Improving a natural enzyme activity through incorporation of unnatural amino acids

Article abstract of DOI:10.1021/ja106416g

The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved t

Full text of DOI:10.1021/ja106416g

Published on Web 12/16/2010
Improving a Natural Enzyme Activity through Incorporation of
Unnatural Amino Acids
Isaac N. Ugwumba,†,‡ Kiyoshi Ozawa,‡,§ Zhi-Qiang Xu,†,§ Fernanda Ely,|
Jee-Loon Foo,‡ Anthony J. Herlt,‡ Chris Coppin,† Sue Brown,† Matthew C. Taylor,†
David L. Ollis,‡ Lewis N. Mander,‡ Gerhard Schenk,|,⊥ Nicholas E. Dixon,§
Gottfried Otting,‡ John G. Oakeshott,† and Colin J. Jackson*,†,‡
Commonwealth Scientific and Industrial Research Organization, Black Mountain, Canberra, Australia,
Research School of Chemistry, Australian National UniVersity, Canberra, Australia, School of Chemistry,
UniVersity of Wollongong, Australia, School of Chemistry and Molecular Biosciences, UniVersity of
Queensland, Australia, Department of Chemistry, National UniVersity of Ireland - Maynooth, Ireland
Received August 1, 2010; E-mail: cjackson@rsc.anu.edu.au
Abstract: The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast
turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the
naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and
to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate
binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with
unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino
acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl
group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product
release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged
4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient
catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to
the difficulty in improving this native activity through screening hundreds of thousands of mutants with
natural amino acids. These results demonstrate that designer amino acids provide easy access to new
and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.
of natural amino acids.7 An alternative, site-specific, approach
Introduction
based on suppressor tRNA allows incorporation of uAAs during
The development of experimental techniques that allow
incorporation of unnatural amino acids (uAAs) into proteins has
opened new avenues in protein engineering and characterization
of protein function, permitting the synthesis of a wide range of
unnatural enzymes with new or improved activities and novel
mechanistic probes.1-4 Methods for translational incorporation
of uAAs can be broadly separated into global and specific
approaches. Global approaches exploit either natural misacy-
lation of tRNA by close structural analogues based on the
substrate promiscuity of existing tRNA synthetases,5 or utilize
chemical misacylation,6 to allow incorporation of uAAs in place
suppressor read-through of a stop codon, most often amber
(UAG).8,9 This approach can be used in both in vitro protein
translation systems and in in vivo translation systems that have
been modified by the addition of an orthogonal tRNA-amino-
acyl tRNA synthetase pair.10,11
This work describes the incorporation of uAAs into a bacterial
phosphotriesterase (arPTE) that has been cloned from an
Agrobacterium radiobacter strain and catalyzes the hydrolysis
of toxic organophosphate (OP) phosphotriester pesticides such
as paraoxon.12 A 90% identical enzyme (pdPTE) has been
isolated from Pseudomonas diminuta and FlaVobacterium sp.
strains and also exhibits OP degrading activity.13-15 These
* To whom correspondence should be addressed.
† Commonwealth Scientific and Industrial Research Organization.
‡ Australian National University.
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§ University of Wollongong.
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J. Am. Chem. Soc. 1989, 111, 8013–8014.
| University of Queensland.
⊥ National University of Ireland - Maynooth.
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326 J. AM. CHEM. SOC. 2011, 133, 326–333
10.1021/ja106416g  2011 American Chemical Society

An Unnatural Phosphotriesterase
A R T I C L E S
Scheme 1. Hydrolysis of Paraoxon at Basic pH Catalyzed by PTE
Scheme 2. Catalytic Cycle of PTEs
Figure 1. Aromatic interactions between Tyr309, diethyl-methoxyphe-
nylphosphate, and Phe132 in the crystal structure of arPTE (PDB ID: 2R1N).
Amino acid side chains forming face-face and edge-face aromatic stacking
interactions with the aromatic group of the substrate are shown in magenta.
The Fe(II) (orange), Co(II) (pink), and water/hydroxyl (red) molecules in
the active site are shown as spheres.
proteins are of interest because of their rapid evolution into
efficient catalysts of the hydrolysis of synthetic OPs and their
potential use for OP detoxification because these compounds
pose a serious health and environmental risk.16
substrate binding in the PTEs is driven in part by metal-substrate
interactions,20 an important role for aromatic and hydrophobic
enzyme-substrate (ES) interactions is implied by crystal
structures of Michaelis and product complexes, which show the
aromatic ring of the leaving group sandwiched between the
aromatic groups of Tyr309, Trp131, and Phe132 (Figure 1).22,27
Moreover, mutation of these residues to alanine results in an
increase in the Michaelis constant (KM),29,30 suggesting that these
residues are involved in the formation of the Michaelis complex.
However, theoretical studies have either indicated that the
substrate approaches for nucleophilic attack without significant
aromatic interactions with these residues,31,32 or have omitted
these residues altogether.33
There have been several reports of protein engineering
experiments to improve the catalytic function of the PTEs,
involving rational design,29,30 combinatorial libraries,34,35 and
laboratory (directed) evolution.18,27,36-38 Most of these studies
have focused on improving inefficient, promiscuous, activities
of the PTEs, such as the turnover of OPs used as chemical
warfare agents or other slowly turned over insecticides. Attempts
to improve already efficient activities, such as the turnover of
paraoxon, have produced no, or only modest, improvements in
arPTE in our laboratories, despite screening hundreds of
thousands of variants.27,37 The inability to significantly improve
the arPTE-mediated turnover of paraoxon by targeted and
random mutagenesis suggests that there is likely to be little easily
accessible sequence space available for improvement of this
activity using naturally occurring AAs.
