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Characterization of a highly ecient antibiotic-


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degrading metallo-b-lactamase obtained from an


Cite this: DOI: 10.1039/c7mt00195a
uncultured member of a permafrost community
Marcelo Monteiro Pedroso, *a Christopher Selleck,a Charmaine Enculescu,a
Jerey R. Harmer,b Natasa Mitic,c Whitney R. Craig,d Waleed Helweh,e
Philip Hugenholtz,af Gene W. Tyson,af David L. Tierney,d James A. Larrabeee and
Gerhard Schenk *a

Antibiotic resistance is a major global health problem, one that threatens to derail the benefits garnered from
arguably the greatest success of modern medicine, the discovery of antibiotics. Among the most potent
agents contributing to antibiotic resistance are metallo-b-lactamases (MBLs). The discovery of MBL-like
enzymes in microorganisms that are not in contact with the human population is of particular concern as
these proteins already have the in-built capacity to inactivate antibiotics, even though they may not need MBL
activity for their survival. Here, we demonstrate that a microbiome from a remote and frozen environment in
Alaska harbours at least one highly ecient MBL, LRA-8. LRA-8 is homologous to the B3 subgroup of MBLs
and has a substrate profile and catalytic properties similar to well-known members of this enzyme family,
which are expressed by major human pathogens. LRA-8 is predominantly a penicillinase, but is also active
towards carbapenems, but not cephalosporins. Spectroscopic studies indicate that LRA-8 has an active site
structure similar to that of other MBLs (in particular B3 subgroup representative AIM-1), and a combination of
Received 4th July 2017, steady-state and pre-steady-state kinetic data demonstrate that the enzyme is likely to employ a metal ion-
Accepted 18th July 2017 bridging hydroxide to initiate catalysis. The rate-limiting step is the decay of a chromophoric, tetrahedral
DOI: 10.1039/c7mt00195a intermediate, as is observed in various other MBLs. Thus, studying the properties of such pristine MBL-like
proteins may provide insight into the structural plasticity of this family of enzymes that may facilitate functional
rsc.li/metallomics promiscuity, while important insight into the evolution of MBLs may also be gained.

Significance to metallomics
Metal-dependent enzymes (e.g. metallo-b-lactamases (MBLs)) constitute up to 50% of all enzymes and combine the selectivity bestowed upon them by the
complexity of the protein scaold with catalytic eciency that originates from their metallic cofactors. MBLs are major contributors to antibiotic resistance, but
their occurrence is not limited to pathogenic organisms. A metagenomics analysis revealed that microbial communities in frozen environments have several
MBL-like proteins. At least one of these is indeed highly eective in inactivating b-lactam antibiotics, posing an emerging threat to global health, potentially
associated with climate change.

a
School of Chemistry and Molecular Biosciences, The University of Queensland,
St. Lucia, Queensland, 4072, Australia. E-mail: m.pedroso@uq.edu.au,

b
schenk@uq.edu.au
Centre for Advanced Imaging, The University of Queensland, St. Lucia,
Introduction
Queensland, 4072, Australia
c
The successful treatment of common bacterial infections
Department of Chemistry, Maynooth University, Maynooth, Co. Kildare, Ireland
d
Department of Chemistry and Biochemistry, Miami University, Oxford,
has been challenged in an alarming manner. This has been
Ohio 45056, USA achieved by the spread of antibiotic resistance in an increasing
e
Department of Chemistry and Biochemistry, Middlebury College, Middlebury, number of pathogens of clinical relevance, sustained by periods
VT, 05753, USA of haphazard use of antibiotics in health care, combined with
f
Australian Centre for Ecogenomics, The University of Queensland, St. Lucia,
global motility.1 Furthermore, antibiotics are employed in a
Queensland, 4072, Australia
Electronic supplementary information (ESI) available: Fig. S1: Map of recombi-
prophylactic manner in the agricultural industry, often in feed
nant expression system and sequence of LRA-8; Fig. S2: SDS-PAGE gel of purified and water supply.2 Worryingly, a handful of antibiotics used to
LRA-8. See DOI: 10.1039/c7mt00195a treat human pathogens are also used in agriculture, engendering

