Structural, Functional, and Spectroscopic Characterization of
the Substrate Scope of the Novel Nitrating Cytochrome P450
TxtE
Sheel C. Dodani
,
Jackson K. B. Cahn
,
Tillmann Heinisch
,
Sabine Brinkmann-Chen
,
John A.
McIntosh
, and
Frances H. Arnold
Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 210-41,
Pasadena, California, USA
Frances H. Arnold: frances@cheme.caltech.edu
Abstract
A novel cytochrome P450 enzyme TxtE was recently shown to catalyze the direct aromatic
nitration of L-tryptophan. This unique chemistry induced us to ask whether TxtE could serve as a
platform for engineering new nitration biocatalysts, as a replacement for current harsh synthetic
methods. As a first step toward this goal and to better understand the wild-type enzyme, we have
obtained high-resolution structures of TxtE in its substrate-free and substrate-bound forms. We
have also screened a library of substrate analogs for spectroscopic indicators of binding and for
production of nitrated products. From these results, we find that the wild-type enzyme accepts
moderate decoration of the in dole ring, but the amino acid moiety is crucial for binding and
correct positioning of the substrate and therefore less amenable to modification. A carbonyl must
be present to recruit the
α
B
′
1
helix of the protein to seal the binding pocket, and a nitrogen atom is
essential for catalysis.
Keywords
aromatic nitration; crystal-structure determination; cytochromes; enzyme catalysis
Introduction
Nature has evolved specialized protein machinery to carry out a vast array of synthetic
transformations with a high degree of specificity and catalytic proficiency. In an effort to
create new and ‘greener’ chemistries, researchers have borrowed enzymes from their native
biological environments and engineered them to catalyze desired synthetic transformations
over a large reaction space under mild, aqueous conditions.
[
1
–
3
]
One transformation for which the available biocatalysts are limited is aromatic nitration.
Conventional methods for the nitration of organic compounds employ harsh reaction
Correspondence to: Frances H. Arnold,
frances@cheme.caltech.edu
.
The content of this paper is solely the responsibility of the authors and does not represent the official views of any of the funding
agencies.
NIH Public Access
Author Manuscript
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. Author manuscript; available in PMC 2015 October 13.
Published in final edited form as:
Chembiochem
. 2014 October 13; 15(15): 2259–2267. doi:10.1002/cbic.201402241.
NIH-PA Author Manuscript
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conditions, most commonly direct aromatic nitration utilizing nitric and sulfuric acids.
[
4
–
6
]
Oxidation is a common side reaction under these conditions and formation and isolation of
the desired isomer can be intractable depending on the complexity of the substrate. In
contrast, biological nitration typically proceeds through the oxidation of an amine or, to a
lesser extent, through unselective and/or inefficient direct aromatic nitration.
[
7
–
11
]
The cytochrome P450 TxtE from
Streptomyces scabies
is the first enzyme reported to
efficiently catalyze a direct and regioselective aromatic nitration.
[
12
]
This enzyme is
partnered with a nitric oxide synthase and is believed to generate a reactive peroxynitrite
intermediate that subsequently disproportionates to nitrate L-tryptophan, forming 4-nitro-L-
tryptophan as an intermediate in the production of the
S. scabies
phytotoxin thaxtomin A.
The
in vivo
activity can be recapitulated
in vitro
with recombinant TxtE, using molecular
oxygen, a nitric oxide donor, and L-tryptophan as substrates and spinach ferredoxin (Fd)
with ferredoxin NADP
+
reductase (Fr) as artificial electron donors (Figure 1).
[
12
]
Given its unique chemistry, TxtE could be an ideal platform to engineer new aromatic
nitration catalysts that could provide a more versatile and ‘greener’ alternative to traditional
synthetic methods. In order to do this, a better understanding of the wild-type enzyme is
required. To this end, we have assessed the scope of tryptophan-like substrates that TxtE can
bind and nitrate. We have also obtained the first high-resolution structures of substrate-free
and tryptophan-bound TxtE. In light of these results, we offer new insights into the substrate
properties required for catalysis and provide information that can guide future protein
engineering efforts.
Results and Discussion
Structural Determination of Substrate-Free and L-Tryptophan-Bound Forms of TxtE
TxtE has been crystallized previously with the inhibitor imidazole bound in the active
site.
[
13
]
Here, we were able to crystallize the enzyme in a substrate-free state using a mixture
of lithium sulfate and ammonium sulfate as a precipitant. Diffraction data were collected
both with and without the addition of L-tryptophan. The crystals diffracted remarkably well,
yielding resolutions of 1.18 Å and 1.22 Å, respectively. Full crystallographic statistics are
shown in Table 1. As previously reported, the crystal structure of TxtE shows the canonical
fold for a cytochrome P450, a mostly alpha-helical trigonal prism (Figure 2a). The structures
described here are in agreement with the previously reported structure (backbone RMSD
0.237 Å and 0.219 Å, respectively, to chain A of 4L36), while providing a higher resolution
picture of the protein active site in the absence and presence of the native substrate. A
detailed discussion of the fold and active site structure can be found in the report of Yu
et
al.
;
[
13
]
here we will focus on how the enzyme interacts with its substrate, which can only be
seen in the current structures.
Yu
et al
. used AutoDock to propose three L-tryptophan binding poses.
