate and helps to stabilize the putative tetrahedral intermediate formed during substrate turnover (37). To test this hypothesis, Tyr228 was changed to an alanine and to a phenylalanine to afford the Y228A and Y228F mutants, respectively.
Small-scale growth cultures showed that both mutants were over-expressed at levels comparable to those of wild-type L1. Large-scale over-expression and purification of the Y228A and Y228F mutants showed that both mutants were isolatable at levels comparable to those of
wild-type L1. Metal analyses of the mutants showed that both bind nearly two Zn(II) ions (Table
2-2), like wild-type L1 (36), and CD spectra of the mutants were similar to those of wild-type
L1. Steady-state kinetic studies were conducted with both mutants in buffer containing 100 µM
ZnCl2 to ensure that both Zn(II) binding sites were saturated in these studies. Addition of higher
concentrations of Zn(II) did not result in different values for the steady-state kinetic constants in
Tables 2-3 to 2-5.
57
When cephalosporins were used as substrates, the Y228A and Y228F mutants exhibited
Km values that were 6- to 45-fold higher than those observed for wild-type L1 (Table 2-3). The
largest change in Km was observed when cefaclor was used as substrate, and the smallest change
was observed when nitrocefin was used as substrate. The Tyr228 mutants exhibited < 4-fold
change in kcat values for the cephalosporins tested (Table 2-3), suggesting that Tyr228 is not
playing a large role in catalysis. When penicillins were used as substrates, the Tyr228 mutants
exhibited 3- to 13-fold increased Km values and < 2-fold changes in kcat, as compared to the
values ascertained using wild-type L1 (Table 2-4). On the other hand when carbapenems were
used as substrates, the Tyr228 mutants exhibited < 6-fold increases in Km values as compared to
those values for wild-type L1 (Table 2-5). Interestingly, there was a 2- to 8-fold drop in kcat values for the Tyr228 mutants, as compared to values observed for wild-type L1, when using the carbapenems as substrates.
Rapid-scanning Vis spectra of the reaction of the Y228A and Y228F mutants with nitrocefin demonstrated a marked decrease in the amount of intermediate formed with these mutants (Figure 2-3). In reactions with 50 µM mutant and 5 µM nitrocefin, only 0.75 and < 0.30
µM intermediate formed for the Y228F and Y228A mutants, respectively. The concentration of mutants were varied between 25 to 150 µM to ensure that all of the substrate was bound; however, none of the reactions resulted in the detection of intermediate at levels observed for wild-type L1 (data not shown). Stopped-flow fluorescence studies of nitrocefin hydrolysis by
o Y191A and Y191F, at 10 C, resulted in KS values of 85 + 9 and 22 + 6 µM, respectively.
58
Figure 2-4: (A) Stopped-flow fluorescence of wild-type L1 with nitrocefin. In a typical reaction, 5-10 µM wild-type L1was mixed with various concentrations of nitrocefin, and the reaction was monitored for up to 1 second at 10 oC, using the conditions described in Materials and Methods. The data were fitted to a double exponential using SigmaPlot v. 6.10. (B) Plot of observed rate constant versus concentration of nitrocefin. Solid lines were fitted to the data as described in Materials and Methods.
59
Table 2-6: Km and KS values for Wild-type L1 and mutants with nitrocefin as substrate.
Enzyme Km (µM) KS (µM) Wild-type 4 ± 1 38 ± 5 S224A 7 ± 1 39 ± 10 S224D 11 ± 5 213 ± 63 S224K 14 ± 2 33 ± 5 F158A 4 ± 1 N.D. I164A 8 ± 2 31 ± 11 Y228A 23 ± 3 85 ± 9 Y228F 36 ± 16 22 ± 6 N233D 9 ± 2 26 ± 8 N233L 7 ± 2 25 ± 7 N.D.- not determined
2.4.5 Ile164 and Phe158 Mutants.
All crystallographically characterized metallo-β-lactamases have a flexible amino acid chain that extends over the active site (37,42,44-49). Previous NMR studies on CcrA have shown that this loop “clamps down” on substrate or inhibitor upon binding, and there is speculation that the distortion of substrate upon clamping down of the loop may drive catalysis
(50). The crystal structure of L1 showed that there is a large loop that extends over the active site, and modeling studies have predicted that two residues, Ile164 and Phe158, make significant contacts with large, hydrophobic substituents at the 2’ or 6’ positions on penicillins, cephalosporins, or carbapenems (37). To test this prediction, Ile164 and Phe158 were changed from large, hydrophobic residues to alanines to afford the I164A and F158A mutants.
Small-scale growth cultures demonstrated the I164A and F158A mutants were over- expressed at levels comparable to that of wild-type L1 (data not shown). Large-scale over-
60
expression and purification of the mutants resulted in comparable quantities of isolatable
enzymes, which had identical CD spectra as wild-type L1 and bound slightly less Zn(II) than
wild-type L1 (Table 2-2). All steady-state kinetic studies were conducted in buffers containing
100 µM ZnCl2 to ensure that both metal binding sites were saturated during the studies.
When using the cephalosporins, nitrocefin, cefoxitin, and cephalothin, as substrates and
the I164A mutant, there were 2- to 10-fold (only for cefoxitin) increases in Km and 2- to 4-fold
increases in kcat observed (Table 2-3). However when cefaclor was used as substrate, the I164A
mutant exhibited a 3-fold decrease in Km and a 1.5-fold decrease in kcat (Table 2-3). On the other
hand, the Km and kcat values for the I164A mutant when the penicillins or carbapenems were used as substrates were very similar to those numbers exhibited by wild-type L1 (Tables 2-4 and 2-5).
When the cephalosporins were used as substrates for the F158A mutant, the Km values
observed were 7- to 31-fold higher than those determined for wild-type L1, and surprisingly, the
kcat values were 2- to 31-fold higher than those exhibited by wild-type L1 (Table 2-3). As with
the I164A mutant, the changes in Km and kcat for the penicillins and carbapenems were relatively
small, as compared with the values obtained with the cephalosporins (Table 2-4).
Rapid-scanning Vis studies on nitrocefin hydrolysis by I164A and F158A showed a
marked decrease in intermediate accumulation, with the I164A mutant generating < 0.30 µM
intermediate and the F158A producing no detectable intermediate (Figure 2-3). Stopped-flow
o fluorescence studies at 10 C resulted in a KS value of 31 + 11 µM for the I164A mutant. The reaction of F158A with nitrocefin was so rapid, we could not determine a KS value for this mutant.
61
2.5 Discussion
β-Lactam containing antibiotics constitute the largest class of antibiotics, and these compounds are relatively inexpensive to produce, cause minor side effects, and are effective towards a number of bacterial strains. Nonetheless, bacterial resistance to these antibiotics is extensive, most commonly due to the bacterial production of β-lactamases (10,51). In fact, there have been over 300 distinct β-lactamases reported, and most of these enzymes utilize an active site serine group to nucleophilically attack the β-lactam carbonyl, resulting in a hydrolyzed product that is covalently attached to the active site. To combat these enzymes, β-lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam have been given in combination with a β-lactam containing antibiotic to treat bacterial infections (52). One class of β-lactamases that are particularly unaffected by the known β-lactamase inhibitors and have been shown to hydrolyze almost all known β-lactam containing antibiotics including late generation carbapenems at high rates are the metallo-β-lactamases (14-19). Although there are no reports of metallo-β-lactamases isolated from major pathogens (51,53), these enzymes are produced by pathogens such as B. fragilis, S. maltophilia, and P. aeruginosa. It is inevitable that the continued and extensive use of β-lactam antibiotics will result in a major pathogen that produces a metallo-β-lactamase.
Efforts to solve the crystal structure of one of the metallo-β-lactamases with a bound substrate molecule have failed, most likely due to the high activity of the enzymes towards all β- lactam containing antibiotics (37,54). Therefore, computational studies have been used extensively to study substrate binding, the role of the Zn(II) ions in catalysis, the protonation state of the active site, and inhibitor binding (37,42,55-59). All of the substrate binding models have made assumptions before the substrate was docked into the active site (37,42), and some of
62
these assumptions have been shown to be invalid for certain substrates (43). With L1, two key assumptions were made: (1) the bridging hydroxide functions as the nucleophile during catalysis and (2) Zn1 coordinates the β-lactam carbonyl (37). With these assumptions and after energy
minimizations, Ser224 was predicted to hydrogen bond to the substrate carboxylate (37),
reminiscent of the role predicted for Lys224 in CcrA (42). Ullah et al. predicted that Phe158 and
Ile164 form hydrophobic interactions with bulky substituents on the substrate, suggesting that
the loss of these residues would only affect binding of substrates with large aromatic substituents
(37). In the modeling studies on CcrA (42), Asn233 was predicted to interact with the β-lactam
carbonyl on substrate, and mutagenesis studies have supported this prediction (43). Although
Asn233 is sequence conserved in L1 (35), it is located 14 Å away from the modeled position of
the β-lactam carbonyl and was predicted not to play a role in substrate binding to L1 (37). On
the other hand, the substrate-binding model predicted that Tyr228 was in position to offer a
hydrogen bond to the β-lactam carbonyl and participate in an oxyanion hole that was proposed to
form as the substrate was hydrolyzed (37). By using the crystal structure and modeling studies
on L1, Ullah et al. proposed a reaction mechanism for the enzyme (37). To test this proposed
mechanism and the proposed roles of the amino acids discussed above, site-directed mutagenesis
studies were conducted on metallo-β-lactamase L1 and reported herein.
The overlap extension method (60) was used to prepare the site-directed mutants, and a
variety of studies were used to probe whether the single point mutations resulted in large
structural changes in the mutant enzymes. (1) The over-expression levels of mutants were
analyzed with SDS-PAGE to ensure that the mutations did not result in changes in the over-
expression levels of the enzymes. With a few L1 mutants and with other enzyme systems in the
lab, single point mutations often result in depressed levels of over-expression (61). In the case of
63
the mutants described here, all of the mutants over-expressed at levels comparable to wild-type
L1 (data not shown). (2) The total amounts of the mutants isolatable after chromatography were
compared with wild-type L1 levels. We have found, in particular with metal binding mutants of
L1 (G. Periyannan, R.B. Yates, and M.W. Crowder, unpublished results) and glyoxalase II (61), that single point mutations can result in over-expressed mutants being processed into inclusion bodies and unisolatable as soluble proteins. In the case of the mutants described here, all of the
mutants were isolated at levels comparable to wild-type L1. (3) CD spectra were collected for
all mutants and compared to the spectrum of wild-type L1. Although we did not expect a large
change in the secondary structure of L1 upon single point mutations, CD spectroscopy is the most common structural technique to characterize site-directed mutants. All of the mutants described here exhibited CD spectra that were very similar, or identical, to that of wild-type L1
(Figure 2-2). (4) Metal analyses on the mutants were used to probe whether point mutations caused a significant change to the metal binding site as to preclude metal binding. The crystal
structures of the metallo-β-lactamases reveal a complex and far-reaching hydrogen-bonding network around the metal binding sites, and disruption of this network is predicted to affect metal binding (37,42,44,45,48,49,62,63). With all of the mutants described here except the
S224K mutant, each mutant binds wild-type or near-wild-type levels of Zn(II) after purification.
The S224K mutant exhibited a 50% reduction in metal binding (Table 2-2), and we postulate this
is due to electrostatic repulsions between the newly introduced Lys with Zn2. In spite of the mutants binding significant amounts of Zn(II), we included 100 µM ZnCl2 in all of the kinetic buffers to ensure saturation of the metal binding sites and to facilitate direct comparison of the kinetic data. (5) All mutants were stable to multiple freeze/thaw cycles and to prolonged storage
(> 3 weeks) at 4 oC, retaining > 95% of their activity. With these five lines of evidence, we were
64
confident that none of the point mutations resulted in large structural changes in L1 and that any
kinetic differences could be attributed to the changed amino acid.
As a first approximation of substrate binding, we examined the steady-state kinetics of 4
cephalosporins, 2 penicillins, and 3 carbapenems (Tables 2-3 to 2-5) and compared the Km values of the mutants with those of wild-type L1. The substrates tested were chosen because they exhibited low Km values in previous kinetic studies (36), and we believed that we could
saturate the enzymes with substrate even if there was large change in binding with the point
mutations. The Tyr228 mutants exhibited increased Km values for 8 of the 9 substrates tested,
with the smallest changes in Km observed when the carbapenems were used as the substrate.
This result supports the proposed role of Tyr228 in substrate binding. In contrast, the results on
the Ser224 mutants suggest that this residue is not important in substrate binding, since the
S224A and S224K mutants did not exhibit any significant increases (by a factor of > 10) in Km
for any of the substrates tested. Only when Ser224 was replaced with an Asp residue was there
significant increases in the observed Km value for 6 of the 9 substrates tested, and the largest
changes were exhibited when the penicillins were used as substrates. This result supports the
observation of differential binding modes of substrates to the β-lactamases, depending on the
structure of the substrate (43,64,65). The only remaining mutants that exhibited significant
changes in the Km values were the I164A and F158A mutants. The I164A mutant exhibited
increased Km values only when using cefoxitin as the substrate, suggesting an interaction of the isoleucine group with the methoxy group on cefoxitin. The F158A mutant exhibited higher Km
values when using the cephalosporins as substrates, suggesting an interaction of the
cephalosporins’ substituents with the phenylalanine on the loop that extends over the active site.
65
None of the other mutants exhibited vastly different values for Km with any of the substrates
tested.
An examination of the kcat values of the mutants revealed some surprising results. The
S224D mutants displayed decreased kcat values for 7 of the 9 substrates tested. Since similar
results were not observed with the S224K and S224A mutants, we do not propose a catalytic role
for Ser224. Instead, we predict that the insertion of an aspartic acid into the active site at
position 224 results in a change in the hydrogen bonding network in L1; this hydrogen bonding
network is extensive in all metallo-β-lactamases that have been characterized
crystallographically (37,42,44,45,48,49,62,63). The N233D mutant also exhibited greatly
reduced kcat values for biapenem and meropenem but not for imipenem or any of the other
substrates tested. This mutation is also predicted to affect the hydrogen bonding network around the active site, and apparently, interactions of the enzyme with the 4’ substituent of the carbapenems has an effect on catalysis. More surprisingly are the increases in kcat of the F158A
mutants. We are uncertain why the mutation of residues on the loop that extends over the active
site would affect kcat, since substrate and product binding have been predicted to be very fast in the reaction of nitrocefin with L1. However, we do note that the kcat/Km values of wild-type L1
and F158A differ by a factor less than 2.
The inability to propose a consistent binding model also supports the recent proposal that
different substrates of L1 are hydrolyzed by different mechanisms and further suggests that using
steady-state kinetic constants may not be a valid way to probe substrate binding to L1. In
addition, the minimal kinetic mechanism of nitrocefin hydrolysis by L1 has been reported, and
this mechanism predicts that Km does not accurately reflect substrate binding. By using this
mechanism (39), Km is equal to {(k-1 + k2)k3k4} / {k1(k3k4 + k2k4 + k2k3}. To probe more directly
66
the reaction, stopped-flow absorbance studies were conducted, and the substrate decay rates (390
nm) were studied as a function of nitrocefin concentration. While nitrocefin is a nontypical
substrate, as a result of the dinitro-substituted styryl substituent (40), it is the substrate about
which the most is known about its hydrolysis mechanism. Therefore, kinetic studies with
nitrocefin as substrate allowed for us to evaluate the effect of point mutations on the reaction mechanism of L1. There was no clear dependence on substrate decay rates with nitrocefin concentration (data not shown). We did note though that the amount of intermediate formed
during the reactions varied considerably depending upon which mutant of L1 was used in the
study. All of the mutants exhibited decreases in intermediate formation, and the S224D, S224K,
F158A, N233D, and N233L mutants yielded rapid-scanning data consistent with no detectable
intermediate. These same mutants exhibited vastly differing Km values. Clearly there is no
correlation of Km with the presence of the reaction intermediate. Apparently, the ability to
observe the intermediate is not governed entirely by the choice of substrate (40), and it also
depends on precise arrangement of active site residues. It is also possible that the site-directed
mutants could be utilizing a different mechanism to hydrolyze nitrocefin (66).
Recently, Spencer and co-workers reported that stopped-flow fluorescence studies can be
used to monitor the reaction of L1 with nitrocefin and that an initial binding step can be directly
monitored (40). By increasing the concentration of nitrocefin, the rate of the initial binding step increased to a maximum, and fitting of these data yielded a binding constant (called KS herein)
for nitrocefin. Each of the L1 mutants were studied using the stopped-flow fluorescence studies,
and the resulting data were fitted as reported by Spencer et al. (40) (Table 2-6). All of the
mutants exhibited KS values identical, within error, to wild-type L1, except the S224D and the
Y228A mutants. The placement of a negative charge at position 224 drastically affects
67
nitrocefin binding and results in a 6-fold decrease in binding affinity (Table 2-6). To a lesser
degree, the aromatic portion of Tyr228 must have an effect on the binding site as the KS value for nitrocefin binding to this mutant is decreased by a factor of 2; however, the hydroxyl group
probably does not form a hydrogen bond to the substrate as proposed. By using nitrocefin as
substrate and Km values alone, a completely different conclusion is reached regarding important
substrate binding residues. The results presented here suggests that none of the residues in this
study are essential for tight nitrocefin binding, possibly because other parts of the active site
accommodate the loss of certain binding contacts.
Spencer et al. also reported stopped-flow fluorescence studies when using cefaclor and
meropenem as substrates, and Ks values for these substrates were reported to be 710 + 180 and
272 + 112 µM, respectively (40). However in our hands, the rates of substrate hydrolysis were
so fast when using wild-type L1 that we could not use substrate concentrations high enough to
saturate the enzyme. Similarly, we could not determine Ks values for penicillin G or ampicillin
because the observed rates of hydrolysis at low substrate concentrations were too fast to observe
data, even at 10 oC.