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greater selective pressure in a more widely disseminated address antibiotic resistance. Examples of such MBL-like proteins
fashion.2,3 are MIM-1 and MIM-2, produced by the marine microorganisms
Antibiotic resistance determinants are frequently located on Novosphingobium pentaromativorans and Simiduia agarivorans,
transposable genetic elements. Through horizontal gene transfer respectively.26 Although MIM-1 and MIM-2 are both ecient MBLs
urban and environmental sources of antibiotic resistance are they also have potent lactonase activity, an activity that is associated
easily connected.46 In particular, uncultured bacteria in soil with quorum sensing.27 This observed functional promiscuity
environments have been identified as an important reservoir of provides one explanation why MBL activity may be found in
diverse antibiotic resistance determinants,7 including some that pristine environments. Similarly, a metagenomics study with
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have not been subjected to anthropogenic influence.8 The latter uncultured bacteria collected from soil from a remote region in
has recently also been demonstrated by an analysis of the gut and Alaska identified a group of 14 candidate genes (LRA; b-Lactam
skin microbiome of the Amazonian tribe of the Yanomami, a Resistance in Alaskan soil genes) predicted to encode b-lactamases,
people that has not been in previous contact with the outside six SBLs and eight MBLs.8 When expressed in Escherichia coli each
world.9 Antibiotic resistance in the absence of human inter- of these 14 candidates conferred resistance to the host organism
vention offers invaluable insights regarding the origin and to a number of b-lactam antibiotics, in some cases to clinically
evolution of antibiotic resistance, which may assist in future relevant levels. The most ecient candidate is LRA-8, which
efforts to counteract and prevent this global threat to modern displays minimum inhibitory concentrations (MICs) greater
healthcare.7,9 than 512 mg mL1 for the four antibiotics tested from the
b-Lactamases are a large family of enzymes, capable of penicillin family. LRA-8, which is homologous to MBLs from
inactivating most commonly used b-lactam antibiotics such the B3 subgroup (Fig. 2), also confers resistance towards several
as penicillins, cephalosporins and carbapenems (Fig. 1). This cephalosporins (with MICs ranging from 32 to 128 mg mL1; no
family is divided into four classes, A, B, C and D.10,11 Classes A, carbapenems were tested).8 With the current trend in global
C and D are serine-b-lactamases (SBLs) which employ an active warming, this raises the possibility that genes encoding LRAs
site serine residue as nucleophile to initiate the hydrolysis of may enter the human population, potentially exacerbating the
the b-lactam bond.12,13 SBLs have narrow substrate profiles in spread of antibiotic resistance. Here, we expressed and purified
addition to a number of clinically available inhibitors.12,14,15 LRA-8 and demonstrated that it is a potent MBL with catalytic
Class B constitutes the group of metallo-b-lactamases (MBLs) parameters matching those of its counterparts from pathogenic
which require one or two zinc(II) ions for the hydrolysis of the organisms.
b-lactam bond.1619 The MBLs can be further divided into as
many as four subgroups, i.e. B1B4, depending on sequence
similarity and the number of zinc(II) ions required for Results and discussion
catalysis.2024 In contrast to their SBL counterparts, MBLs have
a high degree of mutational frequency at their active site, LRA-8 is a potent MBL
conferring substrate promiscuity.22 Moreover, their rapid In order to establish if LRA-8 can act as an MBL, kinetic assays
dissemination and a lack of clinical inhibitors represent a were carried out with several b-lactam substrates that represent
threat to the human population.5,14,25 three major groups of antibiotics that are commonly used
At an increasing rate antibiotic-degrading activities are in medical treatments (Fig. 1). The data are summarised
identified in metagenomic analyses of organisms found in in Table 1. LRA-8 is capable of rapidly hydrolysing the
environments that have not been challenged by human activities. penam substrates ampicillin (kcat B 500 s1) and penicillin
In particular the MBLs play a significant role in this context since G (kcat B 150 s1). These substrates do not bind very tightly
they are catalytically highly ecient while no clinically useful to the enzyme with Km values of B870 mM and B320 mM,
inhibitor is currently available. Insight into the structure and respectively, resulting in catalytic eciencies (kcat/Km ratios)
function of MBL-like proteins identified in such environments, of B0.5 s1 mM1 for both.
including the role(s) metal ions play in promoting the catalytic The carbapenem representative (biapenem) is turned over
proficiency of these enzymes, may thus also inform novel strategies slightly more eciently (kcat/Km B 0.8 s1 mM1), largely due to
to design potent inhibitors that may be of clinical relevance to its enhanced binding interactions (i.e. low Km value). Among
the cephalosporins tested, only the non-clinical nitrocefin was
hydrolysed; no activity towards cefuroxime or cephalothin was
detected. This may suggest a distinct specificity that may guide
future inhibitor design/drug development studies. However,
with a kcat in the range of that measured for the penam
substrates and a Km similar to that of biapenem the catalytic
eciency towards nitrocefin is the highest among the sub-
strates tested here. It should also be pointed out that while for
Fig. 1 Chemical structures of representative antibiotic types, each containing
some MBLs substrate inhibition was observed at substrate
the defining four-membered b-lactam ring. The R groups are indicative of concentrations above respective Km values no such inhibition
modifications for respective antibiotics. was observed for LRA-8.26,31,33

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Fig. 2 Top: Partial sequence alignment including LRA-8 and the well-characterised B3 MBLs L1, AIM-1 and FEZ-1 using ENDscript software; the
secondary structure elements were obtained from the FEZ-1 structure.28 Highly conserved residues are highlighted in red and homologous residues are
in yellow. Metal binding residues are indicated with the appropriate label. Bottom: Active site of AIM-1 (PDB 4AWY).29,30 The grey spheres represent zinc
ions with the numbers indicating metal ion binding sites 1 and 2, respectively.