[
13
]
The binding
orientation observed in our study closely resembles their first two proposals. As shown in
Figure 2b, the tryptophan (which is bound to approximately 80% occupancy) binds with its
indole side chain facing into the distal face of the heme, displacing the water that ligates the
heme (this water is still visible in the tryptophan-bound structure, refined at 20%
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occupancy). The amino acid moiety faces toward the B-C loop containing the pair of B
′
helices. Figure 3 shows a schematic of the interactions involved in tryptophan binding. The
alpha amine of the amino acid is directly coordinated by Asn293 and Thr296 of the pre-
β
3
loop, and also forms a water-mediated hydrogen bond with Glu394. Thr296 is also involved
in coordination of the carboxylate, as is Glu394 through a different bridging water molecule.
The carboxylate also interacts with Arg59, which forms the start of the
α
B
′
1
helix, and
Tyr89 at the end of the
α
B
′
2
-
α
C loop. Residues Met88 and Phe395 define the hydrophobic
cleft for the substrate’s indole moiety. The C-4 position of the indole ring, the site of
nitration, faces away from the heme and toward the hydroxyl of Tyr89 and the solvent
channel that was identified previously
[
13
]
and confirmed in our structures. In the substrate-
free structure, three ordered waters define this solvent channel as reported,
[
13
]
whereas in the
tryptophan-bound structure a tube of indistinct density is visible that could not be modeled
as waters. The binding of tryptophan also displaces two waters present in the substrate-free
structure, one that fills the amine-binding pocket and one that takes the place of the
carboxylate oxygen
trans
to it. The imidazole-bound structure of Yu
et al
. contains both
these waters, as well as another that takes the place of the second carboxylate oxygen.
[
13
]
Interestingly, in the substrate-free structure, the density for the
α
B
′
1
helix was insufficiently
clear to model residues 61–72, which suggests that this portion of the protein is disordered.
The density can be resolved in the substrate-bound structure, though at 80% occupancy
(mirroring that of the tryptophan itself) and with considerably elevated B-factors, suggesting
that it is still fairly flexible. Rearrangements of the B-C loop are known to be crucial for
substrate binding in other P450s.
[
14
–
15
]
Thus we suggest that substrate binding-induced
organization of this loop also occurs in TxtE. This hypothesis is further supported by the
change in the rotamer observed at Arg59, which anchors the
α
B
′
1
helix and provides a key
interaction with the substrate carboxylate. Upon binding of L-tryptophan, Arg59 moves to
coordinate to both the substrate carboxylate and the propionate of the heme, as shown in
Figure 4, resulting in an almost 4-Å shift of Gly60, and presumably imparting enough order
to the helix to make it crystallographically resolvable. In the imidazole-bound structure, the
α
B
′
1
helix is fully resolved, with Arg59 facing downward in a conformation similar to that
observed in this tryptophan-bound structure. As shown in Figure 4c and discussed above, a
more complete network of waters, anchored by the heme-bound imidazole, takes the place
of the tryptophan heteroatoms and may be responsible for the recruitment of Arg59 and the
helix.
An additional detail visible in our high-resolution structures is that Asn293 has two
rotamers. The major rotamer (60% in the substrate-free structure, 80% in the tryptophan-
bound structure) is involved in binding the amine, but the other rotamer (shown in dashed
lines in Figure 3) forms a hydrogen bond with the indole nitrogen of tryptophan, and may be
involved in the catalytic cycle.
[
12
]
The observed binding mode of tryptophan provides a starting point for our survey of the
enzyme’s substrate preferences (Figure 1). The indole side chain is held in place through
nonspecific hydrophobic interactions. Thus, we hypothesized that the side-chain might be
amenable to substitutions and moderate decorations, subject to the electronic requirements
of the reaction itself. The amino acid moiety of the substrate is involved in a complex
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network of hydrogen bonds involving both the amine and the carboxylate with amino acids
of four different loops of the protein and with the propionates of the heme cofactor. With
this structural information in hand, we have explored to what extent the native substrate can
be modified while maintaining productive nitration chemistry.
Overview of the Spectroscopic and Functional Characterization of TxtE Substrate Scope
Consistent with previous reports,
[
12
]
we observed that recombinant TxtE carries out the
regioselective nitration of its native substrate L-tryptophan to generate 4-nitro-L-tryptophan,
in purified enzyme form as well as in cell lysate (Figure S2). Binding and reactivity were
assessed for each of 28 substrates tested (Tables 2–5). Because TxtE is a cytochrome P450,
the binding mode of a small molecule can be inferred by determining the spectral shift of the
Soret peak from a differential UV-visible spectrum (Figure S13–S15).
[
16
]
A type I shift from
420 nm to 390 nm is observed when the incoming substrate displaces the axial water ligand
from the heme iron. This substrate binding results in conversion from low to high spin iron,
which is essential for initiating the catalytic cycle.
[
16
]
L-tryptophan binds TxtE and produces
a type I shift (Figure S13).
[
12
]
Alternately, a type II spectral shift, from 420 nm to 425–435
nm, is characteristic of a ligand that directly coordinates to the iron center and in most cases
prevents oxygen-driven catalysis.