2.6 Conclusion
The results presented herein indicate that none of the active site residues identified with
computational studies are essential for tight substrate binding. These data also indicate that the
use of Km values to describe substrate binding to L1 is unreliable and that there is no correlation
between intermediate accumulation and substrate binding affinity. These results demonstrate
that new computational studies are now needed to probe substrate binding to L1, and these
studies are currently underway. The results presented herein can be used to guide these new
68
computational studies, which will lead to the design of potential inhibitors and hopefully a way to combat penicillin resistance in bacteria.
69
2.7 Acknowledgements
The authors acknowledge NIH R29 AI40052 (to M.W.C) for funding this work, NSF DBI-
0070169 for funding the CD spectropolarimeter, and NSF CHE-0076936 for funding the stopped-flow UV-Vis spectrophotometer used in these studies. RBY was a recipient of a 2001
Miami University Summer Scholars award. The authors would like to thank Drs. James Spencer and Tim Walsh for helpful conversations.
70
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76
Chapter 3
Metal binding Asp120 in metallo-β-lacatamse L1 from
Stenotrophomonas maltophilia plays a crucial role in
catalysis†
James D. Garrity, Anne L. Carenbauer, Lissa R. Herron, and Michael W. Crowder*
Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford,
Ohio 45056
*Corresponding author: phone: (513) 529-7274 fax: (513) 529-5715 e-mail: [email protected]
†This work was supported by the National Institutes of Health (AI40052 and GM40052). Funds to purchase the CD spectrapolarimeter (DBI-0070169) and the Stopped-flow UV-Vis fluorescence spectrophotometer (CHE-0076936) were provided by the National Science
Foundation.
Running title: Asp120 mutants of metallo-β-lactamase L1
77
Abbreviations1
CcrA, metallo-β-lactamase from Bacteroides fragilis; ICP-AES, Inductively Coupled Plasma –
Atomic Emission Spectroscopy; IPTG, isopropyl-β-thiogalactoside; L1, metallo-β-lactamase from Stenotrophomonas maltophilia; LB, Luria-Bertani; MTCN, MES-TRIS-CHES-NaCl buffer.
78
3.1 Summary
Metallo-β-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal binding aspartic acid appears to be common to most dinuclear, metal containing, hydrolytic enzymes; particularly those with a β-lactamase fold. In an effort to probe the catalytic and metal binding role of Asp120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses,
CD spectroscopy, and pre-steady state and steady state kinetics. The D120C, D120N, and
D120S mutants were shown to bind 1.6 ± 0.2, 1.8 ± 0.2, and 1.1 ± 0.2 moles of Zn(II) per monomer, respectively. The mutants exhibited 10-1000 fold drops in kcat values as compared to wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pKa values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme.
This work also confirms that Asp120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate- limiting proton transfer in the hydrolysis reaction.
79
3.2 Introduction
The ability of bacteria to acquire resistance to antibiotics is a serious problem that
continues to challenge modern society (1). Excessive and often misuse of antibiotics in the clinic
and for agricultural purposes has resulted in tremendous selective pressure for antibiotic resistant
bacteria (2). These bacteria utilize a variety of methods to become resistant, including
modification of cell wall components to prevent antibiotic binding, production of efflux pumps
that transport the antibiotic out of the cell, and the production of enzymes that hydrolyze and
render the antibiotic ineffective (1, 2).
The most common and least expensive effective antibiotics currently used are the β- lactams, such as carbapenems, cephalosporins, and penicillins (3, 4). These antibiotics are mechanism-based inhibitors of transpeptidase, a bacterial enzyme required for the production of a strong viable cell wall (5-7). In response to their widespread use, an increasing number of bacterial strains have acquired the ability to produce β-lactamases, enzymes that hydrolyze and render β-lactam antibiotics ineffective. There are over 300 distinct β-lactamases known, and
Bush has classified these into four distinct groups based on their molecular properties (8, 9).
One of the more troubling of these is group 3 (class B), the metallo-β-lacatamases, which are
Zn(II) dependent enzymes that hydrolyze nearly all known β-lactams and for which there are no or very few clinically useful inhibitors (10-17). To date, there are no reports of a metallo-β-
lactamase being isolated from a major pathogen (18, 19); however these enzymes are produced
by a variety of minor pathogens such as B. fragilis, P. aeruginosa, and S. maltophilia, and the
continued extensive use of β-lactam antibiotics will inevitably result in the production of a
metallo-β-lactamase by a major pathogen.
80
There is significant diversity within the metallo-β-lactamases and Bush, based on their amino acid sequence identity and substrate affinity, has further divided them into three subgroups (9). A similar grouping scheme based on structural properties has also been offered
(20). The diversity of these subgroups is best exemplified by their vastly differing efficacies towards non-clinical inhibitors; these differences lead to the prediction that finding a single inhibitor for all metallo-β-lactamases may not be possible (17, 21-29). To address this problem, we are currently characterizing a representative enzyme from each of the metallo-β-lactamase subgroups with the goal of identifying common structural and mechanistic similarities that can be targeted for the generation of clinically useful inhibitors. This work describes out efforts on metallo-β-lactamase L1 from Stenotrophomonas maltophilia.
S. maltophilia is an important pathogen in nosocomial infections of immunocompromised patients suffering from cancer, cystic fibrosis, drug addiction, and AIDS and in patients with organ transplants and on dialysis (30-32). This organism is inherently resistant to most antibiotics due to its low outer membrane permeability (33) and to β-lactams due to the production of two chromosomally expressed β-lactamases, a group 2e β-lactamase called L2 and a group 3c β-lactamase called L1 (34, 35). L1 has been cloned, over-expressed, and partially characterized by kinetic and crystallographic studies (36, 37). The enzyme exists as a homotetramer of ca. 118 kDa in solution and in the crystalline state, tightly binding two Zn(II) ions per subunit. The Zn1 site has 3 histidine residues and 1 bridging hydroxide as ligands, and the Zn2 site has 2 histidines, 1 aspartic acid, 1 terminally bound water, and the bridging hydroxide as ligands (Figure 3-1).
Efforts to solve the crystal structure of one of the metallo-β-lactamases with a bound substrate molecule have failed, most likely due to the high activity of the enzymes, even in the
81
crystalline state, towards all β-lactam containing antibiotics (37, 38). Therefore, computational studies have been used extensively to study substrate binding, the role of the Zn(II) ions in catalysis, the protonation state of the active site, and inhibitor binding (37, 39-44). All of these models have made assumptions before the substrate was docked into the active site (37, 43), and some of these assumptions have been shown to be invalid for certain substrates (45). With L1, three key assumptions were made: (1) the bridging hydroxide functions as the nucleophile during catalysis, (2) Zn1 coordinates the β-lactam carbonyl, and (3) Zn2 coordinates the amide nitrogen of the β-lactam ring (37).
One of the residues identified through computational studies to be catalytically important in L1 is the aspartic acid at position 120 (the standard numbering scheme for class B β- lactamases is utilized herein (20)). From the crystal structure, Asp120 clearly coordinates Zn2,
with its unbound oxygen located directly under the bridging group in the active site (37). This is
a geometry shared by many dinuclear metal-containing hydrolytic enzymes, including other
metallo-β-lactamases and dioxygenases (46). Therefore we believe that the findings of this work
are applicable beyond L1 from Stenotrophomonas maltophilia. In addition to its role as a metal
binding ligand, it has been hypothesized that Asp120 electrostatically interacts with the bridging
hydroxide, properly orienting it for nucleophilic attack on the substrate (37). This work
describes our efforts to test this prediction and further our understanding the role of Asp120 in
both metal binding and substrate turnover. To probe the importance of this residue, three mutant
enzymes were generated. Asp120 was changed to a cysteine, an asparagine, and a serine to
create D120C, D120N, and D120S, respectively (Figure 3-1). Cysteine was substituted to allow
for continued binding of Zn2 but eliminate any interaction of the residue with the bridging
hydroxide/water. Asparagine was chosen as a chemically different but structurally similar
82
surrogate for aspartic acid, allowing for continued binding of Zn2 and providing a moiety for interaction with the bridging hydroxide/water. Replacement of aspartic acid with serine was intended to remove both the metal binding ability of the residue at this position and any ability to interact with the bridging hydroxide/water.
83
Figure 3-1: Pictorial representation of the active site of wild-type L1 rendered using Chem Draw Ultra v. 5.0 (top) and Rasmol 2.6 (bottom). Drawing indicates the proposed interaction of Asp120 with the bridging hydroxide and changes affected by each of the L1 mutants. The coordinates for the Rasmol figure were obtained from the Protein Databank using the accession number 1sml.
84
3.3 Experimental Procedures
E. coli strains DH5α and BL21(DE3) were obtained from Gibco BRL and Novagen,
respectively. Plasmids pET26b and pUC19 were purchased from Novagen. Primers for
sequencing and mutagenesis studies were purchased from Integrated DNA Technologies.
Deoxynucleotide triphosphates (dNTP's), MgSO4, thermopol buffer, Deep Vent DNA
polymerase, and restrictions enzymes were purchased from Promega or New England Biolabs.
Polymerase chain reaction was conducted using a Thermolyne Amplitron II unit. DNA was
purified using the Qiagen QIAQuick gel extraction kit or Plasmid Purification kit with QIAGEN-
tip 100 (Midi) columns. Wizard Plus Minipreps were acquired from Promega. Luria-Bertani
(LB) media in powder form was purchased from GIBCO. Isopropyl-β-thiogalactoside (IPTG),
Biotech grade, was procured from Anatrace. Phenylmethylsulfonylfluoride (PMSF) was
purchased from Sigma. Protein solutions were concentrated with an Amicon ultrafiltration cell
equipped with YM-10 DIAFLO membranes from Amicon, Inc. Dialysis tubing was prepared
using Spectra/Por regenerated cellulose molecular porous membranes with a molecular weight
cut-off of 6-8,000 g/mol. Q-Sepharose Fast Flow was purchased from Amersham Pharmacia
Biotech. Nitrocefin was purchased from Becton Dickinson, and solutions of nitrocefin were filtered through a Fisherbrand 0.45 micron syringe filter. Cefaclor, cefoxitin, and cephalothin were purchased from Sigma; penicillin G and ampicillin were purchased from Fisher.
Imipenem, meropenem, and biapenem were generously supplied by Merck, Zeneca
Pharmaceuticals, and Lederle (Japan), respectively. All buffers and media were prepared using
Barnstead NANOpure ultrapure water.
85
A. Carenbauer generated the mutants for this study. The over-expression plasmid for L1, pUB5832, was digested with NdeI and HindIII, and the resulting ca. 900 bp piece was gel purified and ligated using T4 ligase into pUC19, which was also digested with NdeI and HindIII, to yield the cloning plasmid pL1pUC19. Mutations were introduced into the L1 gene by using the overlap extension method of Ho et al. (47), as described previously (48, 49). The oligonucleotides used for the preparation of the mutants are as follows: D120C forward,
CACgCACACgCCTgCCATgCCggACCggTg; D120C reverse
CACCggTCCggCATggCAggCgTgTgCgTg; D120N forward,
CACgCACACgCCAACCATgCCggACCggTg; D120N reverse,
CACCggTCCggCATggTTggCgTgTgCgTg; D120S forward,
CACgCACACgCCAgCCATgCCggACCggTg; D120S reverse,
CACCggTCCggCATggCTggCgTgTgCgTg. The ca. 900 bp PCR products were digested with
NdeI and HindIII and ligated into pUC19. The DNA sequences were analyzed by the
Biosynthesis and Sequencing Facility in the Department of Biological Chemistry at Johns
Hopkins University. After confirmation of the sequence, the mutated pL1pUC19 plasmid was digested with NdeI and HindIII, and the 900 bp, mutated L1 gene was gel purified and ligated into pET26b to create the mutant over-expression plasmids. To test for over-expression of the mutant enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated over- expression plasmids, and small scale growth cultures were used (48). Large-scale (4 L) preparations of the L1 mutants were performed as described previously (36). Protein purity was ascertained by SDS-PAGE.
The concentrations of L1 and the mutants were determined by measuring the proteins'
-1 - absorbances at 280 nm and using the published extinction coefficient of ε280nm = 54,804 M •cm
86
1 (36) or by using the method of Pace (50). Before metal analyses, the "as isolated" protein
samples were dialyzed versus 3 X 1 L of metal-free, 50 mM HEPES, pH 7.5 over 96 hours at 4
°C. A Varian Inductively Coupled Plasma Spectrometer with atomic emission spectroscopy
detection (ICP-AES) was used to determine the metal content of multiple preparations of wild
type L1 and L1 mutants. Calibration curves were based on four standards and had correlation
coefficient limits of at least 0.9950. The final dialysis buffer was used as a blank. The emission
line of 213.856 nm is the most intense for zinc and was used to determine the Zn content in the
samples. The errors in metal content data reflect the standard deviation (σn-1) of multiple
enzyme preparations. A second analysis of metal content was preformed on enzyme samples
that were incubated for one hour, on ice, in buffer containing a final concentration of 100 υM
ZnCl2. These "metal saturated" samples were then dialyzed versus 2 X 1 L metal free buffer for
a total of 4 hours, and metal content analyzed by ICP-AES as described above.
Circular dichroism samples 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 diluted with
final dialysis buffer to ~75 µg/mL. A JASCO J-810 CD spectropolarimeter operating at 25 oC was used to collect CD spectra.
L. Herron contributed to repetitions of steady-state kinetics. Assays were conducted at
o 25 C in 50 mM cacodylate buffer, pH 7.0, containing 100 µM ZnCl2 on a HP 5480A diode array
UV-Vis spectrophotometer. The changes in molar absorptivities (∆ε) used to quantitate
-1 -1 products were (in M cm ): nitrocefin, ∆ε485 = 17,420; cephalothin, ∆ε265 = -8,790; cefoxitin,
∆ε265 = -7,000; cefaclor, ∆ε280 = -6,410; imipenem, ∆ε300 = -9,000; meropenem, ∆ε293 = -7,600;
biapenem, ∆ε293 = -8,630; ampicillin, ∆ε235 = -809; and penicillin G, ∆ε235 = -936. When
possible, substrate concentrations were varied between 0.1 to 10 times the Km value, and changes
87
in absorbance (∆A) versus time data were measured for a period of 60 seconds for each substrate
concentration. In kinetic studies using substrates with low Km values (cefoxitin, nitrocefin, and
cephalothin) or with small ∆ε values (penicillin and ampicillin), substrate concentrations were
varied between ~ Km and 10 times Km, and as much of the linear portion of the ∆A versus time
data as possible was used to determine the velocity. Steady-state kinetics constants, Km and kcat, were determined by fitting initial velocity versus substrate concentration data directly to the
Michaelis equation using Igor Pro (36). The reported errors reflect fitting uncertainties. All steady-state kinetic studies were performed in triplicate with recombinant L1 from at least three different enzyme preparations.
pH dependence studies were performed as described above but using a buffer system containing 25 mM MES, 50 mM TRIS, 25 mM CHES, 10 mM NaCl, and 100 µM ZnCl2
(MTCN). Buffers for each pH tested were made from a common 10X stock of MTCN buffer.
The pH of each was then adjusted to the desired value using either 6 M HCl or 10 M NaOH, and
the appropriate volume of aqueous ZnCl2 was added to reach a concentration of 100 µM. Km
and kcat were determined as described above, and log plots of those values versus pH were generated using Igor Pro.
Steady-state kinetic assays were conducted at 25 oC in 50 mM cacodylate buffer, pH 7.0,
containing 100 µM ZnCl2 and ranging in D2O concentrations from 0 to 100 %, on a HP 5480A
diode array UV-Vis spectrophotometer. Steady-state kinetics constants, Km and kcat, were determined by fitting initial velocity versus substrate concentration data directly to the Michaelis equation using Igor Pro (36). Plots of kcat versus % D2O were generated using Igor Pro. The
errors reported are generated by Igor Pro as a result of Chi square minimization.
88
Rapid-scanning Vis spectra of nitrocefin hydrolysis by L1 and the L1 mutants were
collected on a Applied Photophysics SX.18MV stopped-flow spectrophotometer equipped with
an Applied Photophysics PD.1 photodiode array detector and a 2 mm path length optical cell.
The wild-type L1 experiment consisted of 25 µM enzyme and 5 µM nitrocefin in 50 mM
cacodylate buffer, pH 7.0, containing 100 µM ZnCl2, the reaction temperature was thermostated
at 25 oC, and the spectra were collected between 300 and 725 nm. Data from at least three
experiments were collected and averaged. Absorbance data were converted to concentration data
as described previously by McMannus and Crowder (51). Due to weaker binding and slower
turnover of substrate with the L1 mutants, enzyme concentrations of 50 µM were used with 5
µM nitrocefin utilizing the same buffer system and experimental conditions as in the wild-type
L1 experiment. Stopped-flow fluorescence studies of nitrocefin hydrolysis by L1 were performed on an Applied Photophysics SX.18MV spectrophotometer, using an excitation wavelength of
295 nm and a WG320 nm cut-off filter on the photomultiplier. These experiments were conducted at 10 oC using the same buffer as in the rapid-scanning Vis studies. Fluorescence data
were fitted to kobs = {(kf [S]) / KS + [S])} + kr as described previously (52) or to kobs = kf[S] + kr
by using CurveFit v. 1.0.
89
3.4 Results
Wild-type L1, D120C, D120N, and D120S were over-expressed in Escherichia coli and
purified as previously described (36). This procedure produced an average of 50-60 mg of >95%
pure, active protein per 4L of growth culture. Circular dichroism spectra were collected on
samples of wild-type and each of the mutants to ensure the proteins produced using the pET26b
over-expression system had the correct secondary structure. The CD spectra (data not shown) of wild-type L1 and the mutants were identical. Metal analyses on multiple preparations of wild- type L1 demonstrated that the enzyme binds 1.9 ± 0.2 Zn(II) ions per monomer (Table 3-1), in agreement with previous results (36). Metal analysis on multiple preparations of D120C,
D120N, and D120S showed 1.6 ± 0.2, 1.8 ± 0.2, and 1.1 ± 0.2 Zn(II) ions per monomer, respectively.
