An OPAA enzyme mutant with increased catalytic efficiency on the nerve agents sarin, soman, and GP

Article abstract of DOI:10.1016/j.enzmictec.2017.11.001

The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215 sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L, showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT, which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer. Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography coupled with atmospheric pressure chemical ionization–mass spectrometry.

Full text of DOI:10.1016/j.enzmictec.2017.11.001

EnzymeandMicrobialTechnologyxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Enzyme and Microbial Technology
journal homepage: www.elsevier.com/locate/enzmictec
An OPAA enzyme mutant with increased catalytic efficiency on the nerve
agents sarin, soman, and GP
Sue Y. Baea, James M. Myslinskib, Leslie R. McMahona, Jude J. Heighta, Andrew N. Bigleyc,
Frank M. Raushelc, Steven P. Harveya,⁎
a
U.S. Army Edgewood Chemical Biological Center, 5183 Blackhawk Rd., RDCB-DRC-C, Aberdeen Proving Ground, MD, 21010-5424, USA
Excet Inc., 5183 Blackhawk Rd., RDCB-DRB-M, E3400, Aberdeen Proving Ground, MD, 21010-5424, USA
Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, TX, 77842, USA
b
c
A R T I C L E I N F O
A B S T R A C T
Keywords:
OPAA
Mutagenesis
Sarin
Soman
GP
The wild-type OPAA enzyme has relatively high levels of catalytic activity against several organophosphate G-
type nerve agents. A series of mutants containing replacement amino acids at the OPAA Y212, V342, and I215
sites showed several fold enhanced catalytic efficiency on sarin, soman, and GP. One mutant, Y212F/V342L,
showed enhanced stereospecificity on sarin and that enzyme along with a phosphotriesterase mutant, GWT,
which had the opposite stereospecificity, were used to generate enriched preparations of each sarin enantiomer.
Inhibition of acetylcholinesterase by the respective enantioenriched sarin solutions subsequently provided
identification of the sarin enantiomers as separated by normal phase enantioselective liquid chromatography
coupled with atmospheric pressure chemical ionization–mass spectrometry.
LC-APCI-MS
1. Introduction
against only a few lethal doses, treatments can easily be overwhelmed
by agent exposure [8,9]. This concern has led to the effort to develop
Sarin, soman (GD and, GP (Fig. 1) are all G-type chemical nerve
agents; members of a class of organophosphates that exert their toxic
effect by phosphorylating the catalytically active serine in the active
site of acetylcholinesterase and preventing it from catalyzing the
breakdown of acetylcholine in the neuromuscular junction. These, and
other compounds are included in the Chemical Weapons Convention
which prohibits their development, production, stockpiling, and use in
the 192 (as of 17 Oct, 2015) signatory nations [1]. However, not all
countries have signed the Convention and reports of the use of chemical
weapons have continued to appear [2–4]. Two websites provide reg-
ularly updated lists of reports of chemical weapons use, many of which
have been verified by the Organization for the Prohibition of Chemical
Weapons [5,6].
Catalytic enzymes are currently being investigated for their poten-
tial as in vivo medical countermeasures for nerve agent poisoning [7].
Current therapies comprise symptomatic treatment such as atropine (a
muscarinic antagonist) and benzodiazepines (to control seizures), in
addition to etiologic treatment of the poisoned acetylcholinesterase
with 2-PAM (an oxime reactivator). Given the high toxicity of nerve
agents and the fact that existing treatments typically confer protection
catalytic antidotes which might confer greater protection than would be
possible with stoichiometric approaches. The particular advantage of
circulating enzymes is that they can detoxify organophosphates in the
blood before they enter the tissue where they bind to acet-
ylcholinesterase [10]. For that reason, they have the potential to
function either alone or synergistically with existing treatments [7].