Table 1 Kinetic parameters of LRA-8. Data were collected in 50 mM are summarised. The table also contains information for two
TrisHCl, pH 8.5, containing 150 mM ZnCl2, using substrates representative MBL-like proteins identified in non-pathogenic marine organisms,
for major b-lactam groups
i.e. MIM-1 and MIM-2 from N. pentaromativorans and S. agarivorans,
kcat (s1) Km (mM) kcat/Km (s1 mM1) respectively. LRA-8 is approximately one order of magnitude
Ampicillin 490  40 870  140 0.55  0.15 less ecient than these other MBLs, largely due to weaker
Penicillin G 150  15 320  75 0.45  0.10 binding (i.e. larger Km values) of the substrates. Nonetheless,
Biapenem 35 5 40 5 0.8  0.2 LRA-8 displays a similar preference for penam-type substrates
Cefuroximea
Cephalothina (i.e. ampicillin, penicillin G) as these MBLs from the B3 subgroup.
Nitrocefin 90 5 50 5 1.8  0.5 Currently there is no clinically useful inhibitor for MBLs.14
a Captopril is, however, an in vitro inhibitor that has been useful
No hydrolysis was observed using substrate concentrations ranging
from 10 mM to 1280 mM. in functional studies of MBLs; while L-captopril is a drug used
to treat hypertension and in some cases of heart failure, in
particular the stereoisomer D-captopril is a potent inhibitor of
In Table 2 corresponding catalytic parameters for two several MBLs.34 Reported inhibition constants (Ki values) range
well-characterised MBLs from the B3 subfamily, AIM-1 and L1, from B6 mM for the B3 subgroup MIM-1 to B70 mM for the

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Table 2 Kinetic parameters for the reactions of two known MBLs (AIM-1 and L1) and two MBL-like proteins (MIM-1 and MIM-2) with dierent b-lactam
substrates and standard deviations are shown, where available. Each of these proteins belongs to the B3 subgroup of MBLs

AIM-1 L1 MIM-1 MIM-2


kcat kcat/Km kcat kcat/Km kcat kcat/Km kcat kcat/Km
Ampicillin 150  5 6.2 580  20 1.9 665  40 2.9 230  5 1.6
Penicillin G 590  30 5.3 410  20 5.5 125  5 2.4 120  2 3.3
Biapenem 235  60 8.1 65  5 0.9 55  5 1.0 5.5  0.3 0.06
Cefuroxime 170  5 4.8 55  10 0.4 30  1 1.8 40  2 0.7
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Data were reproduced from Leiros et al.,29 Selleck et al.,31 Crowder et al.32 and Miraula et al.26 for AIM-1, L1, MIM-1 and MIM-2, respectively.
The kcat values are in (s1) and the values of kcat/Km are in (s1 mM1).

MichaelisMenten-type behaviour was observed for each


substrate at each pH in the range between 6.0 and 11.0
(data not shown). For each substrate kcat increases as the pH
increases until a maximal value is reached; a further increase in
pH does not lead to further changes (Fig. 4; top panel).
This behaviour is characteristic of a monoprotic system with
only one relevant protonation equilibrium. The data were thus
fit to eqn (3) (see Materials and methods section). It is noted
that biapenem is most rapidly hydrolysed only at a pH larger
than 9.0 (kcat,max B 125 s1 versus 35 s1 at pH 8.5; see Table 1).
In contrast to kcat, the pH dependence of the kcat/Km ratio varies
between dierent substrates (Fig. 4; bottom panel); while no
Fig. 3 Inhibition of LRA-8 activity by D-captopril using penicillin G as dependence is apparent for penicillin G, a single protonation
substrate. The inhibitor concentrations are zero (circle), 100 mM (squares),
equilibrium is detected for the reaction with biapenem. These
150 mM (triangles) and 300 mM (diamonds). Individual data points were
recorded in triplicates and the averaged values and standard deviations are data were thus also fit to eqn (3). For the reaction with
indicated. The inhibition constants were obtained by a fit of the experi- nitrocefin very high pH values lead to a decrease in the catalytic
mental data to eqn (2) (see Materials and methods section). eciency. The resulting bell-shaped profile is characteristic
of a diprotic system and was consequently fitted to eqn (4)
(see Materials and methods section).
B2-subgroup CphA.26 D-Captopril also inhibits LRA-8 activity,
albeit somewhat weaker than other MBLs (Fig. 3); nonetheless,
similar to other MBLs studied to date the mode of inhibition is
competitive (Kic B 110 mM, Kiuc 4 7 mM). Thus, in summary
the above substrate preference and D-captopril inhibition data
confirm that LRA-8 is an ecient MBL with catalytic para-
meters resembling those of well-characterised B3-type MBLs
such as AIM-1 or L1.

Mechanism of action of LRA-8


Studying the pH dependence of the kinetic parameters of an
enzyme-catalysed reaction is a powerful approach to gain insight
into the details of the mechanism of action. In particular,
mechanistically relevant protonation equilibria may be uncovered
that allow the identification of residues that are important for the
chemical step (i.e. the hydrolytic step in the case of MBLs),
substrate binding, or both. While relatively few pH dependence
studies have been reported for MBLs, it appears that these
enzymes operate most eciently at pH values close to neutral.17
Furthermore, dierent substrates may lead to dierences in
observed pKa values, possibly a reflection of dierent binding
interactions and subtle mechanistic variations. Here, penicillin G,
biapenem and nitrocefin, substrates that represent three major Fig. 4 pH dependence of kcat (top panel) and kcat/Km (bottom panel) for
classes of b-lactams substrates (Fig. 1), were used to investigate the LRA-8-catalysed hydrolysis of penicillin G (blue), biapenem (black) and
the eect of pH on both kcat and kcat/Km (Fig. 4). nitrocefin (red).