Many studies of the structure and function of the bacterial
PTEs have been described, with crystal structures of arPTE and
pdPTE revealing very close structural similarity (backbone rmsd
of 0.34 Å) and nearly identical active sites with a binuclear
metal ion center.17,18 Each metal ion plays a specific role in the
reaction (Scheme 1), with the more solvent exposed ꢀ-metal
ion apparently involved in substrate coordination and the
R-metal ion involved in stabilizing a hydroxide ion as the
initiating nucleophile.19-23 Paraoxon is known to be hydrolyzed
via an SN2-type reaction with net inversion of configuration at
the phosphorus center,24 and the rate of product release (k5)
has been identified as the slowest and rate-limiting step of the
catalytic cycle (Scheme 2).20,25-27
Discussion of the catalytic power of enzymes often focuses
on the chemical step,28 but the subtle interactions that facilitate
substrate binding and product release are also important; without
fast rates for formation of the Michaelis complex and product
release, the efficiency of the chemical step can be obscured.
Because the chemical step is not rate-limiting for PTE-catalyzed
paraoxon hydrolysis, the PTEs are a valuable model system to
study these physical steps in the catalytic cycle. Although
(15) Serdar, C. M.; Gibson, D. T.; Munnecke, D. M.; Lancaster, J. H. Appl.
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Protein Eng. 2003, 16, 241–241.
In this study, we describe the incorporation of unnatural
7-methyl- and 7-hydroxycoumarinyl amino acids (Mco and Hco;
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L. R.; Ollis, D. L. Biochem. J. 2010, 429, 313–321.
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J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 327

A R T I C L E S
Ugwumba et al.
The ꢀ-ketoester (5.3 g, 12 mmol) was added slowly to a solution
of resorcinol (6.6 g, 60 mmol) in methanesulfonic acid (48 mL) at
room temperature. After stirring for 1 h, 310 mL of diethyl ether
was added and the mixture cooled to -35 °C. The mixture was
stirred for 90 min to give a yellow precipitate, which was filtered
off. The residue was dissolved in 100 mL of water and lyophilized
overnight. It was then dissolved in 100 mL of water at pH 1.3 and
1 M NaOH solution added until the pH of the mixture was 3.5
when a pink solid separates. The mixture was cooled in ice for
1 h, filtered and the residue washed with three portions of ice-water
at pH 3 followed by three portions of acetone to give 1.9 g of crude
L-(7-hydroxycoumarin-4-yl)ethylglycine as the zwitterions, HPLC
purity approximately 90%. The amino acid was further purified by
dissolving in 50 mL of water adjusted to pH 1 with methanesulfonic
acid. Aliquots (4.2 mL) were injected onto a Waters Symmetry (7
µm, C18 150 mm ×19 mm) semipreparative HPLC column and
eluted at 8.5 mL/min with 1:9 acetonitrile-water containing 0.1%
trifluoroacetic acid for 15 min followed by a 10 min wash with
100% acetonitrile. Fractions containing the trifluoroacetate salt of
the amino acid were pooled and rotary evaporated (12 mbar, bath
40 °C). The residue was dissolved in 15 mL 1 M sodium hydroxide
at 0 °C and 36% HCl added to adjust the pH to 3.5. The white
solid was filtered off, washed three times with ice-water at pH
3.5, and dried under high vacuum for 60 h at room temperature.
The yield of the target compound as the zwitterion was 1.2 g (38%),
HPLC purity 99%. HPLC analysis was performed with a Waters
Symmetry (5 µm C18 150 mm ×4.6 mm) HPLC column, eluted
at 1 mL/min with a 20 min linear gradient from 10 to 100%
acetonitrile in water containing 0.1% trifluoracetic acid, and
monitoring at 323 nm. At 22 °C, the compound eluted at 7.0 min.
Stock solutions (50 mM) of Hco in 100 mM KOH were made and
1 mL aliquots stored at -80 °C until needed.
Preparation of L-(7-methylcoumarin-4-yl)ethylglycine (Mco).
Synthesis of L-(7-methylcoumarin-4-yl)ethylglycine hydrochloride
(Mco) was performed as follows. The ꢀ-ketoester derived from
Z-Glu-OBzl (3.25 g, 7.4 mmol), prepared as previously described40
was added in portions to a 4 mL stirred solution of 37 mmol
m-cresol in 30 mL of methanesulfonic acid at room temperature.
After 3.5 h, the deep yellow reaction mixture was poured into 90
mL of ice water and extracted twice with 100 mL of diethyl ether.
The pH was adjusted to 4.6 with sodium hydroxide (18.5 g in 30
mL water) followed by 1 M sodium hydroxide. A small amount of
a pink oily solid was filtered off and the volume of the filtrate was
reduced to approximately 120 mL using a rotary evaporator
(condenser -10 °C, bath 40 °C) and the product was allowed to
stand for 3 days. The white solid that separated was filtered off,
washed with ice water, and dried in air. This material (325 mg)
was slurried with water and 1 M HCl added until it fully dissolved
(total volume approximately 15 mL). The acidified solution was
extracted with two half volumes of distilled dichloromethane. The
aqueous phase was then rotary evaporated (condenser -10 °C, bath
30 °C) to remove traces of organic solvent and lyophilized to give
353 mg of 4-(7-methylcoumarinyl)-2-ethylglycine hydrochloride as
a cream colored solid in 16% yield. Stock solutions (50 mM) of
Mco in 100 mM KOH were made and 1 mL aliquots stored at
-80 °C until needed.