Organophosphorus Acid Anhydrolase (OPAA; EC 3.1.8.2) is one of
the enzymes described for the catalytic defluorination of G-agents. The
wild-type (WT) OPAA enzyme has relatively high levels of activity
against soman and cyclosarin [11,12]. WT OPAA has slight activity
against Russian VX; some mutants have greater activity and, in the case
of one mutant, enhanced stereospecificity such that both enantiomers of
Russian VX are catalyzed at similar rates [13]. WT OPAA activity
against sarin is significantly less than seen with soman and cyclosarin
[15]. In this study we initially used site-directed mutagenesis in an
effort to increase the catalytic efficiency of OPAA against sarin. One of
those mutants, FL, proved particularly promising. Therefore, we studied
its kinetics against soman and a more recently described nerve agent,
GP [16]. Finally, enzymatic reactions were used to generate en-
antioenriched preparations of sarin which were used along with
⁎
Corresponding author.
E-mail addresses: sue.y.bae.civ@mail.mil (S.Y. Bae), sjames.m.myslinski.ctr@mail.mil (J.M. Myslinski), leslie.r.mcmahon.civ@mail.mil (L.R. McMahon),
jude.j.height.civ@mail.mil (J.J. Height), a_bigley@tamu.edu (A.N. Bigley), raushel@chem.tamu.edu (F.M. Raushel),
steven.p.harvey6.civ@mail.mil, sgibaltimore@comcast.net (S.P. Harvey).
https://doi.org/10.1016/j.enzmictec.2017.11.001
Received 22 March 2017; Received in revised form 27 October 2017; Accepted 7 November 2017
0141-0229/PublishedbyElsevierInc.
Pleasecitethisarticleas:Bae,S.Y.,EnzymeandMicrobialTechnology(2017),https://doi.org/10.1016/j.enzmictec.2017.11.001

S.Y. Bae et al.
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Table 1
Enzyme genotypes.
Source
Genotype
Abbreviation
OPAA
Y212F/V342L
FL
OPAA
Y212F/V342I
FI
OPAA
Y212F/V342Y
FY
Phosphotriesterase
H254G/H259W/L303T
GWT
Fig. 1. Structures of sarin/GB (propan-2-yl methylphosphonofluoridate), soman/GD (3-
methylbutan-2-yl methylphosphonofluoridate), and GP (2, 2-dimethylcyclopentyl me-
thylphosphonofluoridate).
harvested and the enzyme was purified by ammonium sulfate fractio-
nation and the 45–65% pellet obtained was redissolved, passed through
a size exclusion column and the active fractions were pooled and loaded
on a Q Sepharose column followed by a 0.2–0.6 M NaCl gradient elu-
tion.
acetylcholinesterase inhibition assays to assign the respective sarin
enantiomers separated and identified by chiral liquid chromatography
coupled with atmospheric pressure chemical ionization–mass spectro-
metry (LC-APCI-MS).
The resulting sample was apparently homogeneous by poly-
acrylamide gel electrophoresis.
2. Material and methods
The wild-type opaa amino acid sequence is as follows:
Sarin and soman were chemical agent standard analytical reference
material from our stocks. Sarin was 97.4% weight percent pure by acid
base titration (traceable to the National Institute of Standards and
Technology, or NIST through potassium phthalate). Purity by gas
chromatography/thermal conductivity detection (GC/TCD) was 97.9%.
Purity by 31P nuclear magnetic resonance (NMR) was 97.7 wt%. Soman
was 95.3 wt% pure by 31P NMR relative to a triethylphosphate internal
standard and traceable to NIST through the internal standard with
purity of 99.87 wt% relative to a NIST traceable standard of di-
methylsulfone via quantitative 1H NMR measurements. The purity of
the soman by GC/FPD was 98.95%.
GP was synthesized in a two-step procedure from 2,2-dimethylcy-
clopentanone. First, a reduction of the ketone by LiAlH4 in Et2O yielded
racemic 2,2-dimethylcyclopentanol after acid workup. Then reaction of
2,2-dimethylcyclopentanol with methylphosphonyl difluoride in the
presence of trimethylamine in Et2O yielded the desired 2,2-di-
methylcyclopentyl methylphosphonofluoridate after filtration to re-
move triethylammonium hydrofluoride, removal of the solvent, and
distillation under reduced pressure.