Table 3-1: Metal analysis, and Ks and kH/kD values with nitrocefin of wild-type L1 and L1 mutants. Enzyme K (µM) Zn (II) Content S Solvent Isotope with (moles Zn (II) / k /k nitrocefin H D mole monomer) Wt 1.9 ± 0.2 38 ± 5 2.08 ± 0.03 D120C 1.6 ± 0.2 37 ± 6 1.47 ± 0.05 D120N 1.8 ±0.2 97 ± 14 5.36 ± 0.22 Unable to D120S 1.1 ± 0.2 1.87 ± 0.06 determine.
90
Steady-state kinetic constants Km and kcat were determined for wild-type L1 and each of
the mutants with nine substrates. These values are presented in Table 3-2. When using
nitrocefin as substrate and 50 mM cacodylate, pH 7.0, as the buffer, wild-type L1 exhibited a kcat
-1 value of 38 ± 1 s and a Km value of 12 ± 1 µM. The inclusion of 100 µM ZnCl2 in the assay
buffer resulted in slightly lower values of Km and higher values for kcat (36). The inclusion of
higher concentrations of Zn(II) did not further affect the steady-state kinetic constants. Four
cephalosporins; cefaclor, cefoxitin, cephalothin, nitrocefin, three carbapenems; biapenem,
imipenem, and meropenem, and two penicillins; penicillin G and ampicillin, were utilized as
representatives of the three major classes of β-lactam containing antibiotics. L1’s preference for
penicillins and carbapenems over cephalosporins, as exemplified by the kcat values, is in
agreement with previous studies and supports L1’s placement in the β-lactamase Bc family (9).
For all substrates tested, the mutant enzymes exhibited reduced kcat values as compared to wild-
type L1, with D120N being the most active of the three. A general trend of activity was
observed for all substrates tested: wild-type L1 > D120N > D120C and D120S (Table 3-2).
There does not seem to be an observable trend with respect to the activities of D120C and
D120S. In most cases they exhibited kcat values very similar to one another. D120N exhibited
an activity fourteen to thirty-seven times lower than wild-type L1 towards carbapenems.
However this was generally ten or more times greater than the activity of either D120C or D120S with the same substrates. With the exception of cefoxitin, D120N exhibited kcat values seven to sixteen times lower than wild-type L1, but fourteen (or more) times greater than that of D120C or D120S toward cephalosporins. While the over-all trend with kcat values holds true for
cefoxitin, it would appear that none of the
91
Table 3-2: Steady-state kinetics constants for wild-type L1 and L1 mutants with (2a) carbapenems, (2b) cephalosporins, and (2c) -1 -1 penicillins. Units for kcat / Km are s · µM NA = no activity
Biapenem Imipenem Meropenem 3-2a -1 -1 -1 kcat (s ) Km (µM) kcat / Km kcat (s ) Km (µM) kcat / Km kcat (s ) Km (µM) kcat / Km Wt 134 ± 4 32 ± 1 4.2 370 ± 5 57 ± 7 6.5 157 ± 9 15 ± 4 10 D120C 0.30 ± 0.02 45 ± 7 0.0067 0.36 ± 0.02 65 ± 7 0.0055 0.29 ± 0.01 60 ± 4 0.0048 D120N 3.6 ± 0.4 128 ± 2 0.028 15 ± 2 310 ± 64 0.048 10.6 ± 0.1 70 ± 4 0.15 D120S 0.50 ± 0.01 81 ± 4 0.0062 0.63 ± 0.02 94 ± 6 0.0067 0.75 ± 0.04 97 ± 10 0.0077 Cefaclor Cefoxitin Cephalothin Nitrocefin 3-2b -1 -1 -1 -1 kcat (s ) Km kcat / kcat (s ) Km kcat / kcat (s ) Km kcat / kcat (s ) Km kcat / Km (µM) Km (µM) Km (µM) Km (µM) 42 ± 1 13 ± 1 3.2 1.9 ± 0.1 1.1 ± 1.7 82 ± 5 8.9 ± 9.2 41 ± 1 4.1 ± 10 Wt 0.1 1.5 1.0 0.30 ± 98 ± 15 0.0031 0.56 ± 55 ± 8 0.010 0.31 ± 33 ± 3 0.0094 0.18 ± 69 ± 8 0.0026 D120C 0.02 0.04 0.01 0.01 6.3 ± 0.6 550 ± 0.011 1.53 ± 98 ± 9 0.016 8.1 ± 0.3 150 ± 0.054 2.54 ± 68 ± 4 0.037 D120N 76 0.07 11 0.05 0.28 ± 80 ± 8 0.0035 0.34 ± 53 ± 5 0.0064 0.37 ± 46 ± 3 0.0080 103 ± 0.00037 D120S 0.028 ± 0.004 0.01 0.01 0.01 16
Ampicillin Penicillin G 3-2c -1 -1 kcat (s ) Km (µM) kcat / Km kcat (s ) Km (µM) kcat / Km Wt 520 ± 10 55 ± 5 9.5 600 ± 100 38 ± 2 16 D120C <0.01 NA NA <0.01 NA NA D120N 77 ± 10 1332 ± 257 0.058 45 ± 2 384 ± 40 0.12 D120S <0.01 NA NA <0.01 NA NA
enzymes, including wild-type L1, hydrolyze this compound well. On average, D120N
showed ten times less activity towards penicillins than wild-type L1. Interestingly
hydrolysis of penicillins was virtually undetectable with both D120C and D120S. Since
Km values are often used as a first approximation of substrate binding, the Km values
exhibited by the mutants were compared to those of wild-type L1. All three mutants exhibited larger Km values than wild-type L1. Typically with the substrates tested,
D120N had the largest Km values; however, a clear trend for Km values was not apparent.
To probe further the binding of nitrocefin to wild-type L1 and the mutants, stopped-flow fluorescence studies were conducted as previously described (52). The reaction of enzyme with nitrocefin under steady-state conditions at 10 oC resulted in a
rapid decrease in fluorescence followed by a rate-limiting return of fluorescence (data not
shown). Fitting of the data, as described by Spencer et al. (52), yielded KS values for
wild-type L1 and the three mutants (Table 3-1).
Rapid-scanning visible spectra of 25 µM wild-type L1 with 5 µM nitrocefin
demonstrated a decrease in absorbance at 390 nm, an increase at 485 nm, and a rapid
increase and slower decrease in absorbance at 665 nm. These spectra are similar to those
previously reported for wild-type L1 and nitrocefin (51), and the features can be
attributed to substrate decay, product formation, and formation and decay of a ring-
opened, nitrogen anionic intermediate, respectively (51, 53, 54). Under these conditions,
~0.7 µM intermediate was formed during the first 10 milliseconds of the reaction (Figure
3-2), and the rate of decay of this intermediate corresponds to the steady-state kcat (Table
3-2). Extending the time scale of scanning and using 50 µM enzyme, due to weaker
substrate binding by the mutants, and 5 µM nitrocefin, rapid-scanning visible spectra of
93
D120C, D120N, and D120S showed similar changes in absorbance at 390 nm and 485 nm; however, only D120N gave rise to any change in absorbance at 665 nm. This absorbance change was significantly less than wild-type and corresponds to only ~0.1
µM intermediate formation.
M) 6 Wt L1 M) 6 D120C -7 -7
4 4
2 2
Intermediate (10 0 Intermediate (10 0 0 20 40 60 80 100 0 30 60 90 120 150 180 210 -3 Time (10 s) Time (s)
M) 6 D120N M) 6 D120S -7 -7
4 4
2 2
Intermediate (10 0 Intermediate (10 0 0 1 2 3 4 5 6 7 8 0 30 60 90 120 150 180 210 Time (s) Time (s)
Figure 3-2: Formation of ring-opened, nitrogen anion intermediate by wild-type L1 and L1 mutants. Spectra were collected using rapid scanning Vis studies, and the absorbance values at 668 nm were converted to concentration values as described in Materials and Methods. Typical reactions were conducted with 25 µM L1 (50 µM mutants due to reduced activity) and 5 µM nitrocefin in 50 mM cacodylate, pH 7.0, containing 100 µM o ZnCl2 at 25 C.
94
pH dependence studies on wild-type L1, D120C, and D120S with nitrocefin
(Figures 3-3 and 3-4) demonstrate no inflections over the pH range tested (pH 5 to pH
10) indicating no rate significant proton transfers with pKa values in that pH range.
Studies with D120N indicated a possible pKa between 9 and 10; however due to the shape of the data, we were unable to obtain a fit to any standard pKa equations. Proton inventories of wild-type L1, D120C, and D120S (Figure 3-5) yielded straight lines and demonstrate there is one proton in flight during the rate-limiting step of the reaction. The proton inventory of D120N did not yield a straight line. These data were fitted to the
Gross-Butler equation (55) and suggested greater than two protons in flight during a rate- limiting step of the reaction. Consistent with previous work (56-58), solvent isotope studies with nitrocefin yielded kH/kD values for all enzymes that indicate proton transfer
during a nucleophilic reaction (Table 3-1).
95
2 A m /K cat k
or 1 cat k Log
0 3 m
/K 2 cat
k B or cat k 1 Log
0 4 5 6 7 8 9 10 pH
Figure 3-3: pH dependence plots of wild-type L1 with (A) nitrocefin, ∆ kcat, ◊ kcat/Km, and (B) penicillin G, Ο kcat, kcat/Km. Error bars represent standard deviation of multiple trials.
96
1.0 D120C 0.0
-1.0 cat
k -2.0 log -3.0
-4.0
-5.0 2.0 D120N 1.0 cat k 0.0 log
-1.0
-2.0
0.0 D120S -0.2
-0.4 cat k -0.6 log -0.8
-1.0
-1.2 5 6 7 8 9 10 pH
Figure 3-4: pH dependence plots of L1 mutants with nitrocefin. Error bars represent standard deviation of multiple trials.
97
45 0.20 40 Wt L1 D120C
) ) 0.16
-1 35 -1 (s (s 0.12 cat 30 cat k k 25 0.08 20
0 25 50 75 100 0 25 50 75 100 %D2O %D2O 2.0
D120N ) 40 D120S -3
) 1.5 -1 x10 (s
-1 30 cat
1.0 (s k cat
k 20 0.5
0 25 50 75 100 0 25 50 75 100 %D O %D O 2 2
Figure 3-5: Proton inventory plots of wild-type L1 and L1 mutants with nitrocefin. For D120N, the dashed straight line represents a single proton model, the dashed curved line represents a two-proton model, and the solid curved line is the fit obtained using the Gross-Butler equation for a multiple proton model (49). Error bars represent standard deviation of multiple trials.
98
3.5 Discussion
Mutations were introduced into L1 using the overlap extension method (47), and three indicators were analyzed to probe whether the single point mutations resulted in large structural changes in these mutants. Over-expression levels, total amounts of isolatable enzyme after protein purification, and CD spectra of the mutants were compared to those of wild-type L1. All mutants exhibited results virtually identical to
those of wild-type L1 for all three indicators, leading to the conclusion that none of the
point mutations resulted in large structural changes in L1 and that any kinetic and metal
binding differences could be attributed to the changed amino acid.
In enzymes, Asp residues can have three major functions: (1) coordination of
metal ions, (2) hydrogen bonding with active site residues and substrates, and (3)
shuttling protons to and from groups in the active site. Our data and previous studies (37)
clearly show that one of the Asp120 oxygens is involved in metal binding at the Zn2 site.
Consistent with our expectations, D120C and D120N bound Zn(II) at levels comparable
to wild-type L1, 1.6 ± 0.2, 1.8 ± 0.2 and 1.9 ± 0.2 moles Zn(II) per monomer,
respectively (Table 3-1). D120S only bound 1.1 ± 0.2 moles of Zn(II) per monomer, as
was expected with the removal of a metal binding ligand at the Zn2 site in this mutant.
From previous work it was known that recombinant wild-type L1 exhibited its
lowest Km values and greatest kcat values with 100 µM ZnCl2 in the assay buffer (36).
Similarly, the mutants exhibited their lowest Km values and greatest kcat values with 100
µM ZnCl2 in the buffer (A. Carenbauer, data not shown). This indicated that the as
isolated enzymes do not bind their full complement of Zn(II) but can incorporate
available Zn(II) into their active sites. Confirming this notion, ICP-AES analysis of wild-
99
type L1, D120C, and D120N that had been incubated in buffer containing 100 µM Zn(II) revealed increased levels of Zn(II) bound to the enzymes (no increase in the amount of bound Zn(II) was observed with D120S). Subsequently, to ensure saturation of the metal binding sites and to facilitate direct comparison of the kinetic data, 100 µM ZnCl2 was
included in all of the kinetic buffers used.
To compare the activity of the mutants with wild-type L1, we examined the
steady-state kinetics of three carbapenems, four cephalosporins, and two penicillins with
each enzyme. The substrates tested were chosen because they exhibited low Km values in previous kinetic studies (36), and we believed that we could saturate the enzymes with substrate even if there was large change in binding with the point mutations. It is not surprising that the smallest kcat values were obtained with cephalosporins, affirming L1’s classification as a group Bc metallo-β-lactamase (preference towards penicillins and carbapenems) (9). In particular, it would appear that L1 has little preference for
-1 -1 cefoxitin, exhibited by a kcat/Km value of 1.7 s ⋅µM . For all other substrates the mutants
showed significantly altered activities, with decreases in kcat values on the order of a 10-
1000 fold. The greatest reduction from wild-type L1 is seen with D120C and D120S, with decreases ranging from 100-1000 fold in kcat. The difference between D120N and
wild-type L1 is less exaggerated with reductions in kcat values ranging 7 - 37 fold. In
most cases, except for cefoxitin, D120N exhibited a 10-20 fold greater activity than either
D120C or D120S.
We first address the reduction in activity observed with D120S. Our data clearly
indicate that D120S is a mononuclear enzyme, even under metal saturating conditions
(Table 3-1). This is not surprising since there is no evidence to suggest that the pKa of
100
the serine residue would be lowered sufficiently to allow for the deprotonation of the hydroxyl and permit it to coordinate Zn(II). Repeated attempts to determine a Ks for
D120S with nitrocefin, via stopped-flow fluorescence, failed due to a lack of any observable consistent change in fluorescence. Under a wide range of enzyme and nitrocefin concentrations, no consistent detectable change in fluorescence could be observed (data not shown). In contrast to D120S, metal analysis of wild-type L1, D120C, and D120N indicate that all are dinuclear enzymes and not surprisingly had Ks values with nitrocefin fairly similar to one another, 37 ± 6 µM, 38 ± 7 µM and 97 ± 14 µM, respectively. It is logical to conclude then that D120S's reduced catalytic activity is due to its inability to bind substrate. This confirms the proposition of Ullah et al. (37), that in L1, both Zn(II)'s are necessary for tight substrate binding and in turn full catalytic activity of the enzyme. It should be noted that this result is contrary to recently published work (59) asserting that β-lactamases, such as L1 and CcrA are apo- (without metal) or mononuclear enzymes in vivo.
The kinetic data on the D120C mutant indicate however that the presence of both
Zn(II)'s alone, and subsequently the ability to bind substrate, does not render an active enzyme. This suggests a role of the unbound oxygen of Asp120. For all substrates tested, the exception being cefoxitin as previously noted, there is a clear trend observed in the steady state kinetic data: wild-type L1 > D120N > D120C and D120S. The similarity in the kinetic data for D120C and D120S can be attributed to residual hydrolysis of substrate due to template and scaffolding effects (60). The possibility of purely background hydrolysis due to the excess Zn(II) in the buffer was ruled out because reactions of just substrate and buffer show << 1% hydrolysis during normal reaction
101
times (data not shown). Further evidence supporting this claim is seen in the rapid scans
of nitrocefin hydrolysis by wild-type L1 and the mutants. Both D120C and D120S show
minimal to no accumulation of intermediate as seen with wild-type L1 and D120N
(Figure 3-2), suggesting that a mechanism other than that of wild-type L1 is occurring
with these enzymes. This is consistent with hydrolysis due to a template effect (60).
What then is the role of the unbound oxygen of Asp120? At physiological pH, it is highly improbable that an Asp bound to a Zn(II) is protonated and can take part in a direct proton transfer. Our data along with previous information (37, 51) support the idea that the unbound oxygen of Asp120 is interacting, via hydrogen bonding, with the group bridging the two Zn(II) ions. It is expected that at neutral pH’s, the bridging group is a hydroxide. Upon substrate binding, we predict that the bridging hydroxide becomes terminally bound to Zn2 while retaining a hydrogen bond to Asp120. As predicted with
model complexes (61), other Zn(II) and Fe containing proteins (62-65), computational
studies on metallo-β-lactamases (40, 42, 66), and with other metallo-β-lactamases (57,
67, 68), this metal bound hydroxide then serves as the nucleophile that attacks the β-
lactam carbonyl. The kinetic data clearly show that D120N is the only mutant that retains
significant hydrolytic activity. Not surprising since D120N is the only mutant that retains
both of the apparently critical requirements for substrate turnover we investigated;
namely the effective binding of substrate and a residue at position 120 capable of
electrostatically interacting with the putative nucleophile. The reduced kcat values
observed with D120N are likely due to the chemical difference of the moiety interacting
with the nucleophile. It would appear that in this mutant the Asn oxygen is coordinating
Zn2 (69, 70), leaving the lone pair of the amine to interact with the nucleophile. This is
102
supported by the pH dependence and proton inventory data for D120N. With wild-type
L1, there are no observable pKa's between pH 5 to 10 (Figure 3-3), and the proton
inventory with nitrocefin indicates a single proton in flight during a rate-limiting step of the reaction (Figure 3-5). pH dependence plots of D120N with nitrocefin however, indicate the possibility of multiple pKa's between pH 9.5 to 10 (Figure 3-4); and proton
inventories show multiple protons in flight during a rate-limiting step of the reaction.