[
16
,
17
]
Nitration reactions were carried out with
overexpressed TxtE in
E. coli
cell lysates at least in triplicate with a vector (no-enzyme)
control. The spinach ferredoxin/ferredoxin NADP
+
reductase system and the nitric oxide
donor diethylamine NONOate were used as previously described.
[
12
]
Productive chemistry
was assessed qualitatively by the formation of yellow color corresponding to a nitro product
(in most cases) and was confirmed by LC-MS analysis. Some substrates were converted to
tryptophan by other enzymes present in the cell lysate so purified protein was used instead.
Disrupting the Heteroatom Bond Contacts of L-Tryptophan
First, we disrupted heteroatom bond contacts in L-tryptophan (substrate
1
) to identify the
components of the molecule that are required for nitration. Methylation of the indole
nitrogen of L-tryptophan in substrate
2
, which eliminates a labile hydrogen atom and adds
steric bulk, completely abolishes nitration chemistry while still maintaining type I binding
(Table 2). The basicity and bulk of the amino acid nitrogen was altered through acetylation
(substrate
3
) and methylation (substrate
4
). Titration of TxtE with substrates
3
and
4
results
in characteristic type I spectra. Uniquely among the compounds tested in this study, nitration
of substrate
4
is observed even in the absence of TxtE. However, higher rates of conversion
are observed in the presence of TxtE (Figure S3). No nitrated product is detected for
substrate
3
, where acetylation causes a larger shift in both the steric bulk and the pKa of the
nitrogen than methylation alone. Lastly, methyl esterification of the carboxylic acid in the
amino acid moiety (substrate
5
) results in a type II spectral shift and no productive nitration.
These results indicate that alkylation or acetylation of the heteroatom bond contacts in L-
tryptophan alters how these substrates are anchored in the active site of the enzyme. With
the exception of substrate
4
, no nitrated products are observed, suggesting that significant
modification of the L-tryptophan heteroatoms prevents productive catalysis.
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Modification of the Indole Moiety of L-Tryptophan
We next investigated substrates that retained the amino acid moiety but were altered in the
indole side chain with ring decorations and heteroatom substitutions. TxtE tolerates methyl
substitution at the 5- (substrate
6
) and 7- (substrate
7
) positions on the indole side chain,
resulting in nitro product formation, but substrate
7
is nitrated to a lesser extent, perhaps due
to a steric clash above the heme pocket (Table 3, Figure S5). Surprisingly, methyl
substitution at the 4-position (substrate
8
), the site of nitration on the native substrate, does
not prevent the nitration reaction and nitrated product is still observed (Figure S6), which
suggests that the enzyme can display alternative regioselectivity when this position is
occluded. These results prompted us to test the effects of electron-withdrawing substituents
with 5- and 6-fluoro-tryptophan analogs (substrates
9
and
10
), which are both nitrated by
TxtE (Figure S7 and S8).
From here, we explored substitution at the 5-position with hydrogen bonding groups.
Substrate
11
has a methoxy substitution that increases the steric bulk beyond the methyl
substitution alone, but the reaction still proceeds (Figure S9). In contrast, substrate
12
, 5-
hydroxy-L-tryptophan, is not nitrated, although it binds in a type I fashion similar to the
other tryptophan derivatives. Likewise, amino substitution (substrate
13
) prevents nitration.
Thus, TxtE can nitrate substrates with different ring substituents, but the aromatic ring
cannot be decorated with certain polar functional groups as these moieties prevent
productive nitration chemistry, perhaps due to unfavorable interactions with the protein
scaffold, the heme center, or the catalytic intermediates. The regioselectivity for the nitration
of non-native substrates is currently under investigation.
The effects of heteroatom substitution in the indole ring were also tested (Table 4). The
introduction of a nitrogen atom at the 7-position of indole (7-azatryptophan, substrate
14
)
results in no nitration activity. This atom is centered above the heme center, and we
speculate that introduction of the nitrogen atom may interfere with the formation of the
peroxynitrite intermediate or with compound II of the catalytic cycle. Replacement of the
indole nitrogen with a less electronegative sulfur or carbon atom does not lead to nitration
(substrates
15
and
16
), similar to substrate
2
. We also tested other aromatic amino acids,
including L-phenylalanine (substrate
17
) and L-histidine (substrate
18
), but observed no
nitration product. These results suggest that the enzyme requires an indole ring system with
a labile hydrogen atom above the iron center that could stabilize or be abstracted to drive the
catalytic cycle. This reasoning is consistent with a mechanism previously proposed by Barry
et al
.
[
12
]
Modification of the L-Tryptophan Amino Acid Moiety
Finally, we evaluated to what extent the amino acid motif can be removed or modified
(Table 5). L-tryptophan was broken down into three substrates: indole, tryptamine, and
indole-3-propionic acid (substrates
19
,
20
, and
21
). No nitrated product was observed for
any of these. A known type II binder to cytochrome P450s,
[
16
]
tryptamine’s lack of
reactivity is not surprising. Nor is it surprising that indole does not react, as it is quite small
and does not provide any of the hydrogen-bonding atoms. However, indole-3-propionic
acid’s lack of reactivity indicates that the hydrogen bonds to the amine part of the amino
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acid may be essential. Similarly, indole-3-propanol (substrate
22
) does not have an amine
and is not nitrated.