The opaa gene was cloned into the NcoI and the EcoRI sites of the
pSE420 expression vector. The cloned gene lacks the last 77 carboxyl-
terminus amino acid residues of the OPAA enzyme; these residues were
removed in a previous investigation and found to have no discernible
effect on enzymatic activity [15]. The OPAA enzyme with the FL mu-
tations was constructed by DNA 2.0 (Menlo Park, CA) by site-directed
mutagenesis [17]. The mutants have the Y212F mutation in combina-
tion with L, I, or Y substitutions at the V342 site (Table 1). The GWT
mutant of the PTE gene was made by directed mutagenesis performed
using the Quick Change (Agilent Technologies) protocol [18].
Enzyme activity was determined with a fluoride electrode con-
nected to an Accumet XL250 ion selective meter (Thermo Fisher
Scientific, Inc.) calibrated against authentic standards. Assays were
conducted in 2.0 mL of 50 mM bis-tris-propane buffer, pH 8.0, con-
taining 0.1 mM MnCl2 which was added just prior to the assay. Data
were logged every 30 s. Sarin was used at a concentration of only
0.5 mM for stereochemistry assays and for enantioenriched prepara-
tions since the rate of racemization is at least partially a function of the
concentration of fluoride in the sample [19].
Note: GP is extremely toxic and its synthesis is regulated by the
Chemical Weapons Convention.
3. Theory/calculation
All reagents and solvents were HPLC grade. Hexane and isopropyl
alcohol were purchased from Fisher Scientific (Waltham, MA). For the
LC–MS analytical analysis, the MS system was operated in total ion
chromatogram (TIC) mode at m/z 50–300. The analytical separations of
the enantiomers were characterized using an Agilent 1200 LC with
atmospheric pressure chemical ionization–mass spectrometry (LC-
Kinetic parameters were calculated using Biosoft EnzFitter© soft-
ware (Biosoft.com). Activity data were generally collected at substrate
concentrations ranging from 1/3 to three times the Km under conditions
that consumed less than 10% of the substrate. At least six, more typi-
cally ten, different substrate concentrations were used for each curve.
The percent uncertainties of the Vmax and Km values determined from
the software were added together to determine the percent un-
certainties of the kcat/Km values.
APCI-MS) performed on
a Phenomenex Lux Cellulose-1 column,
250 × 4.6 mm, 5 μm with a mobile phase consisting of n-hexane (A)
and isopropyl alcohol (B) and a sample volume of 20 μL. The en-
antiomers were baseline resolved within 15 min with a mobile phase of
95/5 A/B (v/v%) with a flow rate 0.6 mL/min. Samples for analytical
separation were prepared at 0.1 mg/mL.
Acetylcholinesterase inhibition was determined on a plate reader
(BioTek Synergy4) using a modified Ellman Assay, as described in
Results
The OPAA enzyme was prepared as described previously [12].
Briefly, the Escherichia coli host cell containing the cloned OPAA gene
was grown to late log phase in 1 L of Luria Broth in a flask. Cells were
The rationale for selecting the respective OPAA sites for mutation
was described previously [13]. Briefly, the OPAA enzyme active site
comprises a small pocket, a large pocket and a leaving group pocket.
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Table 2
OPAA genotypes and kinetic parameters on sarin.
Enzyme
Genotype
kcat min−1
Km (μM)
kcat/Km (min−1 M−1
)
kcat/Km Mutant/WT
WT
FL
FY
FI
Wild-type
1.20E + 04
4.52E + 04
1.10E + 04
1.63E + 04
4.31E + 02
4.62E + 03
1.80E + 03
1.99E + 03
9.02E + 03
5.31E + 03
5.62E + 03
6.00E + 03
6.32E + 02
1.01E + 03
1.57E + 03
1425.09
1.33E + 06
8.50E + 06
1.96E + 06
2.72E + 06
1.41E + 05
2.49E + 06
8.65E + 05
9.80E + 05
Y212F/V342L
Y212F/V342Y
Y212F/V342I
6
1
2
Structural analysis revealed Y212 to be the largest amino acid in the
small pocket and our initial mutagenesis efforts involved the replace-
ment of all amino acids except isoleucine at the Y212 site. Screening
showed most of these substitutions to be deleterious but Y212F and
Y212V mutants showed enhanced activity on several nerve agents.