Figure 2. 7-Hydroxycoumarinyl (Hco), 7-methylcoumarinyl (Mco), and
tyrosine (Tyr) amino acids incorporated at position 309.
Figure 2) in place of tyrosine 309 in the substrate binding site
of arPTE through the use of coupled in vitro transcription/
translation of arPTE. These unnatural arPTE variants were
constructed to probe the role of aromatic enzyme-substrate
contacts in the catalytic cycle and to test whether uAAs allow
improvement of native catalytic efficiencies that have evolved
with the 20 natural amino acids.
Materials and Methods
Materials, Reagents and Bacterial Strains. Paraoxon was
purchased from Sigma Aldrich (USA). Molecular biology reagents
and enzymes were purchased from Invitrogen (Australia), Finnzymes
(Finland) or New England Biolabs (USA). Oligonucleotides for
cloning of genes into expression vectors (Table 1 of the Supporting
Information) were obtained from Geneworks (Australia). QIAGEN
DNA purification kits were used for all DNA purifications unless
stated otherwise. Calcium chloride competent E. coli cells from
DH10ꢀ and BL21(DE3) strains, Luria broth (LB), LB agar, and
antibiotics (ampicillin, chloramphenicol) were prepared as described
by Sambrook et al.39 The vectors pSUP-CouRS-D8 and pSUP-
MjTyrRS-6TRN were a kind gift from Peter G. Schultz (The Scripps
Research Institute, USA).
Preparation of L-(7-hydroxycoumarin-4-yl)ethylglycine
(Hco). The compound was prepared using a modified version of
the published method.40 Z-Glu-OBzl was purchased from Bachem
and all other reagents from Aldrich or Ajax Chemicals (Sydney,
Australia). A solution of potassium hydroxide (16.8 g, 0.3 mol) in
20 mL water was added slowly to an ice-cooled solution of ethyl
hydrogen malonate (39.7 g, 0.3 mol) in 20 mL water. Magnesium
chloride hexahydrate (30.5 g, 0.15 mol) was then added and the
volume adjusted to 100 mL with water. 500 mL of isopropanol
was added to precipitate potassium chloride and after 1 h, the
mixture was filtered. The filtrate was rotary evaporated (40 mbar,
bath 40 °C) to give 46.3 g of ethyl magnesium malonate as a white
solid in quantitative yield. Carbonyldiimidazole (2.5 g, 15.1 mmol)
was added under nitrogen in portions to a solution of Z-Glu-OBzl
(5.1 g, 13.7 mmol) in dry tetrahydrofuran (50 mL) at room
temperature. After 75 min, ethyl magnesium malonate (4.3 g, 15.1
mmol) was added and the reaction mixture was stirred for 18 h
during which a slow evolution of carbon dioxide occurred. 100
mL of water was added followed by 5 mL of 36% HCl to adjust
the pH to 3. A white gummy solid separated. The mixture was
extracted with three separate portions of 200 mL diethyl ether and
the combined organic extracts washed with 100 mL each of 10%
aqueous sodium bicarbonate, water, and saturated brine. After
drying over sodium sulfate, filtering, and rotary evaporating, the
white solid (5.8 g) was flash chromatographed with 1:1 ethyl
acetate-hexane on a 220 mm × 50 mm column of 40-63 µm
silica to yield the ꢀ-ketoester as a white solid (5.47 g, 90%).
Mutagenesis and Cloning. A Tyr309amber (TAG) mutant was
produced by overlapping PCR with primers PTE_MF, PTE_MR,
PTE-Y309TAG_F, and PTE-Y309TAG_R (Table 1 of the Sup-
porting Information) using the opdA gene (encoding arPTE) as a
template. Tyr309Leu, Tyr309Phe, and Tyr309Trp mutants were
similarly generated from the opdA gene using the primers PTE_MF,
PTE_MR, PTE-Y309L_F, PTE-Y309L_R, PTE-Y309F_F, PTE-
Y309F_R, PTE-Y309W_F, and PTE-Y309W_R (Table 1 of the
Supporting Information). The PCR products were digested and
ligated into pETMCSI vector41 between the NdeI and EcoRI
restriction sites. The resulting construct was verified by DNA
sequencing.
(39) Sambrook, J., Fritsch, E. F. Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold
Spring Harbor, N.Y., 1989.
(40) Wang, J. Y.; Xie, J. M.; Schultz, P. G. J. Am. Chem. Soc. 2006, 128,
8738–8739.