Interestingly, reduction of the carboxylic acid of the amino acid to an alcohol (substrate
23
)
yields a type II binder as seen with substrates
3
and
5
. This indicates that an interaction
between Arg59 and the carboxylate of L-tryptophan is important for binding the substrate in
a configuration that prevents the indole nitrogen or the amino acid amine from coming in
close proximity to the iron center, which results in a type II shift and prevents catalysis.
Further, increasing the steric bulk on the amino acid with substrates
24
and
25
does not
prevent binding but nitrated product is only observed for substrate
24
(Figure S10).
From here, we substituted the heteroatoms in the amino acid moiety of L-tryptophan,
resulting in indole-3-lactic acid (substrate
26
) and L-tryptophan amide (substrate
27
). Both
bind in a type I fashion, but substrate
26
does not get nitrated while
27
does (Figure S11). In
the lactic acid substrate, the hydroxyl group can donate only one hydrogen bond instead of
two or three from the amine (depending on the protonation state), so binding to Asn293 and
Thr296 is necessarily reduced. Substrates
25
and
28,
which also lack an amino acid amine
are also not nitrated. However, substrate
27
can be nitrated as it has the requisite amine and
carbonyl motifs to facilitate binding to TxtE in the correct configuration and induce the
observed conformational change. We next tested indole-3-acetamide (substrate
29
), a
smaller compound that has an amide. It binds in a type I mode and is nitrated, and we
suggest it represents the minimal tryptophan derivative that can be nitrated by wild-type
TxtE (Figure S12). Substrate
29
has a primary amide that we speculate can bind in the
pocket formed by residues Asn293 and Thr296. Additionally, the Arg59 residue is likely
able to hydrogen bond with the substrate carbonyl as it did with the carboxylate, either
directly or in a water-mediated fashion, closing the active site and positioning the substrate
for catalysis. A second heteroatom in the form of a carboxylate (substrate
1
) or an amide
(substrate
27
) is not required. Collectively, these examples demonstrate that hydrogen bond
contacts to residues Asn293 and Thr296 from the amino acid amine gate catalysis, and one
carbonyl interaction with residue Arg59 is required to position the substrate for catalysis.
Determination of Binding Constants for L-Tryptophan and Derivatives
Lastly, we sought to determine to what extent differences in binding affinity were
responsible for the observed pattern of substrate reactivity. Dissociation constants (
K
d
) were
determined for purified enzyme with L-tryptophan (substrate
1
), indole-3-propionic acid
(substrate
21
), indole-3-lactic acid (substrate
26
), tryptophan amide (substrate
27
), and
indole-3-acetamide (substrate
29
) by examination of the differential UV-visible spectra
(Table 6, Figure S16). We measured for the recombinant TxtE a K
d
for L-tryptophan of 39
μM, slightly lower than the previously reported K
d
values of 44 to 60 μM.
[
12
,
13
]
If the L-
tryptophan amine is removed (substrate
21
) or replaced with a hydroxyl group (substrate
26
), binding affinity is maintained or even increased (K
d
~ 11 μM and 45 μM, respectively),
but no nitration is observed. This suggests that while the amine moiety is required for
catalysis as described above, it does not provide a significant improvement in binding
affinity.
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As previously discussed, removal of the carboxylate moiety altogether from the amino acid
motif (substrates
20
and
23
) results in non-productive type II binding. However, the
carboxylate of L-tryptophan can be replaced by a terminal amide to retain productive
chemistry, with substrate
27
binding to TxtE with high affinity of < 1 μM. Substrate
29
is a
combination of these two observations. The nitrogen atom that we hypothesize is required
for productive chemistry is present, and catalysis occurs, however, the carbonyl interaction
with Arg59 is reduced resulting in much lower affinity (K
d
~ 1,800 μM). Taken together, a
type I binder can have variable affinity as determined by interaction with Arg59, but can still
be nitrated as long as it has an indole ring system with a labile hydrogen and a nitrogen atom
that can hydrogen bond to Asn293 and Thr296.
Concluding Remarks
In this report, we have described the first substrate-free and L-tryptophan-bound structures
of the cytochrome P450 enzyme TxtE. The tryptophan ring system sits in a hydrophobic
pocket of the protein, with the indole nitrogen facing toward the distal face of the heme.
Residues Asn293 and Thr296 form hydrogen bonds to the amine of the substrate amino acid,
and the
α
B
′
1 helix reorganizes upon formation of a hydrogen bond between residue Arg59
and the tryptophan carboxylate, anchoring the substrate in a position for catalysis. Through a
spectroscopic and functional substrate analog survey, we have determined that wild-type
TxtE can nitrate non-natural substrates that are structurally similar to L-tryptophan with
decorated indole side chains and modified amino acid moieties. We conclude that the
substrate must meet the following criteria in order to be nitrated: it must bind in a type I
mode, and it must also possess an indole ring system with a labile hydrogen atom and a
properly-positioned hydrogen bond donor (nitrogen atom) and hydrogen bond acceptor
(carbonyl). We find that substrates that bind weakly but meet these criteria can still be
nitrated, while substrates that bind with high affinity but do not meet these criteria cannot be
nitrated. An improved understanding of the molecular basis for TxtE’s substrate selectivity
provides key information to guide future mechanistic investigations and engineering of TxtE
for the nitration of diverse substrates.