Subsequent mutations at other sites in the Y212V background were not
productive so were not pursued further. Mutations at the small pocket-
forming V342 site in the Y212F background, particularly V342L,
showed further activity increases on several nerve agent substrates.
The rationale for the construction of the phosphotriesterase (PTE)
GWT mutant was described previously as well [15]. Briefly, a series of
mutants were screened on p-nitrophenyl derivatives of G-type nerve
agents and promising mutants were subsequently tested in several
nerve agents. GWT was also shown to have a reversed stereospecificity
on cyclosarin compared to the wild type enzyme.
stereoisomers [19]. Separations or toxicities of the GP isomers have not
been reported to our knowledge. Here we sought to separate and
identify, by differential enzymatic activity, the respective sarin en-
antiomers by LC-APCI-MS. Fig. 4a shows the total ion chromatogram
(TIC) of the sarin enantiomers eluting at 11.3 min and 12.9 min and
Fig. 4b shows extracted ion chromatogram (EIC) of the sarin en-
antiomers. The mass spectrum of the enantiomer at 11.3 min was
identical to that at 12.9, minute as expected (Fig. 4c). The mass spec-
trum of the enantiomer at 11.3 min was identical to that at 12.9, minute
as expected.
With substrates such as sarin, a molecule comprising two en-
antiomers, the rates of enzymatic degradation can reveal that the pro-
cess is stereospecific. A single exponential curve would indicate that
both enantiomers are degraded at the same rate; whereas a clear mid-
point deflection in the curve would indicate that an enzyme has a
preference for one enantiomer over the other. Fig. 5 shows such a de-
flection when sarin is degraded by FL, but no apparent stereospecificity
was observed with WT.
4. Results
In order to identify the respective sarin enantiomer peaks, the
phosphotriesterase GWT enzyme (1.6 μg/mL) was added to a solution
(15 mL) of 0.5 mM racemic sarin in 50 mM bis-tris-propane, pH 7.2.
The release of fluoride was monitored by fluoride ion selective elec-
trode and when approximately 80% of the substrate had reacted the
entire contents of the reaction was added to 4 mL of cold ethyl acetate,
shaken gently and the organic fraction was subjected to LC-APCI-MS
analysis (Fig. 6). Of the two peaks, at 11.6 and 13.3 min, the area of the
former was approximately five times that of the latter in the enriched
extract, indicating a distinct preference of the GWT enzyme for the
enantiomer corresponding to the 13.3 min peak. The total concentra-
tion of sarin in the sample was determined by standard curve to be
104.1 μg/mL. A sample of racemic sarin was prepared to the same
concentration (104.1 μg/mL), and these two samples, one racemic and
one enriched for the 11.6 min peak, were used to inhibit acet-
ylcholinesterase.
A series of OPAA mutants was screened by assaying the specific
activity of their crude lysates on 3 mM sarin. Although crude lysate
assays do not account for potential differences in enzyme expression
levels, they are frequently indicative of catalytic efficiency and as such
they can provide a useful screening tool with which to select mutants
for subsequent purification and kinetic analysis. Promising mutants FL,
FY, and FI from the screening were purified and full kinetic parameters
were determined (Table 2 and Fig. 2).
The kinetic data with purified enzymes showed that the catalytic
efficiency of FY actually represented only a marginal improvement over
WT, and FI was about twice WT. FL however, had a kcat/Km value on
sarin approximately six times that of the WT enzyme.
Following the encouraging results seen with FL on sarin, kinetic
parameters for the degradation of soman and GP by FL and WT were
determined. The catalytic efficiencies using these two substrates were
approximately five times and ten times respectively that of WT (Table 3
and Fig. 3).