Why is this electrostatic interaction between the putative nucleophile and residue
120 catalytically important? Currently, there are two proposed reaction mechanisms for
the dinuclear Zn(II) β-lactamases, depending on the substrate. When nitrocefin is used as
the substrate, a ring-opened, nitrogen anionic intermediate is thought to form, and the
protonation of this nitrogen anion is rate-limiting (51, 54, 68). When other substrates are
used, the breakdown of a tetrahedral intermediate, which occurs via the protonation of the
β-lactam ring nitrogen, is thought to be rate-limiting (54).
- Figure 3-6: Proposed role of Asp120 in L1. The interactions of Zn2 with substrate CO2 and N groups are excluded in the structure on the right for clarity.
103
Based on the data in this work, we hypothesize that a solvent H2O molecule binds to Zn2, causing the release of the substrate from Zn2. Asp120 forms a hydrogen bond to this H2O molecule, orienting it to form the bridging hydroxide/water and positioning the proton on this water to be donated to the substrate nitrogen (Figure 3-6). While this conclusion supports Ullah et al.’s assertion (41) that Asp120 is essential for activity; it predicts that the orientation of the bridging group by Asp120 is for proton transfer rather than for nucleophilic attack. And importantly, our hypothesis explains the rate-limiting proton transfers in the aforementioned reaction mechanisms. Since this proton is part of a H2O molecule that partially interacts with two Zn(II) ions and is further acidified through hydrogen bonding with Asp120, the pKa of this proton would be expected to be lower
than 5.0 and not observable in our pH dependence studies. Taken together, these studies
demonstrate the vital role(s) that Asp120 plays in L1 and suggests a model for all
metallo-β-lactamases, both mononuclear and dinuclear Zn(II) enzymes. In addition, this
predicted role could be extrapolated to all metallohydrolases in the metallo-β-lactamase
family.
104
3.6 Acknowledgements
The authors would like to thank James Pauff and Greg Patton for their assistance in preparing the mutants and Silvia McMannus-Munoz for initial pH dependence studies.
105
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111
Chapter 4
Probing the dynamics of a mobile loop above the active
site of L1, a metallo-β-lactamase from
Stenotrophomonas maltophilia, via site-directed
mutagenesis and stopped-flow fluorescence
spectroscopy †
James D. Garrity, James M. Pauff, and Michael W. Crowder*
Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford,
Ohio 45056
*Corresponding author: phone: (513) 529-7274 fax: (513) 529-5715 e-mail: [email protected]
†This work was supported by the National Institutes of Health (AI40052 and GM40052).
Funds to purchase the CD spectrapolarimeter (DBI-0070206) and the Stopped-flow UV-
Vis fluorescence spectrophotometer (CHE-0076936) were provided by the National
Science Foundation.
Running title: Trp mutants of metallo-β-lactamase L1
112
Abbreviations1
CcrA, metallo-β-lactamase from Bacteroides fragilis; ICP-AES, Inductively Coupled
Plasma – Atomic Emission Spectroscopy; IPTG, isopropyl-β-thiogalactoside; L1, metallo-β-lactamase from Stenotrophomonas maltophilia; LB, Luria-Bertani; apo, metal-
free.
113
4.1 Summary
A structural feature shared by the metallo-β-lactamases is a flexible loop of amino acids that extends over their active sites and that has been proposed to move during the catalytic cycle of the enzymes, clamping down on substrate. To probe the movement of this loop (residues 152-164), a site-directed mutant of metallo-β-lactamase L1 was engineered that contained a Trp residue on the loop to serve as a fluorescent probe. It was necessary first however to evaluate the contribution of each native Trp residue to fluorescence changes observed during the catalytic cycle of wild-type L1. Five site- directed mutants of L1 (W39F, W53F, W204F, W206F, and W269F) were prepared and characterized using metal analyses, CD spectroscopy, steady state kinetics, stopped-flow fluorescence, and fluorescence titrations. All mutants retained the wild-type tertiary structure, bound Zn(II) at levels comparable to wild-type, and exhibited only slight (<10- fold) decreases in kcat values, as compared to wild-type L1, for all substrates tested.
Fluorescence studies revealed a single mutant, W39F, to be void of the fluorescence changes observed with wild-type L1 during substrate binding and catalysis. Using W39F as a template, a Trp residue was added to the flexile loop over the active site of L1, to generate the double mutant, W39F, D160W. This double mutant retained all the structural and kinetic characteristics of wild-type L1. Stopped-flow fluorescence and
-1 rapid-scanning UV-Vis studies revealed the motion of the loop (kobs = 27 ± 2 s ) to be
-1 similar to the formation rate of a reaction intermediate (kobs = 25 ± 2 s ).
114
4.2 Introduction
The ability of bacteria to acquire resistance to antibiotics is a serious problem that
continues to challenge modern society (1). Excessive and often misuse of antibiotics in the clinic and for agricultural purposes has resulted in tremendous selective pressure for antibiotic resistant bacteria (2). These bacteria utilize a variety of methods to become resistant, including modification of cell wall components to prevent antibiotic binding, expression of efflux pumps that transport the antibiotic out of the cell, and the production of enzymes that hydrolyze and render antibiotics ineffective (1,2).
The most common, least expensive, and effective antibiotics currently used are the β-lactams, such as carbapenems, cephalosporins, and penicillins (3,4). These antibiotics are mechanism-based inhibitors of transpeptidase, a bacterial enzyme required for the production of a strong viable cell wall (5,6). In response to their widespread use, an increasing number of bacterial strains have acquired the ability to produce β-
lactamases, enzymes that hydrolyze and render β-lactam antibiotics ineffective. There
are over 300 distinct β-lactamases known, and Bush has classified these into four distinct
groups based on their molecular properties (5,6). One of the more troubling of these is
group 3, the metallo-β-lactamases, which are Zn(II)-dependent enzymes that hydrolyze
nearly all known β-lactams and for which there are no clinically-useful inhibitors (7-14).
To date, there are no reports of a metallo-β-lactamase being isolated from a major
pathogen (15,16); however, these enzymes are produced by a variety of minor clinical
pathogens such as B. fragilis, P. aeruginosa, and S. maltophilia, and the continued
extensive use of β-lactam containing antibiotics will inevitably result in the production of
a metallo-β-lactamase by a major pathogen. (2).
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There is significant diversity within the metallo-β-lactamases, and Bush, based on their amino acid sequence identities and substrate affinities, has further divided them into three subgroups (17). A similar grouping scheme based on structural properties has also been offered (18). The diversity of these subgroups is best exemplified by their vastly differing efficacies towards non-clinical inhibitors; these differences lead to the prediction that finding a single inhibitor for all metallo-β-lactamases may not be possible
(14,19-27). To address this problem, we are currently characterizing a representative enzyme from each of the metallo-β-lactamase subgroups with the goal of identifying common structural and mechanistic similarities that can be targeted for the generation of clinically-useful inhibitors. This work describes out efforts on metallo-β-lactamase L1 from Stenotrophomonas maltophilia.
S. maltophilia is an important pathogen in nosocomial infections of immunocompromised patients suffering from cancer, cystic fibrosis, drug addition, and
AIDS and in patients with organ transplants and on dialysis (28-30). This organism is inherently resistant to most antibiotics due to its low outer membrane permeability (31) and to β-lactams due to the production of a chromosomally expressed group 2e β- lactamase (L2) and a group 3c β-lactamase (L1) (32,33). L1 has been cloned, over- expressed, and partially characterized by kinetic and crystallographic studies (34,35).
The enzyme exists as a homotetramer of ca. 118 kDa in solution and in the crystalline state, tightly binding two Zn(II) ions per subunit. The Zn1 site has 3 histidine residues and 1 bridging hydroxide as ligands, and the Zn2 site has 2 histidines, 1 aspartic acid, 1 terminally-bound water, and the bridging hydroxide as ligands (Figure 4-1).
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Efforts to solve the crystal structure of one of the metallo-β-lactamases with a
bound substrate molecule have failed, most likely due to the high activity of the enzymes,
even in the crystalline state, towards all β-lactam containing antibiotics (35,36).
Therefore, computational studies have been used extensively to study substrate binding,
the role of the Zn(II) ions in catalysis, the protonation state of the active site, protein
dynamics, and inhibitor binding (35,37-42).
The crystal structure of L1 reveals a series of amino acids that form a flexible
loop, a structural feature shared with other metallo-β-lactamases, which extends over its
active site (residues 152-164). It has been suggested that this loop plays an important
role during the catalytic cycle of the enzyme, clamping down on substrate, perhaps
inducing strain to assist in hydrolysis or helping to stabilize a reaction intermediate (35).
To monitor the motion of this loop in L1, we proposed to introduce a Trp residue on the
loop to act as a fluorescent probe. However, this simple approach was complicated by
the presence of five Trp residues in wild-type L1: Trp39, Trp53, Trp204, Trp206, and
Trp269, at 6.2 Å, 25.2 Å, 18.8 Å, 39.8 Å, and 19.6 Å from the active site, respectively
(Figure 4-1). Spencer and coworkers had demonstrated previously that the quenching of
fluorescence and subsequent return to original values in wild-type L1 could be used to
monitor substrate binding and catalysis (43). It was therefore necessary to determine if
these fluorescent changes could be pinpointed to a single Trp residue. To this end, five mutant enzymes were generated in which each Trp residue, in turn, was changed to a Phe residue, resulting in five mutant enzymes: W39F, W53F, W204F, W206F, and W269F.
Because of its proximity to the active site and predicted edge/face interaction with
His263, a metal binding ligand (35); we hypothesized that if indeed the observed
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fluorescence changes were due to a single Trp residue, it was likely Trp39. If this
strategy was successful, a second mutation could be introduced into W39F, replacing
Asp160, a residue located on the flexible loop, with a Trp to act as a fluorescent probe,
resulting in the double mutant W39F, D160W. This work describes our efforts to
determine if we could generate an enzyme free of fluorescence changes during substrate
binding and catalysis and subsequently create a double mutant that could be used to
monitor the dynamics of the flexible loop in L1.
Figure 4-1: Structure of L1 monomer showing the positions of key residues involved in this study. Figure was created with Rasmol v2.60.
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4.3 Experimental Procedures
E. coli strains DH5α and BL21(DE3) were obtained from Gibco BRL and
Novagen, respectively. The plasmid pET26b was purchased from Novagen. Primers for sequencing and mutagenesis studies were purchased from Integrated DNA Technologies.
Quickchange Mutagensis kit was purchased from Stratagene. DNA was purified using the Qiagen QIAQuick gel extraction kit or Plasmid Purification kit with QIAGEN-tip 100
(Midi) columns. Wizard Plus Minipreps were acquired from Promega. Luria-Bertani
(LB) media in powder form was purchased from GIBCO-BRL. Isopropyl-β- thiogalactoside (IPTG), Biotech grade, was procured from Anatrace. Protein solutions were concentrated with an Amicon ultrafiltration cell equipped with YM-10 DIAFLO membranes from Amicon, Inc. Dialysis tubing was prepared using Spectra/Por regenerated cellulose molecular porous membranes with a molecular weight cut-off of 6-
8,000 g/mol (44). Q-Sepharose Fast Flow was purchased from Amersham Pharmacia
Biotech. Nitrocefin was purchased from Becton Dickinson, and solutions of nitrocefin were filtered through a Fisherbrand 0.45 micron syringe filter (34). Cephalothin and penicillin G were purchased from Sigma and Fisher, respectively. Meropenem was generously supplied by Zeneca Pharmaceuticals. All buffers and media were prepared using Barnstead NANOpure ultrapure water.
The over-expression plasmid for L1, pUB5832, was mutated using Stratagene’s
Quickchange Mutagenesis kit. The following oligonucleotides were used for the preparation of the mutants: W39Ffor,
CACCgTggACgCCTCgTTCCTgCAgCCgATggCACCgC; W39Frev, gCggTgCCATCggCTgCAggAACgAggCgTCCACggTg; W53Ffor,
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CTgCAgATCgCCgACCACACCTTCCAgATCggCACCgAggACCTg; W53Frev,
CAggTCCTCggTgCCgATCTggAAggTgTggTCggCgATCTgCAg; W204Ffor,
CCCCgggCAgCACCgCgTTCACCTggACCgATACCCg; W204Frev,
CgggTATCggTCCAggTgAACgCggTgCTgCCCgggg; W206Ffor,
ggCAgCACCgCgTggACCTTCACCgATACCCgCAATggC; W206Frev,
gCCATTgCgggTATCggTgAAggTCCACgCggTgCTgCC; W269Ffor,
CATCCgggTgCCAgCAACTTCgACTACgCCgCgggTgCC; W269Frev,
ggCACCCgCggCgTAgTCgAAgTTgCTggCACCCggATg, W39F, D160Wfor,
CTgCACTTCggCTggggCATCACCTAC; and W39F, D160Wrev,
gTAggTgATgCCCCAgCCgAAgTgCAg. The DNA sequences were analyzed using a
Perkin Elmer ABI 5300 Genetic Analyzer. To test for over-expression of the mutant
enzymes, E. coli BL21(DE3)pLysS cells were transformed with the mutated over- expression plasmids, and small scale growth cultures were used (45). Large-scale (4 L) preparations of the L1 mutants were performed as described previously (34), with the exception of W53F and W206F. With these two mutants we observed lower levels of isolatable protein using the established protocol and therefore reduced the temperature from 37 °C to 25 °C during cell growth and induction and subsequently were able to obtain amounts of isolatable protein comparable to those of wild-type L1. Protein purity was ascertained by SDS-PAGE.
Extinction coefficients for the mutants were determined utilizing the BCA protein assay kit purchased from Fisher, with wild-type L1 used to create the calibration curve.
The concentrations of L1 and the mutants were determined by measuring the protein absorbances at 280 nm and using the published extinction coefficient of ε280nm = 54,606
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M-1•cm-1 for wild-type L1 (34) and the experimentally determined extinction coefficients
for the mutants. Analysis of metal content was performed on enzyme samples that were
incubated for one hour, on ice, in 50 mM HEPES, pH 7.5, containing a final
concentration of 100 µM ZnCl2. Weakly-bound metal was removed by dialysis versus 2
X 1 L metal-free (chelexed) 50 mM HEPES, pH 7.5. A Varian Liberty 2 Inductively
Coupled Plasma spectrometer with atomic emission spectroscopy detection (ICP-AES) was used to determine the metal content of multiple preparations of wild-type L1 and L1 mutants. Calibration curves were based on four standards and had correlation coefficient limits of at least 0.9950. The final dialysis buffer was used as a blank. The emission line of 213.856 nm is the most intense for zinc and was used to determine the Zn content in the samples. The errors in metal content data reflect the standard deviation (σn-1) of
multiple enzyme preparations. Circular dichroism samples 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 diluted with final dialysis buffer to ~75 µg/mL. A JASCO J-
810 CD spectropolarimeter operating at 25 oC was used to collect CD spectra (34,45).
J. Pauff contirbuted to repetitions of steady-state kinetics. Assays were conducted
o at 25 C in 50 mM cacodylate buffer, pH 7.0, containing 100 µM ZnCl2 on a HP 5480A
diode array UV-Vis spectrophotometer. The changes in molar absorptivities (∆ε) used to
-1 -1 follow the reactions were (in M cm ): nitrocefin, ∆ε485 = 17,420; cephalothin, ∆ε265 = -
8,790; meropenem, ∆ε293 = -7,600; and penicillin G, ∆ε235 = -936. When possible,
substrate concentrations were varied between 0.1 to 10 times the Km value, and changes
in absorbance (∆A) versus time data were measured for a period of 60 seconds for each
substrate concentration. Steady-state kinetics constants, Km and kcat, were determined by
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fitting initial velocity versus substrate concentration data directly to the Michaelis
equation using Igor Pro (34). The errors reported are generated by Igor Pro as a result of
Chi square minimization. All steady-state kinetic studies were performed in triplicate with recombinant L1 and L1 mutants from at least three different enzyme preparations.
Fluorescence spectra of 2 µM wild-type L1 and L1 mutant samples were obtained at 25 °C, using an excitation wavelength of 295 nm on a Perkin Elmer LS 55
Luminescence Spectrometer. Apo (metal-free) enzymes for fluorescence titrations were prepared by dialysis of samples versus 5 x 1L of 50 mM HEPES, pH 7.0, containing 10 mM phenanthroline, followed by dialysis versus 5 x 1L of metal-free (chelexed) 50 mM
HEPES, pH 7.0, and metal analyses were used to verify that the enzymes were metal free.
Data from fluorescence titrations were fitted to:
fx( ) =+Finitial (∆F/[E])(0.5)((KD +[E] +x) −((KD +[E] +x) exp 2 −4xFinitial)) where Finitial is the initial fluorescence reading for each sample, [E] is the apo-enzyme concentration, KD is the dissociation constant, f(x) is fluorescence reading, and x is
concentration (M) (46).
Stopped-flow fluorescence studies of nitrocefin hydrolysis by wild-type L1 and
L1 mutants were performed on an Applied Photophysics SX.18MV spectrophotometer,
using an excitation wavelength of 295 nm and a WG320 nm cut-off filter on the
photomultiplier. These experiments were conducted at 2 oC and 25 oC, depending on the
enzyme studied, using the same buffer as in the rapid-scanning Vis studies. Rapid-
scanning UV-Vis studies were performed under identical conditions, using the above-
mentioned instrument and utilizing an Applied Photophysics diode array detector.