Experimental Section
General
Substrates
7
,
8
, and
10
were purchased from MP Biomedicals. Substrate
16
was purchased
from AK Scientific. Substrates
13
,
15
,
24
, and
27
were purchased from Chem-Impex
International. All other reagents and chemicals were purchased from Sigma-Aldrich and
were used as received unless otherwise stated. If the substrate was purchased in the clean L-
enantiomeric form, it is noted in the chemical structure (Tables 2–5). For tryptophan both
the L-enantiomer and the racemic mixture were tested. Low-resolution mass spectral
analyses were carried out using an Agilent 1290 UHPLC system with a 6140 quadrupole
detector and an Eclipse Plus 2.1 × 50 mm C18 column (1.8 micron particle size).
TxtE Cloning, Expression, and Purification
The gene encoding
Streptomyces scabies
87.22 TxtE was codon optimized for
Escherichia
coli
(
E. coli
) using Gene Designer 2.0 (DNA 2.0), and then obtained as gBlocks from
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Integrated DNA Technologies. The gBlocks were amplified using Phusion High Fidelity
DNA polymerase (Thermo Scientific) according to manufacturer’s instructions and Gibson
cloned into pET28b+.
[
18
]
The His6-tagged construct was used to transform XL1-Blue
chemically competent
E. coli
(Stratagene). After DNA sequencing (Laragen, Inc.), the
correct construct was used to transform BL21 (DE3)
E. coli
for protein expression.
For expression, Hyper Broth
™
(700 mL, 50 μg/mL kanamycin sulfate, 1-L flask)
supplemented with glucose nutrient mixture (35 mL) (Athena Enzyme Systems) was
inoculated with an overnight culture (35 mL, Terrific Broth, 50 mL in a 125-mL flask, 50
μg/mL kanamycin sulfate). After 3.5 to 4 hours of incubation at 37 °C and 225 rpm, the
culture was cooled to 22–25 °C over approximately one hour. Then expression was induced
with the addition of isopropyl beta-D-thiogalactopyranoside (IPTG, 0.5 mM final
concentration, Research Products International Corp.) and 5-aminolevulinic acid
hydrochloride (0.25 mM final concentration, Acros Organics) was added for the
biosynthesis of heme. After 20–24 hours, the cells were harvested by centrifugation at 4 °C,
4,000 g for 10 min and the pellets were stored at −20 °C.
During protein purification, all samples were kept at 4 °C. The cell pellet was resuspended
in Ni-NTA buffer A (25 mM Tris-HCl, 100 mM NaCl, 30 mM imidazole, pH 7, 4 mL/g of
cell wet weight) and lysed by sonication (Sonicator 3000, Misonix, Inc.). The lysate was
clarified by centrifugation at 15,000 g for 30 min, and the supernatant was loaded on a pre-
equilibrated Ni-NTA column (GE Healthcare) using an AKTA purifier FPLC system (GE
Healthcare). After washing with two column volumes (cv) of Ni-NTA buffer A, TxtE was
eluted with a linear gradient (10-cv gradient) of buffer B (25 mM Tris-HCl, 100 mM NaCl,
300 mM imidazole, pH 7). The fractions that showed an absorbance at 280 nm and 417 nm
were combined and buffer exchanged with 20 mM Tris-HCl pH 7.5 on a 10- or 30-kDA
MWCO filter (EMD Millipore). The protein was aliquoted into PCR tubes, flash frozen over
powdered dry ice, and stored at −80 °C until further use.
For qualitative binding assays and screening nitration reactions,
E cloni
® EXPRESS BL21
(DE3) Competent Cells (Lucigen) were transformed with pET28b+ and the pET28b+_TxtE
construct and the proteins were expressed as described above. Cells were aliquoted into
portions (40 mL) and harvested by centrifugation at 4 °C, 4,000 g for 10 min 20 to 24 hours
after induction and stored at −20 °C until further use.
Protein concentration
The concentration of TxtE was determined from ferrous carbon monoxide binding
differential spectra using the reported extinction coefficient forcysteine-ligated P450 (
ε
=
91,000 M
−1
cm
−1
).
[
19
]
For crystallography, total protein concentration was determined with
the Quick Start
™
Bradford Protein Assay (Biorad).
Crystallization and Data Collection
High-throughput screening of crystallization conditions for TxtE was conducted at the
Beckman Molecular Observatory at the California Institute of Technology, and the optimal
conditions were further refined using focused screens. Crystals for diffraction were grown in
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a sitting-drop format in 24-well plate (Hampton Research) by mixing 1 μL of 15 mg/mL
protein in 20 mM Tris buffer (pH 7.5) with 1 μL of the well solution containing 0.1 M
sodium citrate buffer (pH 5.6), 1.25 M Li
2
SO
4
, and 0.5 M (NH
4
)
2
SO
4
. Moderately sized
red-brown orthorhombic crystals appeared within a few days. To obtain substrate-bound
structures, single crystals were transferred into 5-μL drops of mother liquor containing 5–10
mM of L-tryptophan and allowed to equilibrate for 1.5 – 4 hours at room temperature. All
crystals were cryo-protected in mother liquor containing 25% (v/v) glycerol prior to flash-
freezing in liquid nitrogen. Diffraction data were collected using a Dectris Pilatus 6M
detector on beamline 12-2 at the Stanford Synchrotron Radiation Laboratory at 100 K.
