Improving nature's enzyme active site with genetically encoded unnatural amino acids

Article abstract of DOI:10.1021/ja061099y

The ability to site-specifically incorporate a diverse set of unnatural amino acids (>30) into proteins and quickly add new structures of interest has recently changed our approach to protein use and study. One important question yet unaddressed with unnatural amino acids (UAAs) is whether they can improve the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the >30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active site and a >2.3-fold improvement over the best possible natural amino acid. Because immense structural and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to improve enzyme active sites and tune a site to multiple substrates.

Full text of DOI:10.1021/ja061099y

Published on Web 08/03/2006
Improving Nature’s Enzyme Active Site with Genetically
Encoded Unnatural Amino Acids
Jennifer C. Jackson, Sean P. Duffy, Kenneth R. Hess, and Ryan A. Mehl*
Contribution from the Franklin and Marshall College, Department of Chemistry, P.O. Box 3003,
Lancaster, PennsylVania 17604
Received February 23, 2006; E-mail: rmehl@fandm.edu
Abstract: The ability to site-specifically incorporate a diverse set of unnatural amino acids (>30) into proteins
and quickly add new structures of interest has recently changed our approach to protein use and study.
One important question yet unaddressed with unnatural amino acids (UAAs) is whether they can improve
the activity of an enzyme beyond that available from the natural 20 amino acids. Herein, we report the
>30-fold improvement of prodrug activator nitroreductase activity with an UAA over that of the native active
site and a >2.3-fold improvement over the best possible natural amino acid. Because immense structural
and electrostatic diversity at a single location can be sampled very quickly, UAAs can be implemented to
improve enzyme active sites and tune a site to multiple substrates.
Introduction
for site-specific in ViVo incorporation into proteins can be used
to tune and improve enzyme activity beyond that available with
natural amino acids, we focused on a critical active-site amino
acid in the prodrug activator nitroreductase (NTR).
Since gaining the ability to modify proteins from the genetic
level, defined amino acid changes have been used to modify
and study protein function. Altering the chemical and physical
properties of proteins and enzymes has been instrumental in
their use as catalysts, therapeutics, and in the activation of
therapeutics. Although amino acid mutagenesis is one of the
most powerful tools for modifying protein function, it is
restrictive because we have not yet truly moved past the 20
common amino acids with their limited structural and chemical
diversity. The ability to site-specifically incorporate a diverse
set of unnatural amino acids (>30) into proteins and quickly
Nitroreductase from Escherichia coli is being used as a
7
,8
prodrug activator in cancer therapy. Currently employed
prodrug CB1954 and new lead prodrug LH7 have been activated
by NTR using antibody-directed and gene-directed enzyme
methods. NTR is a 48 kDa homodimer that reduces nitroaro-
matic compounds by transferring hydrides from NAD(P)H
through a bound FMN cofactor in a Ping-Pong Bi Bi mecha-
nism. CB1954 is reduced to the corresponding cytotoxic
hydroxylamino derivative 1, whereas LH7 activation proceeds
through the hydroxylamino intermediate which releases anionic
cytotoxic mustard 3 (Figure 1a). Activated drug toxicity results
from the formation of poorly repaired, interstrand DNA cross-
links. The current therapeutic limitation of CB1954/NTR drug
delivery system is the high dosage of drug needed because of
the high KM of CB1954 for native NTR.
add new structures of interest has recently changed our approach
to protein use and study.1
-4
One important question yet
unaddressed with unnatural amino acids (UAAs) is whether they
can improve the efficiency of an enzyme beyond that available
from the natural 20 amino acids.