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Table 3 Relevant pKa values for the LRA-8-catalysed hydrolysis of penicillin G,


biapenem and nitrocefin

Penicillin G Biapenem Nitrocefin


pKes 6.5  0.3 8.4  0.2 6.7  0.2
pKe1 6.6  0.4 6.9  0.1
pKe2 10.1  0.2
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According to the model used to derive eqn (3) and (4) the
pH dependence of kcat provides an estimate for catalytically
relevant pKa values for the enzymesubstrate (ES) complex
(i.e. pKes), while the pH dependence of kcat/Km provides an estimate
for pKa values associated with the free enzyme (i.e. pKe).35,36
Relevant pKa values are summarised in Table 3. The residue likely
to be associated with pKes (and pKe1 in the reaction with biapenem
and nitrocefin) is a metal ion-bridging water molecule (m-H2O).
Deprotonation of this water molecule would strongly enhance its
nucleophilicity. Indeed, on related studies with other MBLs the
m-OH moiety was proposed to act as the hydrolysis-initiating
nucleophile.16,17,19,31 It thus appears that LRA-8 is likely to employ
a catalytic mechanism similar to that of well-characterised MBLs.
However, the data also indicate that different substrates may Fig. 5 Rapid-scan UV-Vis spectra from 300800 nm for the reaction of
interact differently with the active site of LRA-8. For the reaction 50 mM of nitrocefin with 70 mM of LRA-8 in 50 mM TrisHCl, pH 8.5, at
with nitrocefin, substrate binding has little effect on the pKa of the 25 1C. Top: The arrows indicate the loss of substrate (at 390 nm) and the
bridging water molecule (pKe1 B pKes), while for the reaction with accumulation of product (at 485 nm). Bottom: The emergence and
disappearance of a spectral feature at B665 nm is shown. The time course
biapenem the acidity of the bridging water is reduced by nearly
for the reactants as shown in Fig. 6 was determined from the baseline-
two orders of magnitude (pKe1 = 6.6 vs. pKes = 8.4). corrected absorbances at 390 nm (substrate), 485 nm (product) and
A similar observation has been reported for other dinuclear 665 nm (intermediate).
metallohydrolases such as purple acid phosphatases or some
organophosphate-degrading triesterases.3739 In those enzymes
the alkaline shift upon substrate binding was interpreted in properties; the intact molecule has a characteristic absorbance
terms of a move of the bridging water ligand into a pseudo- maximum at 390 nm (e = 11 500 M1 cm1), while hydrolyzed
monodentate position, likely accompanied by an elongation nitrocefin (i.e. after opening of the b-lactam ring) has a max-
of the metalmetal distance in the active site. It is thus imum at 485 nm (e = 17 420 M1 cm1).41 The reaction of 70 mM
hypothesised that in LRA-8 substrates such as biapenem and of LRA-8 with 50 mM nitrocefin was monitored with a photo
nitrocefin bind in different modes to the metal ions in the diode array detector under single turnover conditions between
active site, bridging in the case of biapenem and monodentate 300800 nm (Fig. 5). As expected the substrate is consumed
in the case of nitrocefin. No information about the binding of rapidly as product is formed (indicated by arrows at 390 nm
penicillin G can be gained from the pH dependence of the catalytic and 485 nm, respectively, in Fig. 5). An additional peak at
parameters, except that the affinity for this substrate decreases as 665 nm emerged in the first 25 ms of the reaction and decayed
pH is increased, thus offsetting the beneficial effect of the depro- over the subsequent 150 ms (Fig. 5 and 6). The combined
tonation of the bridging water on the catalytic reactivity. spectroscopic features in Fig. 5, characterized by isosbestic
pKe2, only observed for the reaction with nitrocefin (Table 3 points around B430 and B590 nm, are indicative of a reaction
and Fig. 4), is tentatively assigned to the deprotonation of a that involves the transient formation of a reaction intermediate,
water molecule that is bound to only one divalent metal ion in as was previously reported for some MBLs.31,4245 Plots of the
the active site, an assignment that is consistent with expected pKa absorption intensities of the substrate, intermediate and product
values for aquo complexes of transition metal ion complexes.40 versus time are shown in Fig. 6.
In its deprotonated state this water ligand is slowly exchanged, The simplest model for the reaction mechanism employed
thus interfering with ecient substrate binding and/or product by LRA-8 is shown in Scheme 1 and includes the formation of a
release. In agreement with this interpretation this protonation short-lived intermediate (EI complex). A full numeric evaluation
equilibrium is absent in the enzymesubstrate complex, likely of the model would require eight rate constants, four for the
to be due to the replacement of the water ligand by the bound forward and four for the reverse reactions (i.e. ki and ki, with
substrate. i = 14; Scheme 2). Several simplifications may be applied.
To probe the individual steps of the catalytic mechanism Firstly, the rapid association between the enzyme (E) and the
further stopped-flow measurements were carried out. Nitrocefin substrate (S) or product (P) were assumed to be limited by
was used as substrate due to its beneficial spectroscopic diffusion (i.e. k1 and k4 B 108 M1 s1, fixed). Secondly, the

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Scheme 2 Hydrolysis of nitrocefin (top) and kinetic model used for


modelling and fitting of the experimental data.