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4.4 Results
Wild-type L1 and the L1 mutants were over-expressed in Escherichia coli and purified as previously described (34), with the changes for W53F and W206F as noted in the experimental procedures. This procedure produced an average of 50-60 mg of >95% pure, active protein per 4L of growth culture. Circular dichroism spectra were collected on samples of wild-type L1 and each of the mutants to ensure the proteins produced using the pET26b over-expression system had the correct secondary structure. The CD spectra of wild-type L1 and the mutants were similar and showed an intense, broad feature at 190 nm and a smaller feature at 215 nm (data not shown). These features are consistent with a sample with significant α/β content (35). Analyses of the CD spectra were performed on-line using DICHROWEB utilizing an algorithm called CDSSTR
(http://www.cryst.bbk.ac.uk/cdweb/html/home.html) (47,48). Results are shown in Table
4-1. The CD spectra demonstrate no significant changes in the amount of unordered content and only minor differences in α/β content that can be attributed to fitting errors using the CDSSTR program. Metal analyses on multiple preparations of wild-type L1 and the mutants demonstrated that the wild-type L1and W39F, W53F, W204F, W206F,
W269F and W39F, D160W mutants bind 1.9 ± 0.2, 1.9 ± 0.2, 1.6 ± 0.3, 1.8 ± 0.2, 1.5 ±
0.3, 2.0 ± 0.1, and 1.9 ± 0.2 Zn(II) equivalents per monomer (Table 4-1). Within the limits of error, the metal analyses demonstrate that the single point mutations did not result in significant changes in metal-binding ability of the enzymes.
Steady-state kinetic constants, Km and kcat, were determined for wild-type L1 and each of the mutants with four substrates. These values are presented in Table 4-2.
Cephalothin and nitrocefin, meropenem, and penicillin G were utilized as representatives
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Table 4-1: Analysis of CD Spectra using CDSSTR with a 43 protein reference set optimized for 190-240 nm. Metal Analysis of Wt-L1 and L1 mutants. Error reflects the standard deviation (σn-1) of multiple enzyme preparations. Extinction coefficients for L1 mutants determined utilizing a BCA protein assay.
Metal Analysis (M-1•cm-1) % α-helix % β-sheet % β-turn % Unordered ε (mols Zn(II)/mol monomer) WT L1 31 18 21 30 1.9 ± 0.2 54,606 W39F 29 20 20 31 1.9 ± 0.2 41,256 W53F 18 24 26 32 1.6 ± 0.3 46,387 W204F 20 24 24 32 1.8 ± 0.2 41,910 W206F 18 25 24 33 1.5 ± 0.3 36,969 W269F 30 20 20 30 2.0 ± 0.1 54,588 W39F, D160W 30 19 21 30 1.9 ± 0.2 54,600
Table 4-2: Steady state kinetics constants for wild-type L1 and L1 mutants.
Nitrocefin Cephalothin Meropenem Penicillin G k K k K k K k K cat m k / K cat m k / K cat m k / K cat m k / K (s-1) (µM) cat m (s-1) (µM) cat m (s-1) (µM) cat m (s-1) (µM) cat m 600 ± Wt L1 41 ± 1 4.1 ± 1.0 10 82 ± 5 8.9 ± 1.5 9.2 157 ± 9 15 ± 4 10.5 38 ± 2 15.8 100 W39F 38 ± 4 18 ± 5 2.1 33 ± 2 33 ± 7 1.0 33 ± 2 9 ± 2 3.7 96 ± 4 125 ± 18 0.77
W53F 36 ± 1 3.9 ± 0.5 9.2 53 ± 1 4.9 ± 0.5 10.8 76 ± 3 12 ± 2 6.3 402 ± 7 24 ± 3 16.8
W204F 30 ± 1 3.3 ± 0.2 9.1 46 ± 1 5.4 ± 0.7 8.5 66 ± 2 10 ± 2 6.6 397 ± 7 35 ± 4 11.3
W206F 36 ± 1 4.0 ± 0.5 9.0 49 ± 2 14 ± 2 3.5 64 ± 2 11 ± 1 5.8 407 ± 12 31 ± 5 13.1
W269F 21 ± 1 2.1 ± 0.3 10 55 ± 1 6.4 ± 0.7 8.6 34 ± 1 8 ± 1 4.3 376 ± 12 34 ± 6 11.1 W39F, 39 ± 3 23 ± 4 1.7 23 ± 1 95 ± 11 0.24 17 ± 1 6 ± 1 2.8 161 ± 5 356 ± 31 0.45 D160W
of the three major classes of β-lactam containing antibiotics; cephalosporins, carbapenems, and penicillins, respectively. L1’s preference for penicillins and carbapenems over cephalosporins, as exemplified by the kcat values, is in agreement with previous studies (34) and supports L1’s
placement in the β-lactamase Bc family (17). For all substrates tested, the mutant enzymes
exhibited slightly reduced kcat values (< 10-fold) as compared to wild-type L1. Since Km values
are often used as a first approximation of substrate binding (49), the Km values exhibited by the
mutants were compared to those of wild-type L1. All five mutants, with the exception of W39F and W39F, D160W, exhibited similar Km values to wild-type L1. Km values for W39F were
typically 4-fold greater than those of wild-type L1, while those of the double mutant approached
and in one case (cephalothin) exceeded 10-fold that of wild-type L1. Because changes of 10-fold
or greater are the standard to indicate significant differences (50), steady-state kinetics indicate
only W39F and W39F, D160W approach this standard. However, both enzymes retained enough
activity to be suitable for the purpose of this study.
Initial fluorescence scans of wild-type L1 and the L1 mutants (Figure 4-2) revealed that
W39F and W206F exhibited decreased fluorescence emissions at 340 nm, while W53F and
W269F exhibited increases in their fluorescence emissions at 340 nm as compared to wild-type
L1. W204F exhibited a fluorescence spectrum almost identical to that of wild-type L1, as did the
double mutant, W39F-D160W. These spectra indicate that Trp204 is fairly inconsequential to
the overall natural fluorescence of wild-type L1. The decrease in fluorescence at 340 nm
observed with the removal of Trp39 or Trp206 indicate that these residues contribute
significantly to the natural fluorescence of wild-type L1. It appears as though the addition of the
Trp residue at position 160, compensates for the loss of Trp39. The increased fluorescence at 340
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nm observed upon the removal of Trp53 or Trp269 indicates a mutual fluorescence quenching between these residues.
200 W53F W269F 150 W204F W39F, D160W 100 Wt L1 W206F 50 W39F
Fluorescence (PM Volts) 0 300 320 340 360 380 400 420 440 Wave Length (nm)
Figure 4-2: Fluorescence scans of wild-type L1 and L1 mutants. Samples were 2 µM prepared in 50 mM HEPES, pH 7.0, and spectra taken at 25 °C using a Perkin Elmer LS 55 Luminescence Spectrometer and corrected for background fluorescence. Figure was created using the graphing program Igor Pro v4.05A.
Fluorescence titrations of wild-type L1 (excitation wavelength 295) showed an increase in fluorescence emission at 340 nm with the addition of up to ~0.9 equivalents of Zn(II) with no further increased emission upon addition of greater amounts Zn(II). The fluorescence titration data were fitted to a quadratic equation (46) (lines in Figure 4-3), and the resulting KD values were 2-3 orders of magnitude lower than the concentration of apo-enzyme. These results suggest that these plots are active site titrations and not plots to determine Zn(II) binding KD values. It would appear that L1 binds Zn(II) preferentially to one of its Zn(II) binding sites. Fluorescence titrations of the L1 mutants revealed similar trends to that of wild-type L1 with the exception of
W39F. W39F yielded virtually no change in fluorescence emission at 340 nm regardless of the amount of Zn(II) added (Figure 4-3). Observing a smaller than expected change in fluorescence
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at 340 nm of W269F, we noted that this enzyme precipitates with the first few additions of
Zn(II). The addition of EDTA, to all of the samples except W39F, at the end of each titration resulted in a decrease in fluorescence to the initial value (data not shown), demonstrating reversible Zn(II) binding.
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850
800
750
700
650
600 Wt L1 550 W39F W53F 500 Fluorescence @ 340 nm (PM Volts) 0 1 2 3 4 2+ Added Zn (µM)
750
700
650
600
550 W204F W206F W269F 500 Fluorescence @ 340 nm (PM Volts)
0 1 2 3 4 2+ Added Zn (µM)
Figure 4-3: Fluorescence titrations of apo wild-type L1 and L1 mutants. Samples were 2 µM prepared in 50 mM HEPES, pH 7.0, and fluorescence emission at 340 nm monitored at 25 °C using a Perkin Elmer LS 55 Luminescence Spectrometer. Readings were not corrected for background. Figure was created using the graphing program Igor Pro v4.05A.
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To observe fluorescence changes upon the binding of substrate to wild-type L1 and the
mutants, stopped-flow fluorescence studies were conducted as previously described (43). The
reaction of enzyme with nitrocefin under single-turnover conditions at 25 oC resulted in a rapid
decrease in fluorescence followed by a rate-limiting return of fluorescence for all of the single
point mutants except W39F (Figure 4-4). Stopped-flow fluorescence spectra of W39F, D160W
with nitrocefin differed from that of wild-type L1 and the other mutants, in that with the addition
of substrate, there was a rapid quenching of fluorescence that did not return to the value of the
resting enzyme (Figure 4-5).
-3 40
20
0
-20 Wt-L1 W204F W39F W206F -40 W53F W269F Fluorescence (PM Volts)10
0 20 40 60 80 100 -3 Time (s) x10
Figure 4-4: Stopped-flow fluorescence spectra of wild-type L1 and L1 mutants with nitrocefin. Samples were 25 µM enzyme with 5 µM substrate in 50 mM cacodylate, pH 7.0, and spectra taken at 25 °C using an Applied Photophysics SX.18MV spectrophotometer. Figure was created using the graphing program Igor Pro v4.05A.
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-3
50 40 30 20 10 0
0.0 0.2 0.4 0.6 0.8 1.0 Fluorescence (PM Volts)10 Time (s)
Figure 4-5: Stopped-flow fluorescence spectra of W39F, D160W with nitrocefin. Samples were 20 µM enzyme with 20 µM substrate in 50 mM cacodylate, pH 7.0, and spectra were obtained at 2 °C using an Applied Photophysics SX.18MV spectrophotometer. Figure was created using the graphing program Igor Pro v4.05A. The data were fitted with y = yo + A1exp(-invTau1x) + A2exp(-invTau2x).
We found this behavior of W39F, D160W to be substrate-specific for nitrocefin. When the experiment was repeated with meropenem as the substrate, the same quenching was observed, however, the fluorescence returned to the resting-enzyme value (data not shown). This behavior was confirmed with fluorescence scans of W39F, D160W with 1:1 nitrocefin and meropenem. The scans revealed a decrease of emission at 340 nm for the nitrocefin sample, while the meropenem sample was identical to that of the enzyme without substrate (Figure 4-6).
Close inspection of the stopped-flow fluorescence spectra of W39F, D160W with nitrocefin
(Figure 4-5) revealed that the quenching is biphasic in nature, with an initial substrate concentration-dependent rapid phase, followed by a slower phase seemingly independent of substrate concentration.
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500 Free Enzyme 400 1:1 Meropenem 300 1:1 Nitrocefin 200 100
Fluorescence (PM Volts) 320 340 360 380 400 420 440 Wavelength (nm)
Figure 4-6: Fluorescence scans of W39F, D160W, free enzyme and 1:1 solutions with nitrocefin and meropenem. Samples were 10 µM prepared in 50 mM HEPES, pH 7.0, and spectra taken at 25 °C using a Perkin Elmer LS 55 Luminescence Spectrometer and corrected for background fluorescence. Figure was created using the graphing program Igor Pro v4.05A.
In previous studies, rapid scans of wild-type L1 with nitrocefin revealed absorbance changes at three distinct wavelengths. A decay in the absorbance at 390 nm, an increase at 485 nm, and a rapid increase and decay at 665 nm, which can be attributed to substrate decay, product formation, and the appearance and decay of an intermediate, respectively. To date, nitrocefin is the only substrate with which an intermediate can be spectroscopically observed.
To investigate if the fluorescence quenching observed with W39F, D160W could be related to events during catalysis of nitrocefin, stopped-flow rapid scanning UV-Vis and fluorescence spectra of WT-L1 and W39F, D160W with nitrocefin at single turnover conditions were obtained under identical conditions (Figures 4-5 & 4-7). The temperature was reduced to 2 oC to slow the reaction and better our chances to obtain data suitable for kinetic fitting. Fitting the wild-type L1 data from the fluorescence experiments revealed quenching and recovery rates of 106 ± 2 s-1 and
6.5 ± 0.1 s-1, respectively. The rate of quenching with W39F-D160 revealed two phases, 282 ± 2 s-1 and 27 ± 2 s-1. Fitting of the rapid-scanning data for wild-type L1, W39F and W39F, D160
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yielded rates of 69 ± 2 s-1 and 7.1 ± 0.1 s-1, 18 ± 3 s-1 and 6.8 ± 0.8 s-1, and 25 ± 2 s-1 and 4.8 ±
0.8 s-1 for intermediate formation and decay, respectively. These rate data are summarized in
Table 4-3.
0.14
0.12 Wt-L1 0.10
0.08 W39F, D160W 0.06 W39F 0.04
0.02 Absorbance @ 665 nm 0.00
0.0 0.2 0.4 0.6 0.8 1.0 Time (s)
Figure 4-7: Single wavelength versus time plots of wild-type L1, W39F, and W39F, D160W with nitrocefin. Samples were 20 µM enzyme with 20 µM substrate in 50 mM cacodylate, pH 7.0, and spectra were obtained at 2 °C using an Applied Photophysics SX.18MV spectrophotometer. Figure was created using the graphing program Igor Pro v4.05A.
Table 4-3: Summary of fit constants for stopped-flow rapid-scanning and fluorescence data of enzymes with nitrocefin at 2 °C, using IgorPro v 4.0 graphing program. Data were fitted to single or double exponential equations as determined by the best fit of the data.
Intermediate Fluorescence (665 nm)
Formation Decay Quenching Recovery (s-1) (s-1) (s-1) (s-1) Wt- 69 ± 2 7.1 ± 0.1 106 ± 2 6.5 ± 0.1 L1 W39F 18 ± 3 6.8 ± 0.8 NA NA
W39F, 280 ± 2 25 ± 2 4.8 ± 0.8 NA D160W 27 ± 2
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4.5 Discussion
In proteins, fluorescence properties are typically associated with Trp residues and
changes in the local environment of those residues during catalysis. Previous work by Spencer et
al. (43) demonstrated that the binding of substrate to L1 results in a rapid quenching of
fluorescence, followed by slower return to resting values. The initial quenching of fluorescence
was correlated to substrate binding and utilized to determine Ks values for L1 with multiple
substrates. The rate of return to the fluorescence of the resting enzyme was also correlated to the
kcat value, as determined via steady-state kinetics, of the enzyme with that particular substrate. In order to determine if this fluorescence could be attributed to a specific Trp residue, five mutant enzymes were generated in which each Trp residue was changed to a Phe.
Mutations were introduced into L1 following the Stratagene Quickchange mutagenesis protocol, and the resulting enzymes were characterized to probe whether the single point mutations resulted in large structural changes in these mutants. Over-expression levels, total amounts of isolatable enzyme after protein purification, levels of bound Zn(II), and CD spectra of the mutants were compared to those of wild-type L1. All mutants exhibited over-expression and isolatable enzyme levels nearly identical to those of wild-type L1 (data not shown). Metal analyses of wild-type L1 and the L1 mutants showed that all of the enzymes, within error, bound nearly two moles of Zn(II) per monomer (Table 4-1), indicating the structures of the enzymes’ metal-binding sites were unaltered. While slight variations in the CD spectra of the mutants, as compared to wild-type L1, were observed, fitting of the spectra utilizing the CDSSTR algorithm on the DICHROWEB website revealed virtually no change in the amount of unordered structure for all the mutant enzymes (Table 4-1). These lines of evidence lead to the conclusion that none of the point mutations resulted in large structural changes in L1, particularly at the active site,
134
and that any kinetic differences and changes in fluorescence properties could be attributed to the
changed amino acid.
To compare the activity of the mutants with wild-type L1, we examined the steady-state
kinetics of nitrocefin, cephalothin, meropenem, and a penicillin G with each enzyme (Table 4-2).
The substrates tested were chosen to cover the three major categories of β-lactam antibiotics and
because they exhibited low Km values in previous kinetic studies (34). We therefore believed
that we could saturate the enzymes with substrate even if there was large change in binding with
the point mutations. It is not surprising that the smallest kcat values were obtained with
cephalosporins, affirming L1’s classification as a group 3c metallo-β-lactamase (preference
towards penicillins and carbapenems) (17). For all substrates the mutants showed altered
activities; however, decreases in kcat values were typically on the order of 2-3 fold, with W39F
showing the greatest disparity at 3-6 fold reduced activity. In all cases, the decreases were far
less than the standard one order of magnitude change needed to indicate a significant difference.
Therefore, we conclude that none of the point mutations significantly altered the activity of L1.
Initially we utilized the method of Edelhoch (51) to adjust the extinction coefficient of wild-type L1 to reflect the loss of a Trp residue in each of the L1 mutants, resulting in a value of
48,916 M-1•cm-1 for each. However, fluorescence scans of the mutant enzymes indicated that a
change from Trp to Phe did not alter the natural fluorescence of L1 in a consistent manner
(Figure 4-2), indicating that the environment of the Trp residue is critical in determining its contribution to the natural fluorescence of the enzyme. It is logical then to assume that the local environment of a Trp residue is also important to its ability to absorb radiation at 280 nm, and therefore simply assigning the same extinction coefficient to all of the L1 mutants was not valid.
To account for the environmental differences, we utilized a BCA protein assay in combination
135
with absorbance at 280 nm data to individually determine extinction coefficients for each L1 mutant (Table 4-1).