Diffraction datasets were indexed and integrated with XDS
[
20
]
and scaled using SCALA.
[
21
]
Structure Solution
An initial substrate-free TxtE structure (not published) was solved to 1.62 Å by
multiwavelength anomalous dispersion (MAD) using the anomalous signal from the heme
iron K-edge. Three data sets were collected for this crystal, corresponding to the peak, the
inflection point, and a high-energy remote. The heavy-atom location and phasing were
performed with the SHELXC/D/E package.
[
22
]
After Parrot
[
23
]
density modification,
Buccaneer
[
24
]
was used for initial chain tracing and model building, followed by automated
refinement with Refmac5 (CCP4 suite) and manual refinement using Coot.
[
25
]
This model
was then used as a starting model to solve a 1.18 Å substrate-free data set more thoroughly,
using automated refinement with Refmac5 as well as with PHENIX refine
[
26
]
and manual
refinement using Coot.
[
21
]
The higher resolution model was used as a starting model for the
refinement of the L-tryptophan-bound structure. To minimize phase bias, the same R-Free
set was used for all structures solved. Anisotropic B-factors were calculated for both high-
resolution structures.
Qualitative Binding Assays
Cell pellets expressing the pET28b+ vector or the TxtE construct were resuspended in 25
mM Tris buffer (pH 8, 10 mL) and harvested by centrifugation at 4 °C, 4,000 g for 15 min.
The supernatant was decanted and the pellet was resuspended in 25 mM Tris buffer (pH 8.0,
5 mL) and lysed by sonication (Sonicator 3000, Misonix, Inc.). The lysate was clarified by
centrifugation at 4 °C, 20,817 g for 25 min, and the supernatant was transferred to pre-
chilled Eppendorf tubes. The concentration of TxtE was determined as described above.
TxtE protein (100 μL, 1.9 – 2.8 μM) was plated in a half area 96-well plate (Greiner Bio
One). All substrates were prepared in dimethyl sulfoxide (DMSO) or 0.5 M sodium
hydroxide at 25–50 mM and added to a final concentration of 250–500 μM (1 – 2 μL). The
substrate was allowed to bind to the protein for 15 min and then spectra were recorded from
320 to 600 nm on a plate reader (Infinite M200, Tecan). For differential UV-visible spectra,
the TxtE control spectrum was subtracted from the spectrum of TxtE plus substrate.
TxtE-Catalyzed Nitration Reactions
Clarified cell lysate expressing TxtE (70 μL, 1.9 – 2.8 μM) was plated in triplicate and cell
lysate from pET28b+ expressing cells (70 μL) was plated in single replicate into a 96-well
plate (Evergreen Scientific). For substrates
4
,
26
, and
27
, purified TxtE (2 μM) in 25 mM
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Tris buffer, (pH 8) was used. All substrates were prepared in DMSO or 0.5 M sodium
hydroxide at 250 mM and used at a final concentration of 5.4 mM (2 μL). The substrate was
incubated with the protein for 15 min. Lyophilized spinach ferredoxin NADP
+
reductase
(Sigma) was reconstituted in 1 M Trizma (Sigma) at 17 U/mL. Spinach ferredoxin (Sigma)
was used as received at concentrations of 1–3 mg/mL. A solution of spinach ferredoxin
NADP
+
reductase (1 μL/well, ~ 0.17U/mL final concentration) and spinach ferredoxin (1
μL/well, ~ 0.01 – 0.03 mg/mL final concentration) was prepared in 25 mM Tris buffer (pH
8, 8 μL) and added to each well. DiethylamineNONOate sodium salt hydrate (DEANO,
Sigma) was prepared at 100 mM in 10 mM sodium hydroxide and stored at −80 °C and was
further diluted to 10 mM in 25 mM Tris buffer (pH 8). Nicotinamide adenine dinucleotide
phosphate (NADPH, Codexis, Inc.) was prepared fresh at 20 mM in 25 mM Tris buffer (pH
8). A solution of DEANO (10 mM, 515 μM final concentration) and NADPH (20 mM, 1
mM final concentration) were mixed in equal volumes and 10 μL were added to each well.
The plate was covered, wrapped in foil, and the reactions proceeded with shaking. After 90
min, NADPH (5 μL/well, 1 mM final concentration) was added to each well and the
reactions were allowed to continue overnight during which time some solutions became
yellow. Each reaction was applied to a 0.5-mL 3-kDA MWCO filter (Millipore) and
centrifuged at 20,817 g for 45 min. The filtrate (35 μL) was diluted with acetonitrile (70 μL)
and transferred to a vial insert (Agilent Technologies) and analyzed by liquid
chromatography mass spectrometry (LC-MS). For substrate
29
, three reactions were
combined in the workup for LC-MS analysis, because upon dilution with acetonitrile the
yellow color was diluted and the product did ionize well. The extent of reactivity is denoted
in Tables 2–6 as follows: (+) for the products that required an extract ion chromatogram of
the mass chromatogram and (++) for products where the mass was readily detected in the
mass chromatogram.