Fig. 6 Stopped-flow data analysis. The experimental data were analysed


Table 4 Rate constants for the reaction of LRA-8 with nitrocefin. The rate
using the mechanistic model presented in Scheme 2 and the corres-
constants were obtained using the mechanistic scheme illustrated in
ponding fits to the time course for the substrate, product and intermediate
Scheme 2. kcat and Km represent the theoretical values calculated using
are shown in red.
the KingAltman approach to derive rate equations for the reactions
illustrated in Schemes 1 and 2

rate constants k2 and k3 were set to zero because the hydrolysis k1 (M1 s1) 1.0  108 (fixed)
of nitrocefin is essentially irreversible. Furthermore, estimates k1 (s1) 1.0  104
k2 (s1) 485  11
for k2 and k3 were obtained from the rates of substrate depletion k3 (s1) 90  5
and product formation. These approximations were used to k4 (s1) 1.1  0.1  105
simulate the reaction using the program KINSIM.46 These k4 (M1 s1) 1.0  108 (fixed)
kcat (s1) 76  3
simulations indicate that the shape of calculated concentration KM (mM) 18  5
versus time plots (see Fig. 6) is not sensitive towards the
magnitude of k1, k4 and k4, but depends strongly on the values
of k1, k2 and k3. Table 4 summarises rate constants that were calculated from the constants in Table 4 and using the KingAltman
obtained from a reasonable simulation of the experimental approach35 are 76 s1 and 18 mM, also in good agreement with the
data. The parameters in Table 4 indicate that the slowest step steady-state parameters (i.e. B90 s1 and 50 mM).
in the reaction is the conversion of the intermediate to the Thus, LRA-8 is likely to employ a mechanism as depicted in
product, characterized by the rate constant k3 (B90 s1). Scheme 1, similar to that of a number of other MBLs such as
Based on studies with biomimetics and other MBLs it was NDM-1, BcII and L1, with the decay of a reaction intermediate
previously proposed that the structure of this intermediate as the rate-limiting step.43,45
is an anionic adduct where the nucleophilic oxygen forms
a bond to the electrophilic carbon atom of the b-lactam bond Insight into the active site structure of LRA-8
(Scheme 1).16,40,47 The discussion above demonstrates that LRA-8 is an enzyme
It thus follows that breakage of the b-lactam bond is the with catalytic properties characteristic of MBLs. In the absence
rate-limiting step in the reaction. It should be pointed out that of crystallographic information about the structure of this
k3 is of similar magnitude as kcat, the first order rate constant enzyme we employed spectroscopic techniques to compare its
measured under steady-state conditions (B90 s1 at pH 8.5; active site structure with other MBLs for which corresponding
Table 1). Furthermore, the theoretical kcat and Km values data are available. For this purpose the native Zn(II) cofactor

Scheme 1 Proposed hydrolysis mechanism of nitrocefin by LRA-8, modified from a model previously reported for an MBL from Bacteroides fragilis;47
the rate limiting step is the decay of the anionic reaction intermediate. The hydrolysis-initiating nucleophile is a hydroxide activated by the two metal ions
in the active site.

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was replaced with the paramagnetic Co(II) ion, providing the Table 5 Fitted electronic parameters for MCD spectra and VTVH MCD
basis for both Magnetic Circular Dichroism (MCD) and Electron data
Paramagnetic Resonance (EPR) measurements. Similar to the Parameter 464 nm 491 nm 509 nm
B3-type MBL AIM-1 the replacement of Zn(II) by Co(II) leads to 1
J (cm ) 0.29 0.31 0.27
a B50% decrease in catalytic activity and a moderate eect on Da (6-coordinate) (cm1) Z50 Z50 Z50
substrate (penicillin) binding (i.e. kcat B150 s1 and B60 s1 Db (6-coordinate) (cm1)
and Km B300 mM and B350 mM, respectively, for the Zn(II) and E (a,b metal) (cm1) 0.0 0.0 0.0
Co(II) derivatives of LRA-8 at pH 8.5). Similarly, the competitive
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inhibitory eect of D-captopril is also reduced from B100 mM for


Table 6 Summary of ligand field calculations
the Zn(II) derivative to B250 mM for the Co(II) derivative. Despite
these catalytic changes the Co(II) derivative represents an active dd transition (nm)
form of LRA-8, suitable for spectroscopic investigations of the Origin in Oh Observed Calculated
structure and mechanism of this enzyme. In Fig. 7 the MCD
6-Coordinate species 4
T1g - 2
T1g(G) 464 460
spectrum of LRA-8 is shown, together with VTVH MCD 4
T1g - 4
T1g(P) 491 495
data recorded at three of the major transitions. The spectrum
4
T1g - 4
T1g(P) 502 503
4
T1g - 4
T1g(P) 509 517
could be fitted to four transitions between 450 to 550 nm. The
VTVH MCD data at 464 nm, 491 nm and 509 nm were
extracted and analysed as described in detail elsewhere.48 This interpretation was further probed by comparing the
In brief, the data were fit to the dimer model, resulting
EPR spectra of these two enzymes (Fig. 8).31 Their respective
in relevant fitting parameters summarised in Table 5. continuous wave (CW) EPR spectra are similar and characteristic
The bands at 491 nm and 509 nm are the main spin-allowed of a bimetallic Co(II) system with five or six ligands (the data are
4
T1g(P) transitions from the two six-coordinate Co(II) ions not sensitive enough to distinguish between five or six ligands in
in the active site. Both metal ions can also have a spin-doublet the coordination environment of the metal centres).31,44,45 Fig. 8
around 464 nm (Table 6). Consistently, the VTVH MCD analysis also shows that there is little dierence in the spectral features
of the 464 nm band indicates that the Co(II) ion(s) associated upon the addition of the competitive inhibitor D-captopril; the
with this transition is weakly and ferromagnetically exchange- low field shoulder for both spectra exhibit splittings from a
coupled ( J = 0.29 cm1) to another six-coordinate Co(II) ion
cobalt hyperfine interaction, which are essentially identical.
that is associated with either the 491 nm or 509 nm transition While there is a reduction in the intensity of the sharp feature
(which have virtually identical electronic structural parameters; around 340 mT upon the addition of D-captopril, overall the data
Table 5). suggest that the inhibitor interacts only weakly with the active
The only MBL that has previously been analysed by MCD in site metal ions. This interpretation is again in agreement with a
a similar manner is AIM-1,31 where both Co(II) species are also similar observation reported for AIM-1.31
likely to be six-coordinate with a similar weak ferromagnetic The spectra were simulated using XSophe (Bruker Biospin),
coupling. Thus, it is evident that the active site structures of assuming H0 = bB0gS/h  + SDS, where S = 3/2, |D| c |bgBS/h  |.
both LRA-8 and AIM-1 are very similar.