Typical of bioactive compounds, G-agents are chiral molecules and
the enzymes that catalyze their hydrolysis often show significant ste-
reospecificity. Sarin, soman and GP all have a stereogenic phosphorus
atom and soman and GP have an additional stereogenic carbon atom.
Benschop et al. in 1984 published the toxicity values for the four soman
EC50s: ( )-sarin: 4.16
GWT Enriched sarin: 48.1
0.22 nM
9.7 nM
Solutions of racemic sarin and enantioenriched sarin (GWT) were
prepared in phosphate buffer (0.100 M PO4−, pH = 7.5) in a 96-well
plate at varying concentrations and human acetylcholinesterase
(hAChE) (20 μL) was added. The final concentrations of sarin in the
Fig. 2. Comparison of kcat/Km values for WT and mutant enzymes on
sarin (second version of this graph is added in response to reviewer’s
comments – I don’t think we should include it, though).
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Table 3
Kinetic parameters of WT and FL enzymes on soman and GP.
Enzyme and Substrate
kcat min−1
Km (μM)
kcat/Km (min−1 M−1
)
kcat/Km FL/WT
WT on soman
FL on soman
WT on GP
2.13E + 05
6.60E + 04
7.91E + 04
6.90E + 04
1.71E + 04
3.40E + 03
7.44E + 03
3.08E + 03
1.32E + 04
883.16
6.12E + 03
534.924
1.62E + 03
156.395
1.21E + 03
6.78E + 01
1.61E + 07
7.48E + 07
1.29E + 07
1.29E + 08
3.26E + 06
1.71E + 07
3.78E + 06
2.21E + 07
5
FL on GP
10
Fig. 3. Comparison of kcat/Km values for WT and FL enzymes on
soman, sarin and GP.
Fig. 4. A representative (a) TIC, (b) EIC, and (c) MS for racemic sarin.
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nitrobenzoic acid) (DTNB) (1.6 mM; final concentration 1.3 mM) was
added to each well. The rate of hydrolysis of acetylthiocholine to
thiocholine and acetate was measured by monitoring the change in
absorbance of the solution at 412 nm using a plate reader (Synergy4,
BioTek). The path length was previously determined to be 0.479 mm,
and the extinction coefficient for TNB2−, ε412 = 14150 M−1 cm−1
[20].
The effective concentration at which 50% of the acet-
ylcholinesterase enzyme was inhibited (EC50) was determined for the
racemic and GWT enriched samples. EC50s were determined by fitting
the activity data to a sigmoidal dose-response curve model with vari-
able slope using GraphPad Prism version 6.07 for Windows (Fig. 7).
Boter and Van Dijk reported in 1969 that (S)-(−)-sarin was a much
stronger inhibitor of AChE than (R)-(+)-sarin [21]. Against bovine
erythrocyte AChE, the ratio of reactivities was ≥4.2 × 103 whereas the
ratio for electric eel AChE inhibition was 3.6 × 104. Obviously, in both
cases, essentially all the toxicity from sarin derives from the (S)-(−)
enantiomer. The mouse intravenous LD50 values for (S)-(−)-sarin
(41 μg/kg) and racemic sarin (142 μg/kg) follow this same trend
[21,22]. We therefore reasoned that if the GWT enzyme preferentially
degraded the (R)-(+) enantiomer, the GWT-enriched sample should
inhibit AChE somewhat more strongly than racemic sarin, whereas if
GWT preferred the (S)-(−) enantiomer, the inhibition should be cor-
respondingly reduced. The acetylcholinesterase inhibition data show an
approximately 11-fold reduction in inhibition, consistent with the latter
case, and indicating that the 11.6 min peak is (R)-(+) sarin and the
13.3 min peak (which was reduced in the GWT-enriched preparation) is
Fig. 5. Enzymatic and spontaneous hydrolysis of 500 μM sarin. Enzyme activities differ,
therefore concentrations for each enzyme were chosen to provide similar initial hydro-
lysis rates.
solutions were 892, 357, 143, 57.1, 22.8, 9.13, 3.65, and 1.46 nM. This
solution was incubated at 37 °C for 15 min to allow for complete in-
hibition, whereupon a solution (210 μL) containing acetylthiocholine
iodide (1.2 mM; final concentration 1 mM) and 5,5-dithio-bis-(2-
Fig. 6. A representative (a) TIC, (b) EIC (m/z 99.0100), and (c) MS at 11.5 min and 13.2 min for phosphotriesterase GWT-degraded sarin.