Although UAAs have been incorporated via a variety of
methods into enzymes to probe their function, none has been
reported to improve an enzyme’s activity, begging the question
whether additional amino acids would improve current biological
function.5 Since the practicality of obtaining large quantities
of protein containing unnatural amino acids has improved, it is
now feasible to test whether unnatural amino acids can improve
enzyme efficiency, as well as to perform structure activity
relationship studies by systematically altering the protein instead
of the substrate. To prove that the UAAs currently accessible
To improve the efficacy of the NTR/CB1954 system by
increasing the activity of NTR with CB1954, NTR structural
information helped guide the alteration of all relevant active
,6
7
,9
site residues. Active site residue Phe124 emerged as critical
for substrate binding and was modified to all possible natural
amino acids. These modifications were found to have a range
of effects on CB1954 activation. The F124K and F124N
mutations emerged as the best for improving the activity of NTR
for CB1954. Structural studies have suggested that Phe124 pi
stacks with the aryl ring of the prodrug or NADH when the
active site is occupied. Improved activity with K124 or N124
(
1) Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science 2001, 292, 498-
0
0.
(
2) Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.;
King, D. S.; Horn, D. M.; Schultz, P. G. J. Am. Chem. Soc. 2003, 125,
9
35-9.
(7) Grove, J. I.; Lovering, A. L.; Guise, C.; Race, P. R.; Wrighton, C. J.; White,
S. A.; Hyde, E. I.; Searle, P. F. Cancer Res. 2003, 63, 5532-7.
(8) Hu, L.; Yu, C.; Jiang, Y.; Han, J.; Li, Z.; Browne, P.; Race, P. R.; Knox,
R. J.; Searle, P. F.; Hyde, E. I. J. Med. Chem. 2003, 46, 4818-21.
(9) Lovering, A. L.; Hyde, E. I.; Searle, P. F.; White, S. A. J. Mol. Biol. 2001,
309, 203-13.
(
(
(
3) Xie, J.; Schultz, P. G. Methods 2005, 36,(3), 227-38.
4) Xie, J.; Schultz, P. G. Curr. Opin. Chem. Biol. 2005, 9, 548-54.
5) Judice, J. K.; Gramble, T. R.; Murphy, E. C.; de Vos, A. M.; Schultz, P.
G. Science 1993, 261, 1578-81.
(
6) Gloss, L. M.; Kirsch, J. F. Biochemistry 1995, 34, 12323-32.
11124
9
J. AM. CHEM. SOC. 2006, 128, 11124-11127
10.1021/ja061099y CCC: $33.50 © 2006 American Chemical Society

Improving Enzyme Active Sites with Unnatural Amino Acids
A R T I C L E S
position, we demonstrate that UAAs can tune activity for
different substrates and improve the efficiency of enzymes
beyond that accessible with the natural amino acids.
Results and Discussion
Generation of Natural NTR Variants. The gene encoding
NTR, nfnB, was cloned from E. coli K12 into a standard protein
expression plasmid, pTrcHisA, which added an NH2-terminal
His6-tag to facilitate purification. Site-directed mutagenesis of
nfnB gene in pTrcHisA provided the F124K and F124N
mutants. Site-directed mutagenesis also converted the codon for
Phe124 to a TAG stop codon resulting in pTrc-NTR-124TAG,
which when expressed alone in DH10B cells produces no
purifiable protein. When pTrc-NTR-124TAG is combined in
cells with the orthogonal Methanococcus jannaschii suppressor
Tyr
tRNA/aminoacyl-tRNA synthetase pair (MjTyrRS/tRNA CUA),
a full-length protein is produced with tyrosine incorporated at
Tyr
site 124. The genes for MjTyrRS/tRNA CUA resides in a
13
medium copy plasmid (pDule-Tyr). These expression systems
provided access to large quantities of pure protein depicted as
the natural set of NTR variants in Figure 2a.
Generation of NTR-124-UAA Proteins. Each UAA in
generation 1 (Figure 2a) was incorporated with a unique
orthogonal, evolved Methanococcus jannaschii suppressor
Tyr
tRNA/aminoacyl-tRNA synthetase pair (MjTyrRS/tRNA
CUA).