Determination of Dissociation Constants
A solution of purified TxtE (100 μL, 5 μM) in 25 mM Tris buffer (pH 8) was plated in a half
area 96 well plate (Greiner Bio One) and each substrate was titrated at varying
concentrations. Substrate
1
prepared in 25 mM Tris (pH 8) was added at the following final
concentrations: 0, 12.5, 25, 50, 100, 200, 400, 600, 800, 1,000, and 1,200 μM. The volume
change (6 μL) was the same for each well. Substrate
21
prepared in DMSO was added at the
following concentrations: 0, 10, 50 200, 500, and 750 μM. The volume change did not
exceed 1 μL of DMSO. Substrate
26
prepared in DMSO was added at the following final
concentrations: 0, 10, 50, 100, 200, and 400 μM. the volume change did not exceed 1 μL of
DMSO. Substrate
27
prepared in DMSO was added at the following final concentrations: 0,
0.1, 1, 10, 100, 250, and 500 μM. The volume change did not exceed 1 μL of DMSO except
for 500 μM where 2 μL of DMSO was added. Substrate
29
prepared in DMSO was added at
the following final concentrations: 0, 10 100, 500, 1,500, 2,500, and 5,000 μM. The volume
change did not exceed 1 μL of DMSO. After at least 15 min of shaking, spectra were
recorded from 325 to 525 nm with 5 or 10-nm step size on a plate reader (Infinite M200,
Tecan) in triplicate. For each substrate concentration, differential UV-visible spectra were
determined by subtracting the TxtE control spectrum from the Txte+substrate. The
difference in the absorbance of each spectrum at the
λ
max
and
λ
min
was calculated in
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triplicate and the average with standard deviation was plotted versus the substrate
concentration. Each data set was fitted to a binding isotherm model using KaleidaGraph.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Dr. Jens Kaiser and Dr. PavleNikolovski of the Beckman Molecular Observatory (Caltech) for assistance
with crystallography and Dr. Scott Virgil and the 3CS Center for Catalysis and Chemical Synthesis (Caltech) for
assistance with LC-MS analyses. The authors thank the Resnick Sustainability Institute (Caltech) and Dow
Chemical Company for support through the Dow-Resnick innovation program. S.C.D. and J.A.M. are supported by
Ruth L. Kirschstein NRSA postdoctoral fellowships from the National Institutes of Health (5F32GM106618 and
5F32GM101792, respectively). J.K.B.C. acknowledges the support of the Resnick Sustainability Institute (Caltech).
T.H. is supported by a postdoctoral fellowship from the Swiss National Science Foundation (Fellowship
PBBSP2_146809). The Beckman Molecular Observatory is supported by the Gordon and Betty Moore Foundation,
the Beckman Institute, and the Sanofi-Aventis Bioengineering Research Program (Caltech). The authors thank Dr.
Fei Sun for sharing of the XL1-Blue
E. coli
strain and helpful discussions.
References
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Wiley & Sons; New York: 2000.
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Loria R, Challis GL. Nat Chem Biol. 2012; 8:814–816. [PubMed: 22941045]
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[PubMed: 24282603]
14. Poulos TL. Proc Natl Acad Sci USA. 2003; 100:13121–13122. [PubMed: 14597705]
15. Pochapsky TC, Kazanis S, Dang M. Antioxid Redox Signal. 2010; 13:1273–1296. [PubMed:
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16. Barry SM, Challis GL. Methods Enzymol. 2012; 516:171–194. [PubMed: 23034229]
17. Peng CC, Pearson JT, Rock DA, Joswig-Jones CA, Jones JP. Arch Biochem Biophys. 2010;
497:68–81. [PubMed: 20346909]
18. Gibson DG. Methods Enzymol. 2011; 498:349–361. [PubMed: 21601685]
19. Omura T, Sato R. J Biol Chem. 1964; 91:542–553.
20. Kabsch W. Acta Crystallogr D Biol Crystallogr. 2010; 66:125–132. [PubMed: 20124692]
21. Evans P. Acta Crystallogr D Biol Crystallogr. 2006; 62:72–82. [PubMed: 16369096]
22. Sheldrick GM. Acta Crystallogr D Biol Crystallogr. 2010; D66:479–485. [PubMed: 20383001]
23. Zhang KYJ, Cowtan K, Main P. Method Enzymol. 1997; 277:53–64.
24. Cowtan K. Acta Crystallogr D Biol Crystallogr. 2006; D62:1002–1011. [PubMed: 16929101]
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25. Emsley P, Cowtan K. Acta Crystallogr D Biol Crystallogr. 2004; 60:2126–2132. [PubMed:
15572765]
26. Adams PD, Afonine PV, Bunkóczi G, Chen VB, Davis IW, Echols N, Headd JJ, Hung LW, Kapral
GJ, Grosse-Kunstleve RW, McCoy AJ, Moriarty NW, Oeffner R, Read RJ, Richardson DC,
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Figure 1.
L-Tryptophan nitration catalyzed by TxtE as described by Barry
et al.
[
12
]
Abbreviations:
spinach ferredoxin (Fd) and spinach ferredoxin NADP
+
reductase (Fr).
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Figure 2.