Two fluorescence studies were conducted with each L1 mutant and compared to wild- type L1 to determine if previously observed properties could be attributed to a single Trp residue.
First, each enzyme was analyzed with stopped-flow fluorescence using nitrocefin as the substrate
(43,52). W53F, W204F, W206F, and W269F all exhibited fluorescence spectra similar in shape to that of wild-type L1 (Figure 4-4), showing the quenching of fluorescence upon the binding of substrate to the enzyme and return of fluorescence during substrate turnover. W39F exhibited no change in fluorescence upon the binding and turnover of substrate. There are two possible explanations for this observation. First, as previously observed with other mutants, substrate does not readily bind to W39F; and therefore, no change in fluorescence can be observed (52).
Second, the Trp residue at position 39 is responsible for the observed fluorescence changes in wild-type L1, and the absence of this Trp residue in W39F is the reason no changes in fluorescence can be observed. In those previous studies, the mutant that did not yield fluorescence changes was also observed to have a nearly 1000-fold decrease in its kcat value and a 25-fold increase in its Km value with nitrocefin as compared to wild-type L1. Steady-state kinetic data on W39F, however, shows no significant change in the kcat or Km values of W39F as compared to wild-type L1 (Table 4-2). It is reasonable then to conclude that the absence of fluorescence changes with W39F is not due to an inability of the enzyme to bind and turnover substrate and can be attributed to removal of the Trp residue at this position.
A second experiment, fluorescence titrations of apo wild-type L1 and each of the L1 mutants with Zn(II), confirms this conclusion. With wild-type L1, a steady increase in fluorescence emission at 340 nm is observed upon the addition of Zn(II). At ~ 0.9 equivalents of
136
Zn(II), the fluorescence emission reaches a maximum, and there is no further increase (Figure 4-
3). The addition of EDTA to the sample at the end of the titration resulted in a decrease in fluorescence to the initial value (data not shown). These results demonstrate reversible Zn(II) binding and indicate that binding to one site is preferred, and only the binding of Zn(II) in this site can be monitored with fluorescence. Each of the L1 mutants, with the exception of W39F, yielded spectra with features similar to that of wild-type L1. The small change noted in the fluorescence at 340 nm of W269F, and failure to reach levels observed in the fluorescence scans, are attributed to precipitation of this enzyme upon the addition of Zn(II). No change in fluorescence was observed during the titration of apo W39F with Zn(II). Based on metal analysis data, it is logical to assume that the added Zn(II) is populating the Zn(II) binding sites in
W39F. Therefore, the absence of fluorescence changes can be attributed to the missing Trp residue at position 39. Furthermore, due to its proximity to the Zn2 binding site and proposed face/edge interaction with His263, it is reasonable to conclude that Zn(II) preferentially binds to the Zn2 binding site in wild-type L1. This result is in contrast to previous studies on β-lactamase
II where the Zn1 site has been shown to be tighter binding (53).
Once Trp39 was established as the residue responsible for fluorescence changes observed during substrate binding and catalysis in wild-type L1, W39F was used as a template to create a double mutant, W39F, D160W, using the methods previously described. The replacement of
Asp160 with a Trp residue afforded the ability to directly monitor the dynamics of a flexible chain of amino acids that extends over the active site of L1 (54). This double mutant was characterized using the same methods as for the single point mutants of L1, and all lines of evidence lead to the conclusion that this second mutation did not result in significant structural changes for this enzyme. W39F, D160W over-expression, isolatable enzyme levels, and levels
137
of bound Zn(II) (Table 4-1) were identical to those of wild-type L1. The CD spectra of this
double mutant was virtually identical to that of wild-type L1, as revealed by the fitting of the spectra utilizing the CDSSTR algorithm on the DICHROWEB website (Table 4-1). We were confident then that any kinetic differences and more importantly changes observed in fluorescence studies could be attributed to the Trp residue engineered into the flexible loop of the protein.
Steady-state kinetic constants, Km and kcat, were determined for W39F, D160W, with the
same representative substrates used with the single point L1 mutants. For all substrates tested,
the double mutant exhibited kcat values similar to W39F, with only slightly reduced values for
cephalothin and meropenem and a value approximately twice as large as with penicillin G.
Importantly, the kcat value with nitrocefin was virtually identical to that of W39F and wild-type
L1. The only noticeable difference in Km values, as compared with W39F, was with penicillin
G, where a 3-fold increase was observed. The Km value with nitrocefin was approximately a 6-
fold increase over that of wild-type L1, it is however identical, within error, to that of W39F.
Therefore it is reasonable to conclude that the increased Km as compared to wild-type L1, can be
attributed to perturbations in the active site due to the change from Trp to Phe at position 39, since there appears not to be any further increase caused by the addition of the Trp residue on the flexible loop portion of the enzyme.
Stopped-flow fluorescence studies of W39F, D160W with nitrocefin showed a biphasic quenching of fluorescence that did not return to initial values (Figure 4-5). The initial decline is rapid, and dependent on nitrocefin concentration, which we interpret to be a binding type event where the loop rapidly closes on substrate as it docks in the active site. The second phase of the quenching is significantly slower and is not substrate concentration dependent. The observed
138
-1 rate for this step (kobs = 27 ± 2 s ) is identical, within error, to the rate of intermediate formation
-1 (kobs = 25 ± 2 s ) observed in rapid-scanning experiments performed under identical conditions
(Figure 4-7). Previous work predicted the break down of this intermediate during a protonation event to be the rate-limiting step of the reaction (55). This conclusion is supported by the data in this work, as the rate of breakdown of the intermediate is noted to be slowest step observed in these studies. We conclude therefore that once bound to the active site, the loop assists in destabilizing substrate to form the intermediate, in a non-rate-limiting event.
Because the fluorescence never rebounds from the quenched state with W39F, D160W, we predict that the loop is somehow locked in the closed position trapping an enzyme-bound product species. This appears to be specific to nitrocefin however since in the same stopped- flow fluorescence experiments using meropenem as the substrate, the observed quenching returns to initial values (data not shown). Fluorescence scans of free enzyme and 1:1 enzyme- substrate solutions using wild-type L1, W39F, and D160W, W39F with nitrocefin and meropenem as substrates (Figure 4-6), confirm this conclusion. However, suppressed fluorescence observed with the 1:1 wild-type L1/nitrocefin solution (data not shown), indicates that this enzyme-bound product is not unique to the double mutant, but is a phenomenon observed with L1 and cephalosporins.
In this work we have demonstrated that the fluorescence properties of L1 can be attributed to a single Trp residue, specifically Trp39. We exploited this fact to create a mutant enzyme with a Trp label on the flexible loop portion of the enzyme, and stopped-flow fluorescence and rapid-scanning UV-Vis were used to demonstrate the motion of this loop is kinetically-linked to the non-rate-limiting formation of a reaction intermediate. Furthermore we have presented evidence for an enzyme-bound product when cephalosporins are used as
139
substrates with L1. This knowledge forms the basis for the development of a clinically useful inhibitor of L1 and other metallo-β-lactamases. It may be possible to engineer a substrate mimic containing substituents able to irreversibly bind to the loop, thereby locking the molecule in the active site, or block the loop and effectively inhibiting the enzyme.
140
4.6 Acknowledgements
The authors would like to thank Lissa Herron and Greg Patton for help with preliminary work investigating suitable amino acid substitutions for this project, and Professor Gary Lorigan for guidance in the use of Igor Pro.
141
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(1997) Antimicrob. Agents Chemo.41, 1460-1462
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Acta Cryst. D53, 485-487
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Manual, Second Ed., 1. 3 vols., Cold Spring Harbor Laboratory Press
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(2002) BMC Biochemistry 3, 4-10
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49. Segel, I. H. (1993) Enzyme Kinetics, John Wiley and Sons, Inc., New York
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146
Chapter 5 First Direct Evidence that the Reaction Intermediate of
Metallo-β-Lactamase L1 is Metal Bound
James D. Garrity,1 Brian Bennett,2* and Michael W. Crowder1*
1Department of Chemistry and Biochemistry, 112 Hughes Hall, Miami University, Oxford, OH
45056
2National Biomedical EPR Center, Department of Biophysics, Medical College of Wisconsin,
8701 Watertown Plank Road, Milwaukee, WI 53226-0509
*To whom correspondence should be addressed: M.W. Crowder
e-mail: [email protected]
phone: (513) 529-7274
fax: (513) 529-5715
Brian Bennett
e-mail: [email protected]
phone: (414) 456 4787
fax: (414) 456 6512
147
Abbreviations1
EPR, electron paramagnetic resonance; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; IPTG, isopropyl-β-thiogalactoside; L1, metallo-β-lactamase from Stenotrophomonas maltophilia; RFQ, rapid-freeze-quench; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
148
5.1 Summary
In an effort to probe the structure of the reaction intermediate of metallo-β-lactamase L1 when reacted with nitrocefin and other β-lactams, rapid-scanning electronic and rapid-freeze quench (RFQ) EPR spectra were obtained using the Co(II)-substituted form of the enzyme.
When using nitrocefin as the substrate, rapid-scanning electronic spectra demonstrate that Co(II)- substituted L1 utilizes a reaction mechanism, similar to that of the native Zn(II) enzyme, in which a short-lived intermediate forms. Rapid-freeze quench EPR spectra of this intermediate demonstrate that the binding of substrate results in a change in the electronic properties of one or both of the Co(II)’s in the enzyme, indicating, for the first time, that the reaction intermediate is metal-bound. The RFQ-EPR studies also demonstrate that other β-lactams, such as cephalothin, meropenem, and penicillin G, proceed through an electronically-similar complex and that the role of metal is similar in all cases. RFQ-EPR spectra of Co(II)-L1 after long reaction times with the substrate and EPR spectra of Co(II)-L1+products show that enzyme/product complexes form during catalysis, indicating that reversible product binding must be considered in all future kinetic mechanisms. Finally, detailed analyses of the data suggest that the metal ion in the Zn2 binding site of L1 binds a solvent molecule and is probably the catalytic site in the enzyme.
149
5.2 Introduction
The ability of bacteria to acquire resistance to antibiotics is a serious problem that
continues to challenge modern society (1). Excessive use, and often misuse, of antibiotics in the clinic and for agricultural purposes has resulted in tremendous selective pressure for antibiotic resistant bacteria (2). These bacteria utilize a variety of methods to become resistant, including modification of cell wall components to prevent antibiotic binding, expression of efflux pumps that transport the antibiotic out of the cell, and the production of enzymes that hydrolyze and render antibiotics ineffective (1,2).
The most common, least expensive, and effective antibiotics currently used are the β- lactams, such as carbapenems, cephalosporins, and penicillins (3,4). These antibiotics are mechanism-based inhibitors of transpeptidase, a bacterial enzyme required for the production of a strong viable cell wall (5,6). In response to their widespread use, an increasing number of bacterial strains have acquired the ability to produce β-lactamases, enzymes that hydrolyze and render β-lactam antibiotics ineffective. There are over 300 distinct β-lactamases known, and
Bush has classified these into four distinct groups based on their molecular properties (5,6). One of the more troubling of these is group 3, the metallo-β-lactamases, which are Zn(II)-dependent enzymes that hydrolyze nearly all known β-lactams and for which there are no clinically-useful inhibitors (7-14). To date, there are no reports of a metallo-β-lactamase being isolated from a major pathogen (15,16); however, these enzymes are produced by a variety of minor clinical pathogens such as B. fragilis, P. aeruginosa, and S. maltophilia, and the continued extensive use of β-lactam containing antibiotics will inevitably result in the production of a metallo-β-
lactamase by a major pathogen. (2).
150
There is significant diversity within the metallo-β-lactamases, and Bush, based on their amino acid sequence identities and substrate affinities, has further divided them into three subgroups (17). A similar grouping scheme based on structural properties has also been offered
(18). The diversity of these subgroups is best exemplified by their vastly differing efficacies towards non-clinical inhibitors; these differences lead to the prediction that finding a single inhibitor for all metallo-β-lactamases may not be possible (14,19-27). To address this problem, we are currently characterizing a representative enzyme from each of the metallo-β-lactamase subgroups with the goal of identifying common structural and mechanistic similarities that can be targeted for the generation of clinically-useful inhibitors. This work describes our efforts on metallo-β-lactamase L1 from Stenotrophomonas maltophilia.
S. maltophilia is an important pathogen in nosocomial infections of immunocompromised patients suffering from cancer, cystic fibrosis, drug addition, and AIDS and in patients with organ transplants and on dialysis (28-30). This organism is inherently resistant to most antibiotics due to its low outer membrane permeability (31) and is particularly resistant to β- lactams due to the production of a chromosomally expressed group 2e β-lactamase (L2) and a group 3c β-lactamase (L1) (32,33). L1 has been cloned, over-expressed, and partially characterized by kinetic and crystallographic studies (34,35). The enzyme exists as a homotetramer of ca. 118 kDa in solution and in the crystalline state, tightly binding two Zn(II) ions per subunit. The Zn1 site has 3 histidine residues and 1 bridging hydroxide as ligands, and the Zn2 site has 2 histidines, 1 aspartic acid, 1 terminally-bound water, and the bridging hydroxide as ligands (Figure 5-1).
151
Figure 5-1: Proposed metal binding site of metallo-β-lactamase L1 when bound to reaction intermediate. Figure rendered with CS ChemDraw Ultra v. 5.0.
152
Efforts to solve the crystal structure of one of the metallo-β-lactamases with a bound
substrate molecule have failed, most likely due to the high activity of the enzymes, even in the
crystalline state, towards all β-lactam containing antibiotics (35,36). Therefore, computational studies have been used extensively to study substrate binding, the role of the Zn(II) ions in catalysis, the protonation state of the active site, protein dynamics, and inhibitor binding (35,37-
42).
Previous studies on metallo-β-lactamase CcrA from Bacteroides fragilis (43) and L1
(44,45) have led to proposals for a mechanisms of catalytic hydrolysis in which intermediates in
catalysis may be stabilized by the active site metal ions (43,46). In the present work, we demonstrate that a chromophoric, substrate-derived species is formed within a kinetically- relevant time. We also show that the electronic structures of the catalytic metal ions are perturbed concomittantly with formation of this intermediate species. For the first time, direct evidence for the involvement of the metal ions in non-rate-limiting formation of a catalytic cycle intermediate is presented. Further insight into the catalytic mechanism is gleaned from studies with three classes of substrate.
153
5.3 Experimental Procedures
E. coli strains DH5α and BL21(DE3) were obtained from Gibco BRL and Novagen,
respectively. The plasmid pET26b was purchased from Novagen. Primers for sequencing and
mutagenesis studies were purchased from Integrated DNA Technologies. Quickchange
Mutagensis kit was purchased from Stratagene. DNA was purified using the Qiagen QIAQuick
gel extraction kit or Plasmid Purification kit with QIAGEN-tip 100 (Midi) columns. Wizard
Plus Minipreps were acquired from Promega. Luria-Bertani (LB) media in powder form was
purchased from GIBCO-BRL. Isopropyl-β-thiogalactoside (IPTG), Biotech grade, was procured
from Anatrace. Protein solutions were concentrated with an Amicon ultrafiltration cell equipped
with YM-10 DIAFLO membranes from Amicon, Inc. Dialysis tubing was prepared using
Spectra/Por regenerated cellulose molecular porous membranes with a molecular weight cut-off
of 6-8,000 g/mol (47). Q-Sepharose Fast Flow was purchased from Amersham Pharmacia
Biotech. 1,10-phenanthroline was purchased from Sigma. Nitrocefin was purchased from
Becton Dickinson, and solutions of nitrocefin were filtered through a Fisherbrand 0.45 micron
syringe filter (34). Cephalothin and penicillin G were purchased from Sigma and Fisher,
respectively. Zeneca Pharmaceuticals generously supplied Meropenem. Where hydrolysis
products of antibiotics were stated to have been used, these were prepared by the addition of
catalytic amounts of L1 to solutions of the antibiotics and the reaction was allowed to proceed to completion. All buffers and media were prepared using Barnstead NANOpure ultrapure water.
Large-scale (4 L) preparations of the L1 were performed as described previously (34).
Protein purity was ascertained by SDS-PAGE. Purified protein was dialyzed versus 6 X 1 L of
50 mM HEPES, pH 7.0, with 5 mM 1,10-phenanthroline, over 36 hours at 4 °C, followed by dialysis versus 6 x 1 L of metal-free 50 mM HEPES, pH 7.0, over 36 hours at 4 °C, to yield apo-
154
L1. Two equivalents of Co(II) were added per apo-L1 monomer using published methods (48).
Co(II) incorporation was confirmed by comparing the EPR spectra of Co(II)-L1 and HEPES buffer containing 1 mM Co(II). The spectra were quite distinguishable, and there was no evidence for Co(II)-HEPES in the Co(II)-L1 samples.
Stopped-flow/rapid-scanning electronic absorption spectrophotometric studies of nitrocefin hydrolysis by Co(II)-substituted L1 were performed on an Applied Photophysics
SX.18MV spectrophotometer, as previously described (49,50).
The RFQ samples were prepared and EPR spectroscopy completed with assistance from
B. Bennett. Samples for EPR studies were generated using a modified Update Instruments
(Madison, WI) rapid freeze quench (RFQ) system. All enzyme and substrate starting sample concentrations were 1 mM (except penicillin G which was 4 mM), prepared in metal-free
(chelexed) 50 mM Hepes, pH 7.0. A model 715 Update Instruments ram controller was used to drive a PMI-Kollmorgen stepping motor (model 010-00205-010) connected to a ram that in turn drove the Update Instrument syringes. The syringes, mixer and tubing were all contained in a watertight bath that was maintained at 2 °C using iced water. Immediately prior to sample collection, the nozzle (and, for the shortest reaction times, the attached mixer) was removed from the bath and held 5 mm above the surface of 2-methylbutane (Fisher) contained in a collecting funnel and maintained at –130 °C by a surrounding bath (Update Instruments) of liquid-nitrogen- cooled 2-methylbutane. Samples were packed into EPR tubes at –130 °C, excess 2- methylbutane was decanted, and samples were stored in liquid nitrogen prior to EPR examination, typically within an hour of sample generation. The RFQ system was calibrated by comparing the development of a low-spin Fe(III) EPR signal and the disappearance of a high spin Fe(III) EPR signal with the associated optical changes at 636 nm using stopped flow
155
spectrophotometry, upon mixing myoglobin with an excess of sodium azide. The shortest, total
effective reaction time that could be achieved with the RFQ system was 10 ms.