-
1
,2,10,11
Tyr
The genes for each synthetase/tRNA CUA pair
reside in a medium-copy plasmid (pDule-UAA), allowing a
standard high-copy protein expression plasmid for use in
production of UAA containing NTR (Figure 2b). All UAA-
proteins were produced only when cells containing both
plasmids pDule-UAA and pTrc-NTR-124TAG were induced to
produce protein in modified minimal media supplemented with
the UAA. The expression of all UAA containing NTR with and
without UAA present in the media was monitored by silver stain
Figure 1. Enzymatic activation of prodrugs with NTR: (a) CB1954
activation. (b) LH7 activation. (c) NTR active site with homodimer
represented as yellow and purple strands (PDB code 1ICR). Bound at the
active site is FMN in green and inhibitor, nicotinic acid, in red. The modified
active site phenylalanine 124 is in blue.
could be explained by NH-pi face stabilizing interaction with
the electron deficient prodrugs but other stabilizing interactions
are possible. Phe124 or the amino acid replacing it not only
aids in directing and facilitating the reaction of the substrate
with bound cofactor, FMN, but it can also interact with the
opposite subunit when the active site is closed, as well as interact
with substituents on the aryl ring which project out of the active
site. The exhaustive analysis of all natural amino acids at site
Phe124 to improve the currently used prodrug CB1954 system,
and the diversity of possible stabilizing interactions that are
possible for this site, directed us to test this site in NTR to
determine whether a protein could be improved with unnatural
amino acids.
13
SDS-PAGE analysis (Supporting Information Figure 1).
Using the chemical diversity of site-specifically incorporated
UAAs, we can combine functionalities and alter electrical factors
of the Phe124 aromatic ring. In exploring the activity of the
first generation unnatural-NTR, we found that the pAF substitu-
tion modestly improved NTR activity above the best natural
amino acids at that position (Figure 3). To further explore and
improve the electronic factors of this para position amine, we
sought the substitutions indicated as generation 2 in Figure 2a,
but evolved synthetases for these amino acids did not exist at
the start of the study. The ptfmF synthetase was evolved using
standard selection methods and was found to also incorporate
12
the p-MF with high selectivity and fidelity. We also discovered
that using the evolved synthetase for pAF we were able to site-
specifically incorporate the pAMF and the pNF with high
fidelity but at lower yields (Figure 2b).
The incorporation of different UAAs at site 124 in NTR was
accomplished by using an orthogonal, evolved Methanococcus
jannaschii suppressor tRNA/aminoacyl-tRNA synthetase pair
Tyr
(
MjTyrRS/tRNA CUA) for each UAA. Using evolved syn-
To verify that discrete incorporation of each UAA only takes
place at site 124, each pure protein was digested by trypsin and
the fragments were analyzed by LC-ESI MS. The only peptide
fragment containing the desired change was the expected F(X)-
ADMHR fragment with a minimum of >50% sequence cover-
age for all protein samples (MS/MS, Mascot search). These
results along with the SDS-PAGE gel analysis confirm the high
fidelity and efficiency of UAA incorporation into proteins with
thetase we were able to site-specifically incorporate additional
UAAs similar in structure to the evolved synthetases UAA,
providing us with a total of eight UAAs to compare with natural
1
,2,10-12
amino acids.
Comparing the catalytic proficiency of the
best natural amino acids with eight UAAs at this critical F124
(
10) Chin, J. W.; Martin, A. B.; King, D. S.; Wang, L.; Schultz, P. G. Proc.
Natl. Acad. Sci. USA 2002, 99, 11020-4.
(
11) Wang, L.; Brock, A.; Schultz, P. G. J. Am. Chem. Soc. 2002, 124, 1836.
12) Jackson, J. C.; Mehl, R. A. Site-specific incorporation of a 19F-amino acid
into proteins as an NMR probe for characterizing protein structure and
reactivity. J. Am. Chem. Soc. 2006, submitted.
(
(13) Farrell, I. S.; Toroney, R.; Hazen, J. L.; Mehl, R. A.; Chin, J. W. Nat.
Methods 2005, 2, 377-84.
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A R T I C L E S
Jackson et al.