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An Unnatural Phosphotriesterase
A R T I C L E S
Expression of Wild-Type arPTE and Variants. Wild-type
arPTE (Tyr309) and Tyr309Leu, Tyr309Phe, Tyr309Trp, Tyr309Hco,
and Tyr309Mco variants were expressed in vitro using a cell-free
protein synthesis system with natural and unnatural amino acids,
as has been described previously.42 The only difference was that
the arPTE variants were expressed overnight at 30 °C and no
components for unnatural amino acid incorporation were included
for the expression of wild-type or natural arPTE variants. The
methods for expression and purification of L-(7-hydroxycoumarin-
4-yl) ethylglycine (Hco) tRNA synthetase (CouRS) and expression
and purification of total tRNA including an amber suppressor tRNA
(Sup-tRNA) have been previously described.42 The soluble protein
fraction was loaded on to a DEAE Fractogel column (Merck
Biosciences, Australia), to which arPTE and variants did not bind
and was collected in the flow-through. The protein was then
dialyzed overnight against 20 mM HEPES, pH 7.0, and loaded onto
a 1 mL resource S column (GE Healthcare, UK). Bound arPTE
was eluted at approximately 150 mM NaCl using a linear gradient
from 0 to 1 M. SDS-PAGE analysis of the eluted arPTE protein
indicated a purity of greater than 95%. Protein concentrations were
determined by measuring absorbance at 280 nm using an extinction
coefficient (ε) of 29 280 M-1 cm-1 for wild-type, 29 410 M-1 cm-1
for Tyr309Hco and Tyr309Mco, 28 130 M-1 cm-1 for Tyr309Leu
and Tyr309Phe, and 33 820 M-1 cm-1 for Tyr309Trp. Relative
concentrations were confirmed using SDS-PAGE.
Wild-type arPTE was also expressed in E. coli BL21(DE3) cells
by autoinduction in terrific broth (TB) medium.43 The activity of
this preparation was essentially identical to that of the in vitro
expressed sample (kcat/KM (in vivo) ) 2.56 × 107 s-1 M-1 versus
kcat/KM (in vitro) ) 2.70 × 107 s-1 M-1; Figure 1 of the Supporting
Information). Cells were harvested by centrifugation and resus-
pended in 20 mM Tris-HCl buffer, pH 8.0 before being lysed using
a French pressure cell (Thermo, USA) at 4 °C and cell debris was
removed by centrifugation at 30 000 × g for 30 min. Purification
was performed as described above.44
Confirmation of Insertion of Hco and Mco by In-Gel
Tryptic Digest LC/MS. The incorporation of Hco and Mco into
arPTE was verified by LC/MS using previously described meth-
odology.45 Protein samples were separated by SDS-PAGE and
visualized by staining with Coomassie blue. Protein bands that
migrated at the correct size (∼36 kDa) were excised from the gel,
diced into 1 mm cubes, transferred to a clean 0.5 mL Eppendorf
tube and washed with a mixture of 150 µL 25 mM ammonium
bicarbonate and 10 µL 45 mM dithiothreitol (DTT) on a heating
block set at 60 °C for 30 min. The wash was discarded and gel
pieces subsequently washed three times with 50 µL of 1:1 mixture
of acetonitrile (ACN) and 25 mM ammonium bicarbonate for 20
min each. The gel pieces were further washed three times for 20
min each with 100% ACN and subsequently dried under vacuum
to remove traces of ACN. Rehydration of gel pieces was ac-
complished with 8 µL of protease solution (3 µL of Promega
(Australia) sequencing grade trypsin in 100 µL ammonium bicar-
bonate solution) followed by incubation on a 37 °C heating block
for 4 h. The set up was transferred to a 37 °C water bath and left
to stand overnight. The condensed fluid under the lid of the
microfuge tube was discarded and the gels were rehydrated with
10 µL of 0.1% formic acid and left to stand on the bench for 30
min. The solution containing peptides that diffused from the gel
pieces was pipetted into a 96-well microplate and analyzed by LC-
ESI- Ion Trap Mass Spectroscopy/Mass Spectroscopy (LC-MS/MS)
using an Agilent 1100 capillary liquid chromatography system with
an Agilent XCT ion trap mass spectrometer. Mass spectral data
sets were analyzed by matching with sequence databases using
Agilent’s Spectrum Mill software (Agilent Technologies, USA) to
generate a peptide map. False positive matches were avoided by
using the software’s stringent autovalidation default settings. Mass
search for Hco- and Mco-containing peptides was performed by
matching the calculated +1, +2, +3, and +4 charged peptide
masses generated using the Protein Prospector tool (University of
California, SF, USA) with those identified from the spectral data.
Modeling of the Tyr309Hco Variant. The possible conforma-
tions of the Hco side chain in the Hco309 variant were examined
manually using the program COOT.46 The molecular topology of
Hco was generated using the PRODRG server,47 as were restraints
for bond angles and lengths. The main chain atoms were super-
imposed with those of Tyr309 in the 2R1N22 structure of the
enzyme-substrate complex and the side chain was rotated to
examine possible stable rotamers in which the Hco residue was
not involved in steric clashes with either the substrate or other amino
acid side chains.
Detection of Diethyl Phosphate and 4-Nitrophenol by
Mass Spectrometry. To establish that diethyl phosphate and
4-nitrophenol were both released from the enzyme as a result of
the hydrolysis of paraoxon by Tyr309Hco arPTE (Hco309), a
reaction mixture containing 10 mM paraoxon in 50 mM Tris-HCl,
pH 8.50, 20% methanol, and 0.2 nM Hco309 was allowed to stand
for 5 min. A control sample was similarly set up but with an enzyme
solution that had been inactivated by heating at 70 °C for 30 min
on a heating block. Standard solutions containing 2 mM pure
samples of diethyl phosphate and 4-nitrophenol (Sigma Aldrich,
USA) were also prepared in the same buffer. The samples were
analyzed by negative mode LC/MS ESI ToF using an Agilent 1100
capillary liquid chromatography system with an Agilent MSD ToF
mass spectrometer (Agilent Technologies, USA). The column used
was an Agilent Zorbax Eclipse XDB-C18 (inner dimensions 3.5
µm × 30 mm) rapid resolution column (Agilent Technologies,
USA). Ten microliters of each sample was injected and separation
was achieved using a gradient (0-90%) of acetonitrile with 0.1%
formic acid with a flow rate of 0.8 mL/min for 5 min. Analysis of
mass spectral data to identify diethyl phosphate and 4-nitrophenol
was performed using Analysts QS software (Agilent Technologies,
USA).