A) The overall fold of TxtE, with simplified helices and loops, including the heme (pink)
and bound tryptophan molecule (light blue). The I-helix runs from the top to the bottom in
yellow, with an interruption for the kink caused by the distal threonine. The B–C loop with
the pair of B
′
helices is in light blue at the top of the structure. B) Structure of the TxtE
active site with L-tryptophan bound. TxtE protein is shown in green, the heme cofactor is
shown in pink, and L-tryptophan is highlighted in sky blue. The polar contacts between the
amino acid and the protein scaffold are shown with yellow dotted lines. The water that
distally ligates the heme (and its hydrogen bond network back to Thr 250) is also shown,
despite being present at an occupancy of 20%. An Fo-Fc omit map of the active site is
shown in Figure S1.
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Figure 3.
A schematic of interactions in the L-tryptophan binding pocket. Hydrogen bonds, direct and
water-mediated, are shown (as determined by contact distances and angles) as dashed lines.
Nonpolar contacts are shown with arches. The minor conformation of Asn293 is shown with
dashed lines. For clarity, hydrogen atoms on heteroatoms are not shown, except on water
molecules.
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Figure 4.
Rearrangement of the TxtE aB’
1
helix upon L-tryptophan binding through residue Arg59. A)
The structure of substrate-free TxtE is shown in lime green; B) The structure of L-
tryptophan-bound TxtE is shown in purple, including indications of the polar contacts made
by Arg59 in its new conformation; C) The previously reported imidazole-bound TxtE
structure (PDB ID: 4L36) is shown in cyan, including the imidazole-anchored network of
waters taking the place of the tryptophan amino acid motif; and D) overlay of (A) and (B),
showing the movement of Arg59 and Gly60.
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Table 1
Crystallographic parameters of substrate free TxtE and L-tryptophan bound TxtE.
Parameter
Substrate-free
+L-tryptophan
PDB ID: 4TPN
PDB ID: 4TPO
Data Collection
Space Group
P2
1
2
1
2
1
P2
1
2
1
2
1
a (Å)
65.7
65.8
b (Å)
79.4
79.7
c (Å)
112.4
112.6
α
,
β
,
γ
(°)
90, 90, 90
90, 90, 90
Resolution (Å)
37.61 – 1.18 (1.24–1.18)
37.69 – 1.22 (1.29–1.22)
R
merge
(%)
4.8 (136.2)
12.6 (99.5)
<I>/<
σ
I>
13.9 (1.1)
5.6 (1.0)
Completeness (%)
94.9 (81.1)
98.0 (90.1)
Redundancy
4.9 (4.4)
3.6 (3.0)
Refinement
No. reflections
173,667
161,654
R
work
/R
free
(%)
16.3/17.1
13.9/16.1
No. atoms
Protein
3014
3112
Ligand
93
96
Water
394
409
RMSD
Bond lengths (Å)
0.018
0.026
Bond angles (°)
1.661
2.299
Ramachandran plot (%)
Favored
337
372
Allowed
11
9
Outliers
1
1
Values in parentheses represent statistics from the highest resolution shell.
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Table 2
Chemical structures, spectral shifts, and nitration chemistry of L-tryptophan derivatives with blocked
heteroatom contacts.
Substrate
a
Structure
Spectral Shift
Nitration
b
1
Type I
++
2
Type I
ND
3
Type I
ND
4
c
Type I
++
d
5
Type II
ND
a
Reaction conditions can be found in the experimental section. Differential UV-visible spectra can be found in Figure S13 and LC-MS
chromatograms for nitrated substrates can be found in Figures S2–S3.
b
Abbreviations: not detected (ND).
c
Purified protein was used.
d
Product formation was observed above background.
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Table 3
Chemical structures, spectral shifts, and nitration chemistry of L-tryptophan derivatives with modified side
chains.
Substrate
a
Structure
Spectral Shift
Nitration
b
6
Type I
++
7
Type I
+
8
Type I
++
9
Type I
++
10
Type I
++
11
Type I
++
12
Type I
ND
13
Type I
ND
a
Reaction conditions can be found in the experimental section. Differential UV-visible spectra can be found in Figure S14 and LC-MS
chromatograms for nitrated substrates can be found in Figures S4–S9.
b
Abbreviations: not detected (ND).
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Table 4
Continuation of Table 3. Chemical structures, spectral shifts, and nitration chemistry of L-tryptophan
derivatives with modified side chains.
Substrate
a
Structure
Spectral Shift
Nitration
b
14
Type I
ND
15
Type I
ND
16
Type I
ND
17
Type I
ND
18
Type I
ND
a
Reaction conditions can be found in the experimental section. Differential UV-visible spectra can be found in Figure S14.
b
Abbreviations: not detected (ND).
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Table 5
Chemical structures, spectral shifts, and nitration chemistry of L-tryptophan derivatives with modified amino
acid motifs.
Substrate
a
Structure
Spectral Shift
Nitration
b
19
Type I
ND
20
Type II
ND
21
Type I
ND
22
Type I
ND
23
Type II
ND
24
Type I
++
25
Type II
ND
26
c
Type I
ND
27
c
Type I
++
28
Type I
ND
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Substrate
a
Structure
Spectral Shift
Nitration
b
29
Type I
++
a
Reaction conditions can be found in the experimental section. Differential UV-visible spectra can be found in Figure S15 and LC-MS
chromatograms for nitrated substrates can be found in Figures S10–S12.
b
Abbreviations: not detected (ND).
c
Purified protein was used.
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