Figure 2. NTR modifications: (a) Natural and unnatural amino acids incorporated at site 124 in NTR. Generation 1: p-aminophenylalanine (pAF),
naphthylalanine (Nap), p-benzoylphenylalanine (pBpa), p-methyoxyphenylalanine (pMOF). Generation 2: p-aminomethylphenylalanine (pAMF), p-
methylphenylalanine (pMP), p-trifluoromethylphenylalanine (ptfmF), p-nitrophenylalanine (pNF). (b) Purified unnatural-NTR yields from modified minimal
media expressions.
amine was separated from the aryl ring by a methylene unit in
pAMF, making the basicity more like Lys and the ring
polarization more like Phe. Unnatural protein pAMF-NTR
produced not only the highest catalytic activity of any amino
acid tested but also the highest KM. Reducing aryl ring
polarization and removing basicity by moving to pMF and ptfmF
showed similar results to Phe-NTR and Tyr-NTR. However,
increasing ring polarization by switching to a para nitro
functionality in pNF-NTR improved both the prodrug binding
and catalytic activity of pAF-NTR, resulting in the best
unnatural-NTR (>30 times better than the native NTR, and
>2.3-fold better than the best natural).
The complexity of this multistep reaction has made it difficult
to elucidate the role of natural amino acids in prodrug binding
and activation, but structural work on natural active site variants
are starting to provide clues. Unfortunately the flexibility of
lysine in the crystal structure of NTR-124 prevents elucidation
of its role in improving catalytic efficiency. Of particular interest
within the improved efficiency for the NTR-UAAs: (NTR-
pAF, NTR-Nap and NTR-pNF) is the significant drop in KM
because poor prodrug solubility and high prodrug dosage prevent
wider use of this system for cancer treatment. We expect that
future catalytic analysis and crystal structure studies with these
NTR variants will help elucidate how improved prodrug binding
is achieved and can be further improved.
Figure 3. Catalytic efficiency of modified NTR enzymes: (a) kcat/KM values
for natural and unnatural NTRs with CB1954 and (b) kcat/KM values for
natural and unnatural NTRs with LH7.
the pDule plasmids. Although it seems obvious that UAAs of
similar shape should be utilized by unnatural synthetases which
have been evolved to discriminate against only the natural amino
acids, this method of quickly expanding the subset of current
orthogonal unnatural synthetases has yet to be embraced.
Kinetic Evaluation of NTR Proteins. The activity of NTR
proteins was monitored spectrophotometrically by observing the
formation of the hydroxylamine products at 420 nm for CB1954
Limited in Vitro kinetic work has been done with LH7 and
NTR but our results show that the improvements seen with
natural amino acids for activating CB1954 are not seen with
LH7 (Figure 3b). Multiple UAA-NTRs showed improved
activation of LH7 over the natural-NTRs tested with a >3-fold
improvement from both ptfmF-NTR and pNF-NTR. We
suspect with LH7 the reduced KM provided by the polarized
aromatics at this site will lead to the highest efficiency for this
prodrug. Interestingly, ptfmF-NTR activates prodrug LH7 but
not CB1954 and could be used simultaneously with CB1954
treatment to access different bystander effects on different
targeted cell types.
studies and following the loss of NADH at 340 nm for LH7
studies.7,8 To ensure the special electronic characteristics of
unnatural amino acids were not unfairly targeted, assay condi-
tions for each prodrug were optimized using native NTR and
kept constant for all proteins. Low prodrug solubility, and the
range of NTR-UAA catalytic efficiencies, prevented the
comparison of individually optimized NTR-prodrug assays. The
CB1954 activities of the natural versions of NTR match well
with previous studies, showing slight improvement with Tyr-
NTR and significant improvement with Lys-NTR and Asn-NTR
Although it is difficult to elucidate the electronic interactions
by which the pAF or pNF is improving NTR activity, the
inability to improve prodrug binding without disrupting reduc-
tion has plagued the development of new prodrugs. Different
modes of stabilization might be accessed with different amino
acids at site 124. We suspect that for the UAAs, a polarized
aromatic ring at site 124 aids in pi stacking with the polarized
aromatic substrate while also aiding in hydride transfer.
(Figure 3a). Combining the amine and aryl ring in pAF-NTR
improved both prodrug binding and catalytic activity resulting
in a higher efficiency than feasible with any natural amino acid.