Fig. 8 X-band (9.36 GHz) CW EPR data recorded in perpendicular (top)


and parallel (bottom) mode. Shown are spectra for LRA-8 (blue) and LRA-8
in the presence of the inhibitor D-captopril (red), and for comparison the
Fig. 7 MCD spectrum (at 1.3 K and 7 T) of LRA-8 at pH 7.0 (top) and fitted corresponding spectra from the enzyme AIM-1 (cyan and magenta). The
VTVH MCD data (bottom) for the 464, 491 and 509 nm transitions. Tem- simulation for a Co(II) high-spin species is shown (black). The measure-
peratures of red, green, blue, yellow, grey, black represent data observed at ments were carried out at 20 K under non-saturating conditions using a
1.4, 3.0, 6.0, 12.0, 24.0 and 48.0 K, respectively. modulation amplitude of 1 mT and modulation frequency of 100 kHz.

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Note that when D 4 0 the MS = 1/2 Kramers doublet lies Protein expression and purifications
lowest and all observed EPR transitions are from this doublet; All media used for the expression of LRA-8 were supplemented
if D o 0 the MS = 3/2 Kramers doublet lie lowest. The with 50 mg ml1 of kanamycin. The LRA-8-encoding construct
simulation parameters were D = 14 GHz, E = 0.95 GHz, with LRA8::pJ411 was used to transform competent E. coli BL21
gx, gy, gz = 2.10, 2.29, 2.18, respectively, with the corresponding (DE3) cells by heat shock. The culture was grown at 37 1C and
linewidths of 1.2, 0.61 and 0.35 (modelled by g-strain). The then transferred into TB medium (1 L) and grown at 37 1C until
model assumes that the magnetic interaction between the two an optical density (OD600) of 0.40.6 was reached. The tempera-
cobalt ions is small (consistent with the MCD data) and not ture was then reduced to 18 1C and the culture were incubated
Published on 18 July 2017. Downloaded by ST LAWRENCE UNIVERSITY on 07/08/2017 14:20:42.

resolved by the EPR spectrum. Not surprisingly, these para- for an additional 48 hours. Subsequently, the cells were
meters are similar to those obtained from the simulation of the harvested by centrifugation at 4 1C. The cells were resus-
corresponding EPR data for the enzyme AIM-1. pended in lysis buffer (50 mM TrisHCl, pH 8.5, 5 mM
Thus, in summary the comparison of the spectral para- b-mercaptoethanol, 150 mM NaCl, a protease inhibitor cock-
meters of LRA-8 and AIM-1 indicate that both enzymes have a tail and 1 mg mL1 lysozyme), and then disrupted by sonica-
conserved active site geometry, as would be expected based on tion. The supernatant was loaded onto a 25 mL HiTrapQ FF
the similarity of their kinetic parameters. Ni(II) affinity column (Ni(II)-IMAC resin; GE Healthcare), equi-
librated with 50 mM TrisHCl buffer, pH 8.5, containing
150 mM NaCl, 5 mM b-mercaptoethanol and 20 mM imida-
Conclusions zole. The protein was eluted isocratically using 500 mM
Antibiotic resistance is a major global health concern, one that imidazole. The enzyme was further purified using a S-300
threatens to derail the benefits garnered from arguably the gel filtration column equilibrated with 50 mM TrisHCl,
greatest success of modern medicine, the discovery of antibiotics. pH 8.5, containing 0.2 M NaCl and 0.15 mM of ZnCl2.
MBLs are a major contributor to the emergence and spread of An average expression resulted in a yield of 12 mg of purified
resistance, partly because there are currently no clinically LRA-8 per litre of culture medium. The purity of the enzyme
useful inhibitors available, and partly because these enzymes was estimated by SDS-PAGE analysis to be at least 95%;
are frequently located on mobile genetic elements that may be the molecular weight of the isolated enzyme is estimated to
easily transferred from one microorganism to another. B34 kDa, in good agreement with both the calculated mass and
The discovery of MBL-like enzymes in microorganisms that the weight recorded by mass spectrometry (Fig. S2, ESI). The
are not in contact with the human population is thus of metal ion content was determined by atomic absorption spectro-
particular concern as these proteins already have the in-built scopy and confirmed the presence of two (2.1  0.2) Zn ions per
capacity to inactivate antibiotics, even though they may not active site. It is noted that no activity was detected in the flow-
need MBL activity for their survival. These enzymes are likely to through of the Ni(II)-IMAC column after loading the enzyme
have dierent primary functions, relevant to the organisms sample, indicating that the full-length recombinant protein
metabolism. Thus, studying the properties of such MBL-like remained intact (i.e. no posttranslational modification did
proteins may provide insight into the structural plasticity of occur), in agreement with the molecular weight estimated from
this family of enzymes that may facilitate functional promiscuity, the SDS-PAGE gel (Fig. S2, ESI).
while important insight into the evolution of MBLs may also be
gained. The structural plasticity may also be a major factor that Steady state enzyme kinetics
has contributed to the current lack of clinically useful inhibitors
All kinetic measurements were carried out with a Cary 60 Bio
for MBLs. In this respect, the demonstration that LRA-8 is indeed
Varian UV-Vis spectrophotometer. The hydrolysis of ampicillin
a potent MBL that is likely to employ a reaction mechanism
(e235nm = 890 M1 cm1), penicillin G (e243nm = 936 M1 cm1),
similar to that of well-known virulent MBLs is of great relevance
cefuroxime (e235nm = 9320 M1 cm1), cephalothin (e293nm =
for inhibitor design, but also for functional investigations into
8790 M1 cm1), biapenem (e293nm = 7600 M1 cm1) and
mechanistic factors that may contribute to promiscuity. Eorts
nitrocefin (e485nm = 17 400 M1 cm1) was monitored in 50 mM
towards elucidating the crystal structure of the enzyme are
TrisHCl (pH 8.5), containing 150 mM ZnCl2 (or 100 mM CoCl2 in
currently in progress.
case of the Co(II) derivative of LRA-8), at 25 1C. The buffers were
treated prior to use with Chelex (Bio-Rad). The MichaelisMenten
Materials and methods parameters (kcat, kcat/Km) were determined by non-linear regression