EPR spectra were recorded using a Bruker EleXsys E500 EPR spectrometer equipped
with an Oxford Instruments ITC4 temperature controller and an ESR-900 helium flow crystat.
Spectra were recorded at 12 K with 1 mW microwave power. A Bruker ER-4116DM cavity was
used, with a resonant frequency of 9.63 GHz (in perpendicular mode), and 10 G (1 mT) field
modulation at 100 kHz was employed. Computer simulations of EPR spectra were carried out by
B. Bennett, using the matrix diagonalization program XSophe (Bruker Biospin GmbH (51)). A
3 1 spin Hamiltonian H = βg.B.S + S.D.S, where S = /2 and D > 0 corresponds to an MS = |± /2〉 ground state Kramers’ doublet was employed. For Co(II), generally |D| >> βgBS and the spectrum is therefore insensitive to the precise value of D; in this work the arbitrary value D = 50 cm-1 was taken. The g tensor was assumed to have at least axial symmetry as this allows for a unique solution to the spin Hamiltonian in terms of the real g values, gz and gxy (or g|| and g⊥), and the rhombic distortion of the axial zero field splitting, E/D (52). The relationship between
the resonance positions, geff.(x,y,z), and these parameters is described in detail elsewhere (53,54).
156
5.4 Results
Rapid-scanning electronic absorption spectrophotometric studies on Co(II)-substituted
-1 o L1. L1 has been reported to exhibit a steady-state kcat of 41 s at 25 C with nitrocefin as the
substrate. Previous stopped-flow studies have shown that progress of the reaction of L1 with
nitrocefin is sufficiently rapid that stopped-flow techniques at 25 oC may not access the entire
catalytic cycle (44,45,50). Rapid kinetic studies were therefore conducted at 2 oC. Rapid-
scanning spectrophotometry at 2 oC on 100 µM Zn(II)-containing L1 with 100 µM nitrocefin as the substrate reveal the time-dependent presence of three distinct species: (1) at 7 ms after
mixing, there is a small feature at 390 nm corresponding to substrate nitrocefin and a large
feature at 665 nm that was previously assigned to a reaction intermediate (45), (2) at 36 ms after
mixing, the feature at 665 nm is slightly less intense and there is a shoulder at 485 nm, and (3) at
1 s, the features at 390 and 665 nm are absent and a feature, which corresponds to the ring- opened product, at 485 nm is present (Figure 5-2A). Single wavelength versus time plots (Figure
5-2B) of the features at 390, 485, and 665 nm were similar in appearance to those previously reported and were individually fitted to exponential equations as described in McMannus et al.(45). The decay of substrate (feature at 390 nm) occurs at a rate of 269 + 7 s-1, which is
similar to the rate of intermediate (feature at 665 nm) formation (250 + 5 s-1). The breakdown of the 665 nm intermediate occurs at a rate of 7.3 + 0.1 s-1, which corresponds to the rate of product
formation (feature at 485 nm) of 7.6 + 0.1 s-1. These rate constants suggest that the same
mechanism that was predicted for L1 at 25 oC operates at 2 oC.
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Figure 5-2: (A) Rapid-scanning electronic absorption spectra of the reaction of 100 µM wild- type L1 with 100 µM nitrocefin at 2 oC. (B) Single wavelength versus time plots of reaction of 100 µM wild-type L1 with 100 µM nitrocefin at 2 oC. The buffer used in both reactions was 50 mM HEPES, pH 7.0.
Figure 5-3: (A) Rapid-scanning electronic absorption spectra of the reaction of 100 µM Co(II)- substituted L1 with 100 µM nitrocefin at 2 oC. (B) Single wavelength versus time plots of reaction of 100 µM Co(II)-substituted L1 with 100 µM nitrocefin at 2 oC. The buffer used in both reactions was 50 mM HEPES, pH 7.0.
Rapid-scanning electronic absorption spectra at 2 oC of Co(II)-substituted L1 with
nitrocefin as the substrate show the same three species that were observed in the studies
with Zn(II)-L1. However at 36 ms, the intermediate at 665 nm converts to a fourth species that
absorbs at 600 nm, and this second intermediate then is converted into product (Figure 5-3A).
Single wavelength versus time plots of these data were individually fitted to exponential
equations (Figure 5-3B). Substrate decay (390 nm) occurred at a rate of 67 + 2 s-1, which is
similar to the rate of formation of the 665 nm intermediate (rate = 54 + 3 s-1) and of the 600 nm
intermediate (rate = 72 + 2 s-1). The breakdown of both intermediates occurred at rates of
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7.0 + 0.1 s-1 (665 nm feature) and 6.7 + 0.1 s-1 (600 nm feature), which is similar to rate of
product formation of 8.8 + 0.1 s-1. Since the feature at 600 nm occurred only in the rapid-
scanning spectrophotometric studies on Co(II)-substituted L1, we assign this feature to an
Co(II)-bound intermediate species.
The electronic absorption spectrum of uncomplexed Co(II)-substituted-L1 reveals a
broad feature at 550 nm with an extinction coefficient of 180 M-1cm-1 that we previously assigned to overlapping Co(II) ligand field transitions (48). We examined whether there was a change in the intensities of these ligand field transitions during the reaction of Co(II)-L1 with nitrocefin; however, the presence of the more intense features at 600 nm and 485 nm effectively prevented the investigation of any spectral changes due to Co(II) ligand field transitions in the
550 nm region.
Rapid-scanning spectrophotometric studies were also conducted in order to study the reaction of Co(II)-substituted-L1 with the other substrates cephalothin, meropenem, and penicillin G. With these substrates, there were no observed features at 665 or 600 nm, which demonstrates that the chromophoric species observed during the reaction with nitrocefin are due to nitrocefin and its reaction products rather than a chromophoric center in the enzyme. In the absence of interference from absorption due to nitrocefin, Co(II) d-d bands could be observed with the other substrates. However, no significant changes in intensities of these ligand field transitions occured during the reaction, suggesting that there is no change in the coordination number of the Co(II) ions during the time period in which these spectra were obtained (55).
Rapid-freeze quench EPR spectra of Co(II)-substituted L1. The EPR spectrum of
Co(II)-substituted L1 is shown in Figure 5-4A and was simulated (Figure 5-4B) assuming a
1 single (or two indistinguishable) paramagnetic species with S = 3/2, D >> βgBS, MS = |± /2〉,
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gx,y = 2.32, gz = 2.35, and E/D = 0.03. The origin of an apparently single component EPR signal, which we term the resting signal, from the dinuclear Co(II)-containing L1 is a matter for speculation but the simulation was useful in modeling multi-component spectra seen under reaction conditions. Variable temperature and power studies were carried out to try to identify other species, but no other Co(II) species were detected between 3.6 and 25 K and at powers up to 200 mW. (The simulation does contain an additional component that we assign to an Fe(III) species and is responsible for the multiple inflection points seen on the derivative feature of the
Co(II) signals; this species, with resonances at g values of 4.74, 3.99, and 3.66 was simulated as
Fe(III) with S = 5/2, D >> βgBS, giso = 1.97, E/D = 0.244. This signal accounted for less than 0.2
% of the total spins in the samples and was not investigated further.)
Co(II)-substituted L1 was also examined by EPR after reaction with nitrocefin at 2 °C for various times. After 39 ms reaction time, the resting signal was replaced with another apparently single component spectrum (Figure 5-4E) that we term the complex signal. The complex signal
1 was simulated (Figure 5-4F) assuming S = 3/2, D >> βgBS, MS = |± /2〉, gx,y = 2.31, gz = 2.42, and
E/D = 0.03. At the shortest reaction time that we could access, 10.4 ms, the spectrum (Figure 5-
4C) could be well modeled (Figure 5-4D) as a mixture of 15 % of the resting signal and 85 % of the complex signal. When rapid-freeze-quenched samples were thawed and refrozen after 60 s, an EPR spectrum that consisted of roughly 50% of the resting signal and 50% of the complex signal was observed. This latter signal was very similar to the EPR signal (Figure 5-4I) exhibited upon the addition of the hydrolysis product of nitrocefin to Co(II)-substituted L1.
To compare with nitrocefin, analogous experiments were performed using the related cephalosporin cephalothin. After reaction times of 10 and 39 ms, the EPR signals obtained
(Figures 5-5B and 5-5C) were indistinguishable from the complex signal that was seen with
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nitrocefin. However, after thawing out a sample and refreezing after 60 s, an entirely new signal
was observed (Figure 5-4D). This signal could be readily simulated (Figure 5-5E), although gz was not resolved and all that can reliably be determined from the simulation is that gxy = 2.28
and E/D is low (< 0.05). A similar signal was observed (Figure 5-5F) upon addition of the
hydrolysis product of cephalothin, though the signal contained a small amount of a second,
poorly defined, broad component, estimated as accounting for 20 % of the spin density by
modeling using the Complex signal.
Two other substrates were examined by EPR of freeze-quenched samples, meropenem
and penicillin G. With meropenem, signals were observed after 10 ms (Figure 5-6B) and 39 ms
(Figure 5-6D) that were visually distinguishable from the complex signals exhibited upon
reaction of Co(II)-substituted L1 with nitrocefin and with cephalothin, but simulation (Figure 5-
6C) showed their EPR parameters to be very similar, with only a slight difference in E/D, 0.05
and 0.03 for meropenem and nitrocefin, respectively. After thawing a sample out and refreezing
after 60 s, the signal changed slightly (Figure 5-6E) but a new signal (Figure 5-6F) was seen
upon addition of the hydrolysis product of meropenem to Co(II)-substituted L1. This signal
could not be well simulated as either a single species or as a mixture of any of the other EPR
signals from Co(II)-substituted L1. Somewhat similar behavior was exhibited upon the reaction
of Co(II)-substituted L1 with penicillin G. A signal (Figure 5-7B) was observed after 10 ms that
superficially resembled the complex signal, though the simulation (Figure 5-7C) indicated
slightly different parameters of gx,y = 2.38, gz = 2.50, and E/D = 0.005. This signal persisted when the sample was thawed and refrozen after 60 s (Figure 5-7D) but, as with meropenem, a different signal was observed (Figure 5-7E) upon adding the hydrolysis product of penicillin G to
Co(II)-substituted L1. An attempt to model the spectrum using a mixture of the complex and
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resting signals was not entirely successful (Figure 5-7F), and it is likely that new species are involved.
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Figure 5-4: EPR spectra and simulations of Co(II)-substituted L1 with nitrocefin as the substrate. Trace A shows the resting EPR signal of Co(II)-L1. Traces C, E and G show the EPR spectra of Co(II)-L1 upon reaction with nitrocefin for 10 ms at 2 °C (C), 39 ms at 2 °C (E) and 60 s at 23 °C (G). Trace I shows the EPR spectrum of Co(II)-L1 upon incubation for 5 min at 23 °C with the L1-hydrolysed product of nitrocefin. Trace B is a computer simulation of A 3 -1 1 assuming S = /2, D >> βgBS (50 cm ), MS = |± /2〉, gx,y = 2.32, gz = 2.35, and E/D = 0.03. Trace 3 -1 1 F is a computer simulation of E assuming S = /2, D >> βgBS (50 cm ), MS = |± /2〉, gx,y = 2.31, gz = 2.42, and E/D = 0.03. Traces B and F also contain a 0.2 % minor component due to Fe(III) 5 -1 with S = /2, D = 20 cm , giso = 1.97 and E/D = 0.244. Traces D, H and J are models for C, G and I, respectively, and consist of (0.15 × B) + (0.85 × F) (D), (0.50 × B) + (0.50 × F) (H) and (0.52 × B) + (0.48 × F) (J). Experimental EPR spectra were recorded at 10 K with 1 mW microwave power at 9.63 GHz. 10 G (1 mT) field modulation at 100 kHz was employed.
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Figure 5-5: EPR spectra and simulations of Co(II)-substituted L1 with cephalothin as the substrate. Trace A shows the resting EPR signal of Co(II)-L1. Traces B, C and D show the EPR spectra of Co(II)-L1 upon reaction with cephalothin for 10 ms at 2 °C (B), 39 ms at 2 °C (C) and 60 s at 23 °C (D). Trace F shows the EPR spectrum of Co(II)-L1 upon incubation for 5 min at 23 °C with the L1-hydrolysed product of cephalothin. Trace E is a computer simulation of D 3 -1 1 assuming S = /2, D >> βgBS (50 cm ), MS = |± /2〉, gx,y = 2.28, gz = 2.35, and E/D = 0.03. Trace G is a model for F and consists of (0.80 × B) + (0.20 × Trace F of Figure 4). Trace G also -1 contain a 0.2 % minor component due to Fe(III) with S = 5/2, D = 20 cm , giso = 1.97 and E/D = 0.244. Experimental EPR spectra were recorded at 10 K with 1 mW microwave power at 9.63 GHz. 10 G (1 mT) field modulation at 100 kHz was employed.
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Figure 5-6: EPR spectra and simulations of Co(II)-substituted L1 with meropenem as the substrate. Trace A shows the resting EPR signal of Co(II)-L1. Traces B, D and E show the EPR spectra of Co(II)-L1 upon reaction with meropenem for 10 ms at 2 °C (B), 39 ms at 2 °C (D) and 60 s at 23 °C (E). Trace F shows the EPR spectrum of Co(II)-L1 upon incubation for 5 min at 23 °C with the L1-hydrolysed product of meropenem. Trace C is a computer simulation of B 3 -1 1 assuming S = /2, D >> βgBS (50 cm ), MS = |± /2〉, gx,y = 2.32, gz = 2.35, and E/D = 0.05. Experimental EPR spectra were recorded at 10 K with 1 mW microwave power at 9.63 GHz. 10 G (1 mT) field modulation at 100 kHz was employed.
165
Figure 5-7: EPR spectra and simulations of Co(II)-substituted L1 with penicillin G as the substrate. Trace A shows the resting EPR signal of Co(II)-L1. Traces B and D show the EPR spectra of Co(II)-L1 upon reaction with penicillin G for 10 ms at 2 °C (B), and 60 s at 23 °C (D). Trace E shows the EPR spectrum of Co(II)-L1 upon incubation for 5 min at 23 °C with the L1- 3 hydrolysed product of penicillin G. Trace C is a computer simulation of B assuming S = /2, D -1 1 >> βgBS (50 cm ), MS = |± /2〉, gx,y = 2.38, gz = 2.50, and E/D = 0.005. Trace F is a model for E and consists of (0.40 × C) + (0.60 × Trace B of Figure 4). Trace F also contain a 0.2 % minor -1 component due to Fe(III) with S = 5/2, D = 20 cm , giso = 1.97 and E/D = 0.244. Experimental EPR spectra were recorded at 10 K with 1 mW microwave power at 9.63 GHz. 10 G (1 mT) field modulation at 100 kHz was employed.
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5.5 Discussion
Kinetic studies on L1 (44,45) and the related enzymes metallo-β-lactamase CcrA from
Bacteroides fragilis (43,46) and β-lactamase II (56) have suggested a common mechanism for the hydrolysis of the chromophoric substrate nitrocefin by these enzymes. The proposed mechanism invokes rapid formation of an intermediate complex, which may involve one or more steps, followed by rate-limiting breakdown of this penultimate species to complete the reaction cycle. Stopped-flow fluorescence studies suggest that the hydrolyses of other substrates of L1, including carbapenems and other cephalosporins, proceed through analogous mechanisms, though the chemical natures of the intermediates may differ in an important respect (44). With nitrocefin, it has been proposed that the rate-limiting intermediate is a ring-opened anionic species, and that this species is stabilized by both interaction with the catalytic site metal ions in
L1 and by the presence of a dinitro-substituted styryl substituent that can accomodate excess electron density through the formation of resonance structures (43,46). However, the available evidence suggests that the β-lactam amide bond remains intact as the stable intermediate forms when using other β-lactams as substrates. The breakdown of these intermediates with other substrates must involve hydrolytic cleavage of the β-lactam amide bond, and thus, the mechanism of hydrolysis of nitrocefin may be unique. In the absence of the need for stabilization of a high energy intermediate, such as the proposed nitrogen anionic species of nitrocefin, it is not immediately clear whether the metal ions would be expected to perform a role in formation of an enzyme-substrate complex. Prior to the present study, no direct evidence for such a role has been presented.
Comparison of the RFQ-EPR and stopped-flow spectrophotometric data for the reaction of Co(II)-substituted-L1 with nitrocefin confirmed the earlier proposals that the metal ions are
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indeed involved in stabilizing the rate-limiting complex. The rate of formation of the well-
studied 665 nm complex was measured at 54 s-1 at 2 °C and that of the appearance of absorbance
at 600 nm, not observed with the native Zn(II) enzyme, at 72 s-1. These data indicate that within
the shortest reaction time accessible in the RFQ-EPR experiment, 10 ms, significant formation of
enzyme-substrate complex would have occurred. Should the metal ions be involved in the
formation of this complex, the consequent perturbation of the electronic structure would be
observable in the EPR spectrum. This was, indeed, the case; the EPR data showed that only 15
% of the enzyme remained in the resting state after 10 ms and that after 39 ms the resting state,
which decayed at a rate of 67 s-1 in the stopped-flow experiments, was undetectable and only the
complex signal remained.