Focusing on the electrostatic and structural aspects of pAF, the
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Improving Enzyme Active Sites with Unnatural Amino Acids
A R T I C L E S
Conclusion
centrifuged at 1000 × g for 5 min, and 750 µL of supernatant was
removed. The remaining 250 µL was resuspended and 62.5 µL was
used to inoculate 55 mL of minimal media, containing 0.01 mM
We have demonstrated that UAAs, with their immense
structural and electrostatic diversity, can provide proteins with
improved catalytic properties not accessible with only natural
amino acids. We report the >30-fold improvement of prodrug
activator nitroreductase (NTR) efficiency with an UAA over
that of the native active site, and a >2.3-fold improvement over
the best possible natural amino acid for currently used prodrug
CB1954. We have also shown that this quick and versatile
approach to modifying proteins can be used to tune an active
site to multiple substrates. Combining improved UAA-enzymes
with analogous autonomous 21 amino acid bacteria could
address the evolutionary fitness of natural 20 amino acid
organisms. Because these unnatural amino acids are incorporated
in ViVo, any protein produced in E. coli, irrespective of size,
can be explored with this method. Further studies are underway
to understand the mechanism of activity enhancement so that
future prodrugs and/or protected metabolites can be paired with
improved unnatural active-sites.
13
riboflavin and 1 mM of unnatural amino acid where applicable.
Minimal media cultures contained 50 µg/mL ampicillin and 10 µg/mL
tetracycline where appropriate. Cell cultures were allowed to grow to
an OD600 ≈ 0.8 at 37 °C with shaking (15-25 h). Cultures were induced
to overexpress protein with 250 µL of 200 mM IPTG and allowed to
express 20-24 h. Overexpression cultures were centrifuged at 6000
×
g for 20 min, and the procedure for protein purification found in the
BD Talon Metal Affinity Resins User Manual (BD Biosciences 1/28/
003) was followed. Approximately 10 µL of the first 500 µL elution
2
of each overexpression was analyzed using a 12% SDS-polyacrylamide
gel (Supporting Information Figure 1). Protein was removed from
elution buffer containing imidizole using Ambersham PD10 columns
and eluted into a 10 mM Tris-HCl buffer, pH 7.00. Protein concentra-
tions were determined using BCA (bicinchoninic acid) Protein Assay
Kit and instructions (Pierce 23225).
Mass Spectrometric Analysis of UAA Proteins. Proteins in 10 mM
Tris buffer were dried overnight on a vacuum-line, resuspended in 0.1
M ammomium hydrogen carbonate, and then incubated at 55 °C for
3
0 min. Sequencing grade trypsin in 0.1 M ammomium hydrogen
Experimental Section
carbonate was added to each protein sample, and the samples were
incubated at 37 °C overnight. Mass spectral analyses were performed
on an Agilent 1100 series LC/MSD SL ion trap mass spectrometer
with electrospray ionization and MS/MS capabilities (see Supporting
Information).
General Methods. Chemical reagents were purchased from Sigma-
Aldrich and used without further purification. Unnatural amino acids
were purchased from Bachem and Peptech. Oligonucleotides, dH10B
cells, and pTrcHisA were purchased from Invitrogen. CB1954 was
donated by Dr. William Denny, Director of the Auckland Cancer
Society Research Centre, The University of Auckland, New Zealand.
LH7 was synthesized according to the procedure by Hu L. et al.8
Overexpression of NTR Containing Natural Amino Acids. The
nfnB gene was amplified by PCR from E. coli K12 genomic DNA
using the following primers: 5′-GTGCTGGGATCCGATATCATTTCT-
GTCGCCTTAAAGCGTCATTCC-3′ forward, 5′ GCTCTTAGGAAT-
TCGCCCGGCAAGAGAGAATTAC-3′ reverse. The amplified nfnB
gene was digested with BamHI and EcoRI and inserted into pTrcHisA
producing pTrc-NTR. QuickChange site directed mutagenesis was
performed on pTrc-NTR with primers 5′-GGTCGCAAGTTCAAAGCT-
GATATGCACCG-3′, 5′-GGTCGCAAGTTCAACGCTGATATGCAC-
CG-3′, 5′-GGTCGCAAGTTCTAGGCTGATATGCACCG-3′ to pro-
duce pTrc-NTR-K124, pTrc-NTR-N124, and pTrc-NTR-124TAG,
respectively. Natural NTR proteins (NTR-native, NTR-124-Lys, and
NTR-124-Asn) were produced in dH10B cells according to the
procedure in the pTrcHisA manual. DH10B cells containing pDule-
Tyr and pTrc-124TAG were grown, and protein was expressed
according to the procedure outlined in Farrell et al. to produce NTR-
Enzyme Assays with Prodrugs. Enzyme assays were performed
spectrophotometrically in 10 mM Tris-HCl buffer, pH 7.00, at 25 °C
in the presence of 100 µM NADH and 4% DMSO and varying
concentrations of a second substrate (Supporting Information Table 1).