The recombinant expression system containing the full-length


LRA-8-encoding sequence, modified by the addition of an Recombinant LRA-8 contains a non-cleavable hexa-histidine tag. However, its
N-terminal hexa-histidine tag, in the vector pJ411 was purchased presence is unlikely to aect the metal ion content and catalytic properties of the
enzyme significantly. This interpretation is supported by the observation that in
from DNA 2.0 (see Fig. S1 (ESI) for a map of the construct). E. coli
the Co(II) derivative of LRA-8 the metal ions exclusively bind in the close proximity
BL21 (DE3) cells used for protein expression were purchased from provided by the catalytic active site (Fig. 7 and 8). Furthermore, the closely related
New England BioLabs. All chemicals used were purchased from enzyme AIM-1 was purified in the absence of a hexa-histidine tag and the
Sigma-Aldrich unless stated otherwise. spectroscopic properties are indeed similar to those of LRA-8.31

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analysis (eqn (1)35) of the experimental data using GraphPad in Scheme 2.47,50,51 The data were then fitted using Reactlab
Prism 6 software. software.
Vmax  S
v (1) Spectroscopic characterization of LRA-8
Km S
The active site structure of LRA-8 was probed using MCD and
Here, v, Vmax and Km represent the measured catalytic rate, the CW EPR. In order to use these methods Zn(II) ions were
maximal rate and the Michaelis constant, respectively, while S replaced by paramagnetic Co(II). The preparation of the apo-
indicates the substrate concentration. enzyme was performed following a previously published
Published on 18 July 2017. Downloaded by ST LAWRENCE UNIVERSITY on 07/08/2017 14:20:42.