Earlier work (43-46) had suggested that the formation of the rate-limiting intermediate
with nitrocefin likely involves the metal ions. However, this mechanism may be unique for
nitrocefin. There is as yet no clear rationale for the role of metal ions in the formation or
stabilization of the rate-limiting intermediate in the proposed closed-ring-intermediate
mechanism proposed for the therapeutic antibiotic substrates of L1 (44). RFQ-EPR studies were
therefore carried out with cephalothin, a non-chromophoric cephalosporin related to nitrocefin but lacking the dinitrostyryl substituent and, therefore, more strictly analogous to cefaclor, an L1 cephalosporin substrate that has been proposed to form a closed-ring rate-limiting intermediate.
After the shortest available reaction time, 10.4 ms, the resting enzyme signal was completely undetectable and a signal indistinguishable from the complex signal of Co(II)-L1-nitrocefin was observed. This signal persisted after 39 ms reaction time. Thus, it is clear that with cephalothin, as with nitrocefin, a perturbation of the electronic structures of one or both of the metal ions occurs in a time consistent with formation of the rate-limiting intermediate. Clearly, then,
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cephalosporins that are proposed to form both open ring and closed ring intermediates interact
with the metal center of L1 in doing so.
In order to further test the generality of the involvement of the metal center of L1 in formation of reaction intermediates, meropenem, a carbapenem, and penicillin G were also examined. Although the literature values of kcat for both meropenem and nitrocefin are reported
to vary somewhat depending on precise conditions (18,44,57), they appear to be generally
comparable whereas the kcat for penicillin G is significantly higher (34,49,50). As with the cephalosporins, both of these substrates cease to exhibit the resting signal after only very short reaction times, indicative of participation of the metal center in formation of the rate-limiting intermediate.
In addition to providing kinetic information, some structural information can often be
3 derived from EPR of S = /2 Co(II) (52). The axial zero-field splitting, D, is generally
sufficiently large that only one of the two Kramers’ doublets is thermally-populated. Signals
3 from the MS = |± /2〉 doublet are effectively diagnostic for tetrahedral-based geometry, whereas
1 signals from the MS = |± /2〉 doublet indicate five- or six-coordinate Co(II). The rhombic
1 distortion of the axial zero-field splitting, E/D where /3 ≥ E/D ≥ 0, is a measure of the amount of
axial electronic symmetry and thus reflects the symmetry of the ligand field. Further information
can be extracted from linewidth measurements. Co(II) often exhibits broad lines due to extensive
strain in both g and in the zero-field splitting tensor, D. These strains are, in turn, due to the freezing out of a continuity of microheterogenous confomers, afforded by vibrational flexibility and often associated with solvent derived ligands, such as water or hydroxyl, with poorly defined orientations. On the other hand, the observation of resolved hyperfine structure and very narrow
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lines indicates a very tightly constrained ligand sphere such as would be provided by polydentate
ligands, including rigid proteins.
All of the signals that were observed from Co(II)-L1 were due to transitions in the MS =
1 |± /2〉 Kramers’ doublet and are due, therefore, to either five- or six-coordinate Co(II). The
signals are all essentially axial, with E/D ≤ 0.05, and this high degree of axial electronic
symmetry is suggestive of an axially symmetric ligand field. The g-values for the resting and
complex signals are close to those computed for an axially symmetric tetragonal system, where
gz = 2.43 and gxy = 2.50 (58), and support the conclusion that the EPR-detected Co(II) is in an axially-symmetric ligand field.
The EPR spectra from Co(II)-L1 also suggest that a solvent water ligand is present on the
EPR-detected Co(II) and that the rate-limiting intermediate is not formed by polydentate chelation of the EPR-detected Co(II) by the substrate molecule. The most intensively studied
Co(II) metalloprotein system is the aminopeptidase (VpAP) from Vibrio proteolyticus (52). The
Co(II) ions in the resting enzyme can adopt an axially-symmetric geometry by virtue of a solvent-derived water ligand. The inherent flexibility of this weak ligand allows it to adopt a conformation that provides the lowest energy, and thus highest symmetry, ligand field geometry
around the Co(II) in VpAP (53,54). This inherent flexibility, however, also resulted in high
strains, which in turn lead to broad lines in the EPR spectrum and the lack of resolved hyperfine
structure. Upon the addition of the inhibitor butane boronic acid, the boronic acid provided a
monodentate ligand to Co(II) and also strongly interacted with the water ligand. Consequently, some geometric and vibrational flexibility was lost, and modest line narrowing and an increase in
E/D were observed in the EPR spectra of VpAP (53,59). Upon replacement of the water with a bidentate ligand, leucine phosphonic acid, Co(II) EPR spectra with very high rhombicities (E/D
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~ 0.3) and well-resolved hyperfine structure were observed due to the rigidity imposed by the protein- and inhibitor-derived coordination sphere (59,60). In the EPR spectra of Co(II)-L1, the high degree of axial symmetry and the lack of resolved hyperfine structure are highly analogous to the resting state of VpAP and argue for the presence of a water ligand of Co(II) in Co(II)-L1.
Upon reaction with substrates and formation of the rate-limiting intermediate in L1, it is entirely possible that the substrate provides a single ligand to the EPR-detected Co(II), but it is highly unlikely that multiple covalent bonds from substrate to a Co(II) ion are formed (Figure 5-1).
Further information on the catalytic cycle of L1 can be obtained by investigation of the interaction of the hydrolysis products of substrates with the enzyme. With each of the four substrates examined, an EPR signal was obtained upon the addition of product to Co(II)-L1 that was different from both the resting signal and from the complex signals obtained at reaction times optimal for generating the rate-limiting intermediate. In the case of nitrocefin and penicillin G, the signals obtained upon adding product to Co(II)-L1 were similar to models generated by adding proportions of the complex and resting signals, though the model was not a particularly good fit to the data for penicillin G. It is unclear, then, whether any new species are involved or whether the product-bound form of the enzyme is very similar indeed to the rate- limiting intermediate but with a poor binding constant. It is interesting that the spectrum from
Co(II)-L1 incubated with penicillin G for 60s, after which all of the penicillin G would be expected to have been hydrolyzed, gave a different spectrum to Co(II)-L1 to which the pre- prepared hydrolysis product was added. It is possible that at the very high concentration of reagents being used substrate inhibition may occur due to transient formation of non- hydrolyzable intermediates. Alternatively, product dissociation and association may be very slow. Although the precise mechanism for the formation of product-related EPR signals is
171
unknown, it is clear that there is some interaction of the hydrolysis products of penicillin G and
nitrocefin with Co(II)-L1. An EPR signal was seen upon adding the hydrolysis product of
meropenem to Co(II)-L1 that could not be modeled by assuming mixtures of resting and
complex signals, though features in the spectra suggest that there is at least some contribution
from the resting signal. The effect of adding the hydrolysis product of cephalothin to Co(II)-L1,
however, was striking. A new species was observed with atypically narrow gxy features but with
gz so broadened as to be unresolvable. The signal was also seen when cephalothin was allowed
to react completely with Co(II)-L1. In the case of cephalothin, then, clear evidence exists for a
distinct product complex that is formed in essentially stoichiometric amounts.
The EPR data clearly indicate that the products of hydrolysis interact with the catalytic
metal ions of Co(II)-L1 to form significant amounts of an enzyme-product complex that, in the
case of cephalothin, represents a distinct chemical species. Importantly, previous kinetic
simulations on L1 assumed reversible binding of products to the resting enzyme (45); the EPR data presented herein support this assumption and emphasize that this step must be included in future kinetic simulations on L1.
Through RFQ-EPR, it has been possible to characterize EPR-detectable Co(II) in Co(II)-
L1. Co(II)-L1 contains two Co(II) ions per monomer, presumably in close proximity in a dinuclear active site. However, the EPR spectrum from the resting signal can be explained in terms of a single paramagnetic species. Upon reaction with cephalothin for 10 to 40 ms, this is converted into the complex signal, which can also be explained in terms of a single paramagnetic species. Finally, upon complete hydrolysis of cephalothin, yet another single species is exhibited in the EPR, corresponding to an enzyme-product complex. In principle, the EPR spectrum can be doubly-integrated, and the spin concentration calculated. In practice, this can be extremely
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difficult for high-spin Co(II), particularly for dinuclear systems. The spectral envelope can be
extremely wide, 500 mT (5000 G) or more, and even small errors in background subtraction can
affect the quantitation significantly. The spectra are also weak in intensity, due to the broad lines
and the dilution of the enzyme by a factor of four as a consequence of the RFQ procedure, and so
contain much noise. Because the spectra are weak, it is often impractical to record spectra under
strictly non-saturating or non-passage conditions, and integration results are therefore not valid.
Finally, in a dinuclear system there is always the possibility, as occurs in VpAP (53), that a
proportion of the molecules contain a strongly spin-exchange coupled pair of Co(II) ions that
exhibit no EPR signal. Quantitation of the signals from Co(II)-L1, therefore, is impracticable.
However, some insight may be available from consideration of the crystal structure of L1 (35)
and the EPR data. The two Zn ions in L1 are each ligated by three amino acid side chains and
are bridged by a solvent-derived oxygen atom (presumably –OH or H2O). In the case of Zn1, the
amino acids are all histidines, furnishing a four-coordinate site, whereas Zn2 has two histidine
and one aspartate ligand. Zn2, then, has the possibility of being five-coordinate. All the EPR
signals observed from Co(II)-L1 can be assigned to either five- or six-coordinate Co(II). Four-
3 coordinate distorted tetrahedral Co(II) gives rise to EPR signals in the MS = |± /2〉 doublet.
However, as the distortion is decreased, E/D → 0, gxy → 0 and the transition probability, i.e. the
signal intensity, tends to zero. Therefore, for axially symmetric tetrahedral Co(II), the signal is
3 either very weak indeed or non-existent. Also, MS = |± /2〉 signals from Co(II) are often very fast
relaxers; in the case of VpAP in complexation with thiol-based inhibitors, microwave powers of
3 up to 550 mW at 3.6 K were required to observe MS = |± /2〉 EPR signals (61,62). It is, then,
entirely possible that a tetrahedral site in Co(II)-L1 could go undetected. That we do see clear
substrate and product binding to the EPR-detected Co(II) in Co(II)-L1 leads us to propose that
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the hydrolytic metal ion in the native enzyme is Zn2 rather than Zn1 (Figure 1). This proposal is
consistent with the strong likelihood that a water ligand is present on the EPR-detected Co(II)
ion, analogous to Zn2, which may correspond to the active nucleophile.
The use of stopped-flow spectrophotometry and RFQ-EPR has shown that the formation of
the rate-limiting intermediate in the reaction of L1 with three classes of antibiotic substrate
involves binding at one or both of the active site metal ions. This binding occurs at a
catalytically competent rate and produces spectroscopically-distinct intermediate species with
different classes of substrate; however, the intermediates generated by the “ring-open” substrate
nitrocefin and the other “ring-closed” substrates are electronically-similar, nonetheless, and the
intimate involvement of the metal ions in formation and stabilization of the rate-limiting
intermediate is general and, therefore, relevant to therapeutically-important substrates. A
previously unidentified interaction between the reaction products and the metal site of L1 has
been characterized, and any full description of the kinetics and mechanism of the reaction must
now take into account product binding and dissociation. Consideration of the EPR data along
with crystallographic information (35) leads us to propose that Zn2 is the hydrolytic metal ion in
L1.
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Chapter 6
Conclusions
6.1 The Growing Problem of Antibiotic Resistance.
Disease-causing microbes that have become resistant to drug therapy are an increasing public health problem. Tuberculosis, gonorrhea, malaria, and childhood ear infections are just a few of the diseases that have become hard to treat with antibiotics
(http://www.fda.gov/oc/opacom/hottopics/anti_resist.html). The problem of bacteria developing or acquiring antibiotic resistance is not new. Penicillin resistance emerged before the end of
WWII, only a couple of years after the introduction of penicillin into the clinical realm (1,2).
The ever-increasing number of bacterial strains that are resistant to one or more antibiotics can be attributed to two major causes. First is the over-use and misuse of antibiotics, both in clinical and non-clinical applications. There is a strong correlation between antibiotic use and the presence of antibiotic resistance (3-6). Exposure of a population to antibiotics promotes the acquired antimicrobial resistance of the pathogens in that community. This is especially evident in the amplified frequency of antibiotic resistance associated with the increased use of an antibiotic in hospitals (5). 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 (5).
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 (5,6).
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The excessive use of antibiotics is not limited to the clinical realm. Currently there are
more than 700 household products containing antibacterial agents, up from a few dozen in the
mid-1990s (7). Initially, these products were designed for use in households containing
immuno-compromised patients, such as those suffering from cancer; however, these products are
now routinely found in healthy households. One only needs to walk the aisle at the local store to
find the cornucopia of products claiming to be “antimicrobial.” 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 (7).
The use of antibiotics and surface antibacterials for agricultural and other non-clinical purposes has allowed these antibacterial agents to enter the environment in an uncontrolled manner (8).
Residues of these antibiotics can be found in the environment for long periods of time after they are administered, allowing the bacteria to develop resistance during or after antibiotic treatment
(8).
The second factor contributing to increasing numbers of antibiotic resistant bacterial strains is the remarkable ability of bacteria to adapt to environmental changes. They achieve this through a myriad of strategies, such as chemically altering the metabolic process targeted by the antibiotic or producing substances able to destroy the antibiotic before it reaches its intended target. Additionally, the ability of bacteria to freely exchange genetic information only compounds the problem. Without addressing the proliferation of resistance genes, the inability to effectively treat bacterial infections will only become worse (8).
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”
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(2); but recently, the development of new antibiotics has been slow (9), and history shows that resistant phenotypes are quick to follow (1,2). Also, the financial cost 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 (9), with resistance to the new antibiotic occurring in 1-2 years after introduction into the clinic (5).
Rather than depend solely on the development of new antibiotics, other methods to control antibiotic resistance should be considered. Interestingly, there is more than casual evidence that reducing the use of antibiotics could help impede the spread of antibiotic resistance. The reduced consumption of antibiotics is often followed by the reduction of resistance to that specific drug (5,6). Apparently it is too biologically expensive for the bacteria to maintain resistance in the absence of antibiotics (3). Simply modifying the use of antibiotics could also potentially limit the proliferation of resistance genes and possibly lead to a decline in antibiotic resistance. Additionally, the development of inhibitors for known bacterial resistance proteins, such as to β-lactamases, would allow the continued use of current antibiotics for which resistance has already developed. According to Levy, improvements in antibiotic use and decreasing the proliferation of resistance genes could reverse the problem of antibiotic resistance
(10).
6.2 Direct Inhibition Metallo-β-lactamases
The use of agents that inhibit metallo-β-lactamases, when used in conjunction with existing antibiotics, is one strategy for treating resistant bacterial infections. To be clinically- useful, these agents have to inhibit the enzyme at extremely low concentrations. This strategy has been successfully employed to treat resistant bacteria that produce a serine-β-lactamase (11).
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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 have been designed and tested; unfortunately, none of them have been found to be clinically-useful (12,13).
However, the structural and mechanistic information gained from the study of these inhibitors, as well as using the structural and mechanistic information for L1 and the other metallo-β- lactamases, will be useful aids in the rational design of a clinically-useful inhibitor.
One possible method of inhibition is the design of a molecule that binds tightly and irreversibly to the enzyme, thereby blocking the active site. To accomplish this, it is vital to determine enzyme-substrate points of contact. The work presented in Chapter 2 has shown that amino acid residues predicted to be important in substrate recognition and binding to L1 from computational studies, are in fact not vital and has demonstrated the need for further investigation in this area. Interestingly, the work presented in Chapter 4 concerning the flexible loop of amino acids extending over the active site of L1, raises the possibility that residues on this loop may be key to substrate recognition and binding. In addition, the correlation of the movement of the loop to the formation of an intermediate provides important mechanistic information. Further investigation into specific residues on this loop that interact with substrate, and a determination if the blocking of this loop movement would halt substrate turn-over, should be pursued. It may be possible to engineer a substrate mimic containing substituents able to irreversibly bind to the loop, thereby locking the molecule in the active site, or block the loop and effectively inhibit the enzyme. Additional mechanistic information that could potentially lead to a mechanism-based inhibitor of L1 is the discovery, in Chapter 3, that the metal-binding
Asp120 plays a critical role in catalysis. It may be possible to design a substrate molecule that contains a chemical modification substituent, such as Woodward’s Reagent K, which would be
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released upon hydrolysis of the molecule and bind to Asp120, interfering with regeneration of active site to a catalytically competent form.
Another strategy for the inhibition of L1, and other metallo-β-lactamases, is to design inhibitors that mimic an intermediate of the catalytic process. A thorough understanding of the mechanism, and of course identification of intermediates, is essential to this strategy. In Chapter
5, the first direct evidence for a metal-bound intermediate in the catalytic cycle of a metallo-β- lactamase was presented. Furthermore, the demonstration that this intermediate is common to all the classes of β-lactam containing antibiotics tested indicates, for the first time, the targeting of this intermediate may be useful in the development of a single metallo-b-lactamase inhibitor.
6.3 Indirect Inhibition of Metallo-β-lactamases
While metallo-β-lactamases make up only about 7 % of the known β-lactamases, the diversity of the bacteria that harbor a metallo-β-lactamase are an evolving clinical threat (14,15).
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. Controversy surrounds whether metallo-β-lactamases exist in vivo as apo-, mono-, or di-Zn(II)-enzymes, with some suggesting that metal is only loaded in the enzyme in the presence of substrate (16). What is clear however, is that in vitro, without metal the enzymes are catalytically inactive (17). It has been shown that there is less than 1 “free”
Zn(II) per every two bacterial cells (18). How does the cell regulate the Zn(II) loading of the metallo-β-lactamases? The understanding of how Zn(II) is loaded into enzymes may reveal other possible targets for inhibition of metallo-β-lactamases. 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
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spectroscopic silence. 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 offered and administered.
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6.4 References
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