For CB1954, the reaction was initiated by the addition of 10 µL of
NTR solution to a final concentration of 4 nM. A 5 mM stock solution
of CB1954 was made up in 4% DMSO, 10 mM Tris-HCl pH 7.00
buffer. Due to mild inhibition of NTR by DMSO, it was kept constant
at 4% in all CB1954 assays. The progress of the reaction was monitored
by observing the formation of the hydroxylamine products at 420 nm
-
1
-1
(
with a molar absorbance of ꢀ ) 1200 M cm ) at 10 s intervals for
8
a period of 5 min and normalized for enzyme concentration. CB1954
concentrations varied from 250 to 5000 µM. For LH7, the reaction
was initiated by the addition of 10 µL of NTR solution to a final
concentration of 40 nM. A 25 mM stock solution of LH7 was made
up in 60% ethanol, 10 mM Tris-HCl pH 7.00 buffer. LH7 concentra-
tions varied from 50 to 3000 µM. The progress of the reaction was
monitored at 340 nm by observing the oxidation of NADH. This was
converted to a rate of reduction of LH7 using the molar absorbance of
-1
-1
NADH (ꢀ ) 6200 M cm ), assuming 2 mols of NADH are consumed
1
3
1
24-Tyr. Protein was purified to >95% according to the purification
per LH7, reduced to the hydroxylamine and normalized for enzyme
procedure found in the BD Talon Metal Affinity Resins User Manual
8
concentration. The observed rates (the observed slope of the reaction
(BD Biosciences 1/28/2003).
for 1 min) as a function of substrate concentration were fitted to a
Overexpression of Mutant NTR Containing Unnatural Amino
hyperbolic curve using Wilman Kinetic software to generate K
m
and
Acid. Overexpression of protein containing unnatural amino acid was
performed according to the procedure outlined in Farrell et al.13
Electrocompetent dH10B cells were transformed with plasmid DNA
pTrc-NTR-124TAG (1 µL of 25 ng/µL, enabling resistance to ampi-
cillin) and pDule-UAA (1 µL of 25 ng/µL, enabling resistance to
tetracycline). Plasmid pDule-UAA encodes tRNA and synthetase for
incorporation of a given unnatural amino acid into TAG codon.
Transformed cells were placed into 1 mL of rescue media and incubated
at 37 °C with shaking for 250 rpm. Cells containing both plasmids
were selected on agar plates containing 100 µg/mL ampicillin and 25
mg/mL tetracycline by incubating overnight at 37 °C. All overexpres-
sion cell lines for the production of protein containing unnatural amino
acids were prepared in this manner. A single colony from the selection
plates was used to inoculate 6 mL of 2× YT broth, containing 100
µg/mL ampicillin and 25 mg/mL tetracycline, and grown overnight
with shaking at 250 rpm at 37 °C. Saturated cell culture (1 mL) was
k
cat values for the data (Supporting Information Tables 3 and 4).
Acknowledgment. We thank Peter Schultz for providing
synthetases, William Denny for donating CB1954, and Scott
Van Arman and Peter Fields for their helpful discussions. This
work was supported by F&M Hackman and Eyler funds and
NSF-MCB-0448297, Research Corporation (CC6364), and
ACS-PRF (42214-GB4).
Supporting Information Available: A description of SDS-
PAGE gel silver stain protein analysis, mass spectrometry of
proteins, kinetic assays, and catalytic constants are provided.
This material is available free of charge via the Internet at
http://pubs.acs.org
JA061099Y
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