Enzyme Kinetics and pH-Rate Profiles. Kinetic measurements
of the catalyzed hydrolysis of paraoxon were performed according
to published procedures.19 The catalytic activity of the variants of
arPTE was assayed by measuring the release of 4-nitrophenol/4-
nitrophenolate spectrophotometrically at 347 nm (ε347 ) 5176
M-1cm-1), the isosbestic wavelength for the substituted phenol and
corresponding phenolate. The pH-dependence of the phosphotri-
esterase activity of arPTE was analyzed using 10-1000 µM
paraoxon as the substrate over pH 4.0-10.0 in 0.5 pH unit
increments. Reactions were conducted in a multicomponent buffer
(MCB) composed of 50 mM each of acetate, MES, HEPES, CHES,
and CAPS, to maintain the same ionic strength in the pH buffering
range.48 Activity measurements were performed in a 96 well
microplate at 30 °C using a BMG Polarstar SPECTRAmax M2
microplate reader (Molecular Devices). A 4× arPTE enzyme
solution was prepared in 2 mM Tris-HCl, pH 8.0, containing 0.5
mg/mL bovine serum albumin (BSA). The final 300 µL reaction
mixture contained 75 µL of 4× arPTE enzyme solution, 75 µL of
4 × MCB solution, and 150 µL of 2× substrate solution. All assays
were conducted in duplicate and in 20% methanol, because of the
(41) Neylon, C.; Brown, S. E.; Kralicek, A. V.; Miles, C. S.; Love, C. A.;
Dixon, N. E. Biochemistry 2000, 39, 11989–11999.
(42) Ugwumba, I. N., Ozawa, K., de la Cruz, L., Xu, Z., Herlt, T., Hadler,
K. S. Coppin, C., Brown, S. E., Schenk, G., Oakeshott, J. G. Otting,
G. Assay Drug DeV. Technol. 2010, epub ahead of print: doi:10.1089/
adt.2010.0306.
(43) Studier, F. W. Protein Expression Purif. 2005, 41, 207–234.
(44) Jackson, C. J.; Carr, P. D.; Kim, H. K.; Liu, J. W.; Herrald, P.; Mitic,
N.; Schenk, G.; Smith, C. A.; Ollis, D. L. Biochem. J. 2006, 397,
501–508.
(45) Campbell, P. M.; Cao, A. T.; Hines, E. R.; East, P. D.; Gordon, K. H. J.
Insect Biochem. Mol. Biol. 2008, 38, 950–958.
(46) Emsley, P.; Lohkamp, B.; Scott, W. G.; Cowtan, K. Acta Crystallogr.
Sect. D: Biol. Crystallogr. , 66, 486–501.
(47) Schuttelkopf, A. W.; van Aalten, D. M. Acta Crystallogr. Sect. D:
Biol. Crystallogr. 2004, 60, 1355–1363.
(48) Ellis, K. J.; Morrison, J. F. Methods Enzymol. 1982, 87, 405–426.
9
J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 329

A R T I C L E S
Ugwumba et al.
limited solubility of the substrate. The kinetic parameters, kcat and
kcat/KM, were obtained by fitting the data to the Michaelis-Menten
equation using the curve fitting software, GraphPad Prism (Graph-
Pad Software Inc., USA). The values of kcat and kcat/KM were
determined by fitting the initial velocity data to the equation
ν ) Vmax[S]/(KM + [S])
(1)
where ν is the initial velocity, Vmax is the maximum velocity, S is
the substrate concentration, and KM is the Michaelis constant. In
the case of an enzyme for which product dissociation (k5) is rate-
limiting, KM is defined by eq 2, where k1, k2, k3, and k5 are shown
in Scheme 2.
KM ) (k5(k2 + k3))/(k1(k3 + k5))
(2)
Figure 3. SDS-PAGE analysis of wild-type and variant arPTEs: (A) gel
stained with Coomassie blue, (B) same gel observed by fluorescence (Exλ
) 360 nm and Emλ ) 460 nm); Lane M ) molecular weight marker, 1 )
Tyr309Hco, 2 ) Tyr309Mco, 3 ) wild type arPTE.
pKa values were determined from pH-rate profiles by fitting the
experimental data to eqs 3 or 4 for single-proton and double-proton
profiles, respectively,
c
that wild-type expression used naturally evolved tRNA syn-
thetases, while the incorporation of Hco and Mco relied on a
tRNA synthetase, CouRS, that was engineered to accommodate
Hco.40
log y ) log
(3)
[H]
K1
1 +
(
)
aK2
SDS-PAGE was used to confirm that Hco and Mco were
incorporated into arPTE (Figure 3). Before staining with
Coomassie blue, the gel was imaged by fluorescence with
excitation and detection wavelengths of 360 and 460 nm, which
are near the excitation (367 nm) and emission (455 nm) maxima
of 7-hydroxycoumarin. The full-length Hco- and Mco-containing
proteins, which behaved identically to wild-type arPTE under
Coomassie staining, were clearly fluorescent, consistent with
incorporation of the uAAs. The Hco-containing protein band
had much higher fluorescence than the Mco protein band,
consistent with the 260-fold higher fluorescence of free Hco in
solution compared with Mco at these wavelengths (Figure 2 of
the Supporting Information). Further confirmation for the site-
specific incorporation of Hco and Mco was obtained through
tryptic peptide mass fingerprinting (Figure 4), which identified
peptides from segments of protein both before and after residue
309, indicating the expression of full-length Tyr309Hco and
Tyr309Mco proteins. The coumarinyl-glycine-containing pep-
tides (ILVSHDWLFGFSSHcoVTNIMDVMD and ILVSHD-
WLFGFSSMcoVTNIMDVMD) were identified by peptic ion
masses. The matched peptides covered 44% of the protein,
including the predicted N and C termini.
c 1 +
(
)
[H]
log y ) log
(4)
K2
[H]
K1
(
)
1 +
+
[H]
where [H] is the proton concentration, K1 and K2 represent
protonation equilibria, c and a are the pH independent values of y,
and y is the kinetic parameter which can be either kcat or kcat/KM.