Inhibition assays were conducted in the same manner as procedure31,52,53 by adding 10 mM EDTA in 20 mM Hepes
described above using the substrate penicillin G and various buer, pH 7.0, to the purified enzyme. After 24 h incubation
concentrations of the inhibitor D-captopril. The data were fitted at 4 1C the chelating agent was removed using a desalting
to a general inhibition equation that allows for both a compe- column (Econo-Pac 10DG from Bio-Rad) that was equilibrated
titive and uncompetitive mode of inhibitor binding (eqn (2)).35 with 20 mM Hepes buer, pH 7.0. Subsequently, three equi-
Kic and Kiuc represent the inhibitor dissociation constants for valents of Co(II) were added to the metal-free protein solution
competitive and uncompetitive binding, respectively, and I is and the mixture was incubated over night at 4 1C. The excess of
the inhibitor concentration. Co(II) was removed using again a desalting column, pre-
Vmax  S equilibrated with 50 mM TrisHCl buffer, pH 8.0. The concen-
v     (2)
I I tration of the resulting sample was 1.75 mM with nearly two
Km 1 1 S equivalents of bound Co(II) (1.8  0.1, determined by atomic
Kic Kiuc
absorption spectroscopy). For spectroscopic measurements this
The pH profiles of these parameters were determined using solution was diluted with glycerol to a final concentration
nitrocefin as the substrate. The pH for these assays ranged from of 0.7 mM (i.e. a 3 : 2 glycerol : buffer mixture).
7.0 to 11 using a 20 mM acetate, 20 mM MES, 20 mM HEPES, For MCD measurements an aliquot was transferred to a
20 mM CHES and 20 mM CAPS multi-component buer system. 0.62 cm path length nickel-plated copper sample cell with quartz
The experimental data were fitted using equations ((3) and (4), windows. The MCD system used has a JASCO J815 spectropolari-
respectively) for mono- or diprotic systems as described meter and an Oxford Instruments SM4000 cryostat/magnet. Data
elsewhere.35,36 were collected at 7.0 T and 1.4 K. Variable-temperature, variable-
0 1 field (VTVH) data were collected at increments of 0.5 T from 0
B c C to 7.0 T and at temperatures of 1.4, 3, 6, and 12 K. The
Log y LogB
@
C (3)
HA experimental spectra were plotted as a function of wavenumbers
1
K1 and fitted to a minimum number of Gaussian peaks to achieve
the final composite spectrum using the GRAMS AI software
0 1 package. The data were subsequently analysed as described in
B c C detail elsewhere.31,48,5357
Log y LogB
@
C (4)
H K2 A EPR measurements were carried out with the same sample
1
K1 H used for MCD experiments. Low temperature X-Band CW EPR
spectra were recorded on a Bruker EMX EPR spectrometer
Here, H is the proton concentration, Ki represents the acid equipped with an Oxford Instruments ESR900 helium flow cryostat.
dissociation constant for either the enzymesubstrate complex Spectra were recorded at 9.64 GHz (B0>B1) and 9.38 GHz (B08B1)
(ES) or the free enzyme (E), c is the pH independent values of using an ER4116DM dual-mode cavity, with 10 G (1 mT) magnetic
y, and y is kcat or kcat/Km. field modulation at 100 kHz. Spin Hamiltonian parameters were
Pre-steady state kinetics measurements estimated from computer simulations carried out using XSophe
(Bruker Biospin), assuming H0 = bB0gS/h  + SDS, where S = 3/2,
All pre-steady state kinetics measurements were carried out |D| c |bgBS/h  |, and where D 4 0 implies the MS = 1/2
in 50 mM TrisHCl, pH 8.5, at 25 1C using an Applied Kramers doublet lies lowest and all observed EPR transitions
Photophysics SX-18 spectrometer, coupled with a photodiode are from this doublet, and D o 0 implies the MS = 3/2
array detector. Data were collected under single turnover Kramers doublet lie lowest and all observed EPR transitions
conditions using a solution of 70 mM of LRA-8 in one syringe are from this doublet.
and a solution of nitrocefin (50 mM) in the other. Absorbance
changes were monitored with the photodiode array detector
(for 5 seconds over the wavelength range of 300800 nm) as Statement of contributions
described elsewhere.31,4245,49
Data from at least five reproducible experiments were MP, CS and CE were responsible for most of the data collection
collected, averaged and corrected for the instrument dead time and the initial draft of the paper; JH, WC and DT contributed to
(1.5 ms). The kinetic mechanism for the reaction was then EPR data collection and analysis; NM, WH and JL contributed
analysed using the program KINSIM and the model shown to MCD data collection and analysis; PH and GT contributed to

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target selection and, together with NM, DT and JL, they also 10 L. P. Kotra and S. Mobashery, b-Lactam antibiotics,
contributed to the refinement of the draft manuscript; GS was b-lactamases and bacterial resistance, Bull. Inst. Pasteur,
responsible for the overall project, the outline and refinement 1998, 96, 139150.
of the manuscript and the allocation of individual tasks of the 11 S. Ghafourian and N. Sadeghifard, Extended spectrum beta-
project. lactamases: definition, classification and epidemiology,
Curr. Issues Mol. Biol., 2015, 17, 1121.
12 B. G. Hall and M. Barlow, Evolution of the serine
b-lactamases: past, present and future, Drug Resist. Updates,
Acknowledgements
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2004, 7, 111123.
This research was supported by Project Grants from the NH&MRC 13 I. E. Crompton, B. K. Cuthbert, G. Lowe and S. G. Waley,
(APP1084778) and Australian Research Council (DP150104358) b-Lactamase inhibitors. The inhibition of serine b-lactamases
and a Future Fellowships (FT120100694) awarded to GS. CS by specific boronic acids, Biochem. J., 1988, 251, 453459.
acknowledges financial support in form of an APA Scholarship 14 R. P. McGeary, G. Schenk and L. W. Guddat, The applica-
from the University of Queensland and NM is grateful to the tions of binuclear metallohydrolases in medicine: recent
Science Foundation Ireland for funding in form of a President of advances in the design and development of novel drug leads
Ireland young Researcher Award (SFI-PIYRA). JAL and WH thank for purple acid phosphatases, metallo-b-lactamases and
the National Science Foundation (USA) for financial support from arginases, Eur. J. Med. Chem., 2014, 76, 132144.
grants CHE1303852 and CHE0820965 (MCD instrument). The 15 M. Shahid, F. Sobia, A. Singh, A. Malik, H. M. Khan, D. Jonas
authors are grateful to the reviewers for their constructive support and P. M. Hawkey, b-Lactams and b-lactamase-inhibitors
in the preparation of this manuscript. in current- or potential-clinical practice: a comprehensive
update, Crit. Rev. Microbiol., 2009, 35, 81108.
16 M. W. Crowder, J. Spencer and A. J. Vila, Metallo-b-lactamases:
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