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enzyme. However, FL had an approximately six-fold increased kcat/Km
value compared to WT. Specifically, the Km value of FL was about half
that of WT and the kcat value was about three times that of the WT
enzyme. FY and FI had kcat and Km values that were all comparable to
WT. Clearly, FL was the most promising mutant obtained with respect
to sarin activity, and it showed significant increases in soman and GP
activity as well.
Interestingly, the increased catalytic efficiencies seen on soman and
GP were both caused by a decrease in the Km with the FL mutant, while
the kcat of FL remained essentially unchanged from the WT. Soman and
GP are related in that they both possess a stereogenic center adjacent to
the P-O bond in addition to the chiral phosphorus atom, giving a total of
four stereoisomers.
The stereospecificity of the OPAA FL and PTE GWT mutants proved
useful to us for making enriched preparations of each enantiomer which
permitted assignment of the respective peaks in the LC chromatogram
based on the differential inhibition of acetylcholinesterase, which was
correlated with the published toxicity values for the two enantiomers of
sarin. Similar mutants might also aid the assignment of unidentified
peaks in the chiral separation of soman and/or GP, although the si-
tuation is obviously much more complex given the increased stereo-
chemical complexity of these molecules.
Fig. 7. Acetylcholinesterase inhibition with ( )-sarin and GWT enriched sarin.
(S)-(−)-sarin. These results were corroborated by an OPAA-FL cata-
lyzed sample enriched for the 13.3 min peak which showed little
change in acetylcholinesterase inhibition, as would be expected for a
sample where primarily the (R)-(+) sarin was hydrolyzed. We can also
conclude on this basis that OPAA-FL exhibits a preference for the (R)-
(+) enantiomer while PTE GWT prefers (S)-(−)-sarin (Fig. 8).
5. Discussion
6. Conclusions
Regarding the kinetic determinations on sarin, the mutants FY and
FI revealed little actual change in catalytic efficiency from the WT
Beyond the laboratory, the practical significance of stereospecificity
Fig. 8. A representative (a) TIC, (b) EIC, and (c) MS for OPAA FL-degraded sarin.
6

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depends on the purpose for which the enzymes might be used. If the
enzymes are intended for use as a medical countermeasure where they
must act in blood with agent concentrations likely in the micromolar
range or below, stereospecificity is a critical consideration since an
enzyme with strong stereospecificity might degrade one enantiomer
while leaving the other present at toxic concentrations, thereby con-
ferring little protection. However, the situation is considerably different
if the enzymes are intended for use as a surface decontaminant. Owing
to Le Chatlier’s Principle, decontamination could potentially be per-
formed using enzymes preferring either enantiomer, as G-agents are
known to racemize rapidly in the presence of concentrations of F−
above the low mM range [23]. If the enzymes are to be used as part of a
decontamination solution, small amounts of fluoride can easily be
added to the solution to ensure racemization even at very low agent
concentrations. On that basis, it might be concluded that the enhanced
catalytic efficiency observed with the FL mutant on sarin could be more
beneficial for decontamination than for in vivo treatment, since it comes
with an increased preference for the less toxic (R)-(+) sarin en-
antiomer.
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Acknowledgement
We gratefully acknowledge the Defense Threat Reduction Agency
for their support of this research (CB3742 for SH; CB3998 for JH and
JM, CB3662 for SB and LM, HDTRA1-14-1-0004 for FR and AB).
Appendix A. Supplementary data
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Supplementary data associated with this article can be found, in the
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