Results and Discussion
In Vitro Protein Expression and Incorporation of Unna-
tural Amino Acids in arPTE. To probe the role of aromatic
interactions in ES and EP complexes of arPTE, two unnatural
variants were created: Tyr309Hco and Tyr309Mco (Figure 2).
These unnatural coumarinyl amino acids were chosen so that
the variants would retain aromatic character in this region of
the protein structure, whereas the hydroxyl group of Hco can
be titrated through a pH region of high catalytic activity for
arPTE (pH 8-9), allowing the effect of its protonation state
on activity to be monitored. Mco provided a control, being
completely analogous to Hco with the exception that the
ionizable hydroxyl group is replaced with a methyl group. The
codon for Tyr309 in the wild-type gene encoding arPTE was
mutated to the amber (TAG) stop codon, to be used in
Mechanistic Analysis of Unnatural Phosphotriesterases. The
effects of the Tyr309Hco and Tyr309Mco mutations on the
catalytic behavior of arPTE were examined using pH-rate
profiles. Figure 5 shows pH-rate profiles for the catalyzed
hydrolysis of paraoxon by wild-type arPTE and the unnatural
variants over a pH range of 4.0 - 10.0 (Table 1). All variants
exhibit acidic dissociation constants with pKe and pKes values
between 4.0 and 5.0, consistent with previous results for native
wild-type arPTE,21 which indicates a titration of the rate-limiting
step from catalysis (k3) to a physical step (product release, k5),
as described previously.19 However, unlike wild-type and
Tyr309Mco arPTE, the Tyr309Hco variant undergoes a second
deprotonation event at basic pH. We note that the effect of this
is less significant than the acidic protonation event, as seen in
the lower slope of this curve (ca. 0.2 vs 0.6). The pKa value of
this deprotonation event (8.2-8.4) corresponds to the pKa of
the hydroxyl group of the 7-hydroxycoumarinyl side chain
conjunction with mutant suppressor tRNA, MjtRNATyr
,
CUA
which was enzymatically acylated with either Mco or Hco by
purified engineered tRNA synthetase CouRS.40 The translational
incorporation of this residue was carried out using a cell-free
coupled transcription/translation system that allowed the various
nucleic acid, protein, and chemical components required for
synthesis of the gene to be added in defined quantities,49
allowing optimization of the conditions for maximal protein
yield using a minimum quantity of uAAs; an important
consideration given that they are not commercially available
and required synthesis. Wild-type arPTE was produced in higher
yield in vitro than Tyr309Hco, which was in turn produced in
higher yield than Tyr309Mco (47 µg vs 13 µg vs 4 µg per mL
of cell-free reaction mixture). This was expected considering
(49) Ozawa, K.; Headlam, M. J.; Schaeffer, P. M.; Henderson, B. R.; Dixon,
N. E.; Otting, G. Eur. J. Biochem. 2004, 271, 4084–4093.
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330 J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011

An Unnatural Phosphotriesterase
A R T I C L E S
Figure 4. Confirmation of Tyr309Hco and Tyr309Mco arPTEs expression by MS-TRAP tryptic digest. Peptides that were identified by MS are shown in
red with the digestion site underlined. The 7-hydroxy- or (7-methylcoumarinyl)ethylglycine containing peptide is shown in pink. Unidentified sequence is
shown in black. The position at which the uAA is incorporated (309) is shown in blue (X). The starting Met corresponds to residue 35 because the expressed
protein is lacking a signal peptide.
Figure 5. pH-dependence of the catalytic rate constants for wild-type (black), Tyr309Mco (red), and Tyr309Hco (blue) variants of arPTE for the hydrolysis
of paraoxon at room temperature.
Table 1. Acid Dissociation Constants for Wild-Type arPTE and the
Table 2. Kinetic Parameters for the Hydrolysis of Paraoxon at pH
8.5 by arPTE Variants with Natural and Unnatural Mutations at
Position 309; the Wild-Type Residue is Tyr
Unnatural Variants Mco309 and Hco309
variant
pKe1,2
pKes1,2
variant
kcat
KM
kcat/KM
Tyr309
Mco309
Hco309
4.7, -
4.7, -
4.7, 8.4
4.8, -
4.8, -
5.2, 8.2
s-1
µΜ
s-1 M-1
Tyr309
Leu309
Phe309
Trp309
Mco309
Hco309
510 ( 10
260 ( 9
465 ( 13
436 ( 11
910 ( 20
3990 ( 110
20 ( 1
51 ( 4
73 ( 6
80 ( 7
96 ( 7
138 ( 8
2.5 × 107
5.1 × 106
6.4 × 106
5.5 × 106
9.4 × 106
2.9 × 107
(8.3),50 suggesting that the change in the protonation state of
this hydroxyl moiety causes the change in activity across pH
8-9. This conclusion is also supported by the observation that
the turnover rate of the Mco309 variant does not change over
the same pH range and Mco is structurally identical to Hco
with the exception that the hydroxyl group is replaced by a
methyl group.
In addition to Hco and Mco, we made three other natural
variants of arPTE at this position: Leu309, Phe309, and Trp309.
Kinetic parameters for the six variants at pH 8.5 are reported
in Table 2. For efficient turnover, an enzyme must balance
strong interactions with the substrate (k1[S] . k2) with fast
dissociation of products (k5), around an efficient chemical step
(k3). Because of the similarity between substrate and product
in many reactions, this is not always an easy feat. The data in
Table 2 show that improvement in substrate turnover for
enzymes such as arPTE, where the chemical step is not rate
limiting, depends upon mutations differentially affecting sub-
strate binding and product release. In the case of the PTEs, it is
difficult to assess the effects of mutations by analyzing KM
because it is a combination of k1, k2, k3, and k5 (eq 2). However,
the relative changes of kcat and KM in the variants clearly show
that different mutations affect the various microscopic constants
differently. The comparison between wild-type, Tyr309Mco, and
Tyr309Hco is particularly informative: for Tyr309Mco we see
KM increase much more than kcat, whereas for Tyr309Hco KM
and kcat increase similarly. Thus, for Tyr309Hco the concerted
increase in KM and kcat can be largely attributed to an increase
in k5. In contrast, for Tyr309Mco, the much larger increase in
KM than in kcat suggests that the affinity between enzyme and
substrate (k1 and k2) has been adversely affected, without the
same increase in k5 as is seen in Tyr309Hco. In combination
with the pH profiles that show that the changing protonation
state of Hco309 affects turnover, this analysis establishes that
the residue at position 309 is directly involved in both Michaelis
complex formation and product release.
The possible rotamers of Hco309 are shown in Figure 6. The
effect of this mutation on turnover may be explained by the
fact that the phosphotriester substrates of PTEs are uncharged,
unlike the 4-nitrophenolate leaving group, which is negatively
charged at pH values above 7.1. The negative charge associated
with Hco309 will therefore electrostatically repel the negatively
charged product more than the neutral substrate, thereby
affecting product release more than substrate binding. These
data, which could only be obtained using mutagenesis with
(50) Ferrari, A. M.; Sgobba, M.; Gamberini, M. C.; Rastelli, G. Eur. J. Med.
Chem. 2007, 42, 1028–1031.
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J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011 331

A R T I C L E S
Ugwumba et al.
Figure 6. Potential rotamers (without steric clashes to other amino acid side chains or substrate) of Hco309 relative to the substrate diethyl-
methoxyphenylphosphate and Tyr309 in the wild-type protein based on the crystal structure of the enzyme-substrate complex (PDB accession code 2R1N).
The topology of Hco, bond restraints, and angle restraints were produced using the PRODRG server.47
uAAs, highlight the importance of noncovalent interactions in
ES and EP complexes and the trade-off between efficient binding
and fast turnover in enzymes.
active site residues altogether.33 The experimentally determined
activation energy for the rate limiting step, product release, is
of the order of 12 kcal/mol,27 meaning the energy barrier for
the chemical step must be even lower. The results presented
here, as well as previous analysis of alanine and phenylalanine
mutants of Tyr309,19,29 are more consistent with crystal structure
snapshots obtained during the catalytic cycle22,27 than recent
simulations; because both Tyr309 and the bulkier uAAs analyzed
here support fast substrate turnover and directly affect KM and
kcat, they are unlikely to impede formation of the transition state,
and instead most likely contribute to productive alignment of
the substrate for in-line attack from an alternative nucleophilic
water/hydroxyl to the µ-hydroxo bridge.
In principle, the increased turnover rate observed in the
Tyr309Hco variant could also result from an alteration in the
catalytic mechanism in which the hydroxyl group of Hco acts
as a nucleophile in the hydrolysis of paraoxon because it will
be positioned in the vicinity of the hydrolyzable P-O bond.
Indeed, lysine and tyrosine residues have been shown to be
responsible for the stoichiometric breakdown of organophos-
phates by albumin.51 In this scenario, Hco309 would become
phosphorylated by the substrate, followed by slow regeneration
of the enzyme through water-mediated dephosphorylation.
Because the spectrophotometric assay only detects the release
of 4-nitrophenol/4-nitrophenolate, we performed liquid chro-
matography/mass spectrometry to test for simultaneous release
of both diethyl phosphate and 4-nitrophenolate, to establish
whether both products were released. This was confirmed
(Figure 3 of the Supporting Information), consistent with the
rapid rate of substrate turnover, which is incompatible with any
slow dephosphorylation of Hco that would be necessary to
regenerate the free enzyme following any Hco-mediated hy-
drolysis of paraoxon.
Comparison to Computer Simulation. The altered KM values
of the variants presented here suggest that the residue at position
309 is directly involved in the formation of the Michaelis
complex (k1 and k2 in eq 2) during the catalytic cycle, while
the altered kcat values also suggest that it is involved in
interactions with the 4-nitrophenolate product of paraoxon
hydrolysis during the rate-limiting product release step (k5).
Previous computer simulations suggested that, to acquire the
transition state geometry for nucleophilic attack from the
µ-hydroxo bridge between the metals, paraoxon moves closer
to the binuclear metal center to become properly aligned for
SN2 attack and loses much of its aromatic interaction with
Tyr309,31,32 and that this rearrangement contributes to an energy
barrier in the order of 18.7 kcal/mol, partly due to steric clashes
with Tyr309.32 Other studies have omitted Tyr309 and other
Engineering Enhanced Activity in arPTE with uAAs. The kcat
values for wild-type, Tyr309Mco, and Tyr309Hco arPTEs for
paraoxon hydrolysis at pH 8.5 are 510, 910, and 3990 s-1
,
respectively (Table 2); an 8-fold increase in the turnover rate
for the Tyr309Hco variant over that of the wild-type enzyme.
The magnitude of this increase is even greater at pH values
above 8.5 (11-fold at pH 10). However, although kcat is
increased, the ratio of kcat/KM, also known as the specificity
constant and often considered to be a measure of catalytic
efficiency, is essentially unchanged for the Tyr309Hco variant
compared to the wild-type enzyme. As discussed above and
shown in Scheme 2 and eq 2, the value of kcat/KM is informative
and valuable because it incorporates most of the different rate
constants of the catalytic cycle. However, the present data also
show that kcat/KM is not well suited for evaluating catalytic
improvements in enzymes: despite wild-type and Tyr309Hco
arPTE having equal kcat/KM values, the Tyr309Hco variant is a
substantially more effective catalyst than the wild-type enzyme
at all substrate concentrations above 10 µM (Figure 7), which
is less than one tenth of its KM and much lower than any
substrate concentration that is typically used during laboratory
protein engineering/evolution experiments (100-500 µM). In
field applications, concentrations higher than 10 µM are
regularly encountered in some agricultural waste waters (e.g.,
spent animal and horticultural dip liquors).
(51) Lockridge, O.; Schopfer, L. M. Chem. Biol. Interact. 2010, 187, 344–
348.
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332 J. AM. CHEM. SOC. VOL. 133, NO. 2, 2011

An Unnatural Phosphotriesterase
A R T I C L E S
hundreds of thousands of random mutants27,37 or via rational
mutation of Tyr309 to other hydrophobic residues (Leu, Phe,
Trp; Table 2). Griffiths and Tawfik34 previously isolated a
mutant of pdPTE with a 3.6-fold increase in specific activity at
a concentration of 250 µM paraoxon, although this variant was
identified through the impressive technical achievement of
screening 3.7 × 107 mutants by in vitro compartmentalization;
here we have increased the specific activity of arPTE at 250
µM 5.5-fold with a single unnatural point mutation. These results
highlight the potential that man-made amino acids hold for
accessing functional space that is not available during natural
evolution and engineering with natural amino acids, showing
that the already exciting prospects for de novo design of enzyme
catalysts59-61 may be greatly enhanced through the use of
unnatural amino acids.
Summary
In this work two uAAs, Mco, and Hco, were introduced into
arPTE in place of Tyr309. The use of these uAAs allowed the
detailed analysis of the interaction between enzyme and substrate
during formation of the Michaelis complex and product release,
and established that the residue at position 309 is directly
involved in both of these processes. The data imply that the
complexes seen in recent crystal structures of the enzyme
captured during substrate turnover are most likely accurate and
highlight the importance of nonchemical steps for the mainte-
nance of fast substrate turnover during the catalytic cycles of
enzymes. The potential value of uAAs in protein engineering is
emphasized by the observation that the single site Tyr309Hco
mutation reported here produced a greater increase in activity
than has been obtained from screening of many thousands of
variants created using natural amino acids.
Figure 7. Comparison of reaction velocities of wild-type arPTE and
variants for hydrolysis of paraoxon at pH 7.5 and 8.5.
Others have previously highlighted the unsuitability of kcat/
KM as an accurate measure of catalytic efficiency,52-54 particu-
larly in the context of protein engineering. Put simply, these
results also support the notion that the best means to evaluate
the effectiveness of an enzyme catalyst for a particular reaction
is to determine the rate of substrate turnover, or the maximum
amount of substrate that is turned over, at defined substrate
concentrations. By this measure, it is clear that, for substrate
concentrations greater than 10 µΜ, the Tyr309Hco variant is a
considerably improved catalyst when compared with the wild-
type enzyme.
Impressive improvements in the turnover rates of enzymes
have been achieved through protein engineering using both uAAs
and natural AAs, targeting inefficient promiscuous, or non-native,
activities.55-57 In contrast, improving the efficiency at which
an enzyme catalyzes a reaction with its native substrate is very
difficult to achieve.27,37,58 Despite the fact that paraoxon was
only present in the environment for ca. 40 years before the
discovery of the PTEs,13,14 several mechanistic studies have
shown them to be highly optimized for catalysis of paraoxon
hydrolysis.20,25,27 Their natural evolutionary optimization is also
seen in our inability to significantly increase the rate of paraoxon
turnover by arPTE in the laboratory either through screening
Acknowledgment. We thank Prof. P. Schultz (The Scripps
Research Institute, USA) for the vectors pSUP-CouRS-D8 and
pSUP-MjTyrRS-6TRN, Prof. Y. Mo (University of Western
Michigan, USA) for coordinates from computer simulations and
Dr P. Campbell (CSIRO Entomology, Australia) for assistance with
tryptic digest mass spectrometry. Financial support by CSIRO’s
Emerging Science Initiative (I.U.), and the Australian Research
Council, including an Australian Research Fellowship to K.O., is
gratefully acknowledged.
Supporting Information Available: Analytical and spectral
characterization data for the synthesis of uAAs (Hco and Mco)
and other information. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA106416G
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