Discovery of a regioselectivity switch in nitrating P450s guided
by MD simulations and Markov models
Sheel C. Dodani
a
,
Gert Kiss
b
,
Jackson K. B. Cahn
a
,
Ye Su
a
,
Vijay S. Pande
b,*
, and
Frances
H. Arnold
a,*
a
Division of Chemistry and Chemical Engineering 210-41, California Institute of Technology,
California, 91125, USA.
b
Department of Chemistry, SIMBIOS NIH Center for Biomedical Computation, and Center for
Molecular Analysis and Design, Stanford University, 318 Campus Drive, Stanford, California,
94305, USA.
Abstract
The dynamic motions of protein structural elements, particularly flexible loops, are intimately
linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter
protein stability, substrate binding, and even dramatically impact enzyme function. When these
flexible regions are structurally unresolvable, computational reconstruction in combination with
large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here,
we present a collaborative approach consisting of both experiment and computation that led to the
discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that
simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from
the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is
naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized
enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic
intermediate.
Enzymes rely on finely tuned molecular recognition events to selectively install chemical
functionalities. This is especially true for reactions with highly reactive intermediates such
as those catalyzed by enzymes of the cytochrome P450 superfamily, which activate
molecular oxygen at the heme cofactor to access regio- and stereoselective oxygenation.
1
-
7
While the precise positioning of catalytic groups in the active site is essential for such
modifications, dynamic aspects of secondary structural elements, in particular flexible loops,
are also integral to P450 catalysis.
8
-
10
The functional dynamics of these enzymes are
characterized by open and closed arrangements of the B/C and F/G loops that act like lids
upon substrate binding.
8
-
12
Not only do these loops seal the active site from bulk solvent to
Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research,
subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms
*
; Email: frances@cheme.caltech.edu, ; Email: pande@stanford.edu
Author contributions
S.C.D. and G.K. contributed equally to this work. S.C.D. and G.K. designed the research. S.C.D., G.K., J.K.B.C., and Y.S. performed
research. F.H.A and V.S.P. supervised and provided advice. S.C.D., G.K., and J.K.B.C. analyzed the data. S.C.D., G.K., J.K.B.C., and
F.H.A wrote the text and conceived the figures with input from all of the authors.
HHS Public Access
Author manuscript
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Published in final edited form as:
Nat Chem
. 2016 May ; 8(5): 419–425. doi:10.1038/nchem.2474.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
produce a catalytically productive microenvironment, they are also believed to have a direct
role in substrate recognition and alignment during catalysis.
8
This picture is supported by a host of crystallographic studies that have assigned structural
significance to various positions along the B/C loop as well as the B and F helices.
8
Although the F/G loop has been identified as a specificity-determining region (SDR) on the
basis of mutational studies,
8
,
13
there is only limited structural evidence of its direct
interaction with substrates.
14
-
16
This observation raises the question of whether these
contacts are too transient to be captured by crystallography, or whether it is indeed rare that
residues in these loops can extend sufficiently deep into the active site of P450s to contribute
to substrate positioning during catalysis.
Here we present the discovery of such a functional position in the F/G loop of the nitrating
P450 TxtE from
Streptomyces scabies
and demonstrate its control over regioselectivity and
loop dynamics. TxtE is a rare example of an enzyme that catalyzes a direct and
regioselective aromatic nitration.
17
-
19
It differs from other known P450s by combining nitric
oxide, usually an inhibitor of heme cofactors, with molecular oxygen to generate a putative
ferric peroxynitrite intermediate which then disproportionates to nitrate L-tryptophan,
producing 4-nitro-L-tryptophan in the thaxtomin A pathway.
20
In previous work, we obtained high-resolution structures of TxtE and described a key
rearrangement of the B/C loop that anchors the substrate proximal to the heme, but we were
unable to determine whether such specific interactions extend to the disordered F/G loop.
21
Motivated by this knowledge gap and the unique chemistry of TxtE together with its
potential application as a nitration biocatalyst, we set out to study the role of the F/G loop in
the recognition and positioning of the L-tryptophan substrate. We turned to computational
methods to reconstruct the unresolved loop region (residues 176–183) and characterize its
functional dynamics on a biologically relevant timescale through GPU-accelerated
simulations and modern statistical analysis approaches.
In silico
methods of this type are
particularly powerful when combined with laboratory techniques to overcome limitations in
the experimental data. The sparse structural information on P450 F/G loops, and that of
TxtE in particular, makes TxtE engineering a natural application of this paradigm. Our
discovery process combined these large-scale simulations with mutagenesis and X-ray
crystallography to identify a single position in the F/G loop that interacts directly with the
substrate to control regioselectivity not only in TxtE, but also across other nitrating P450s.
Results and Discussion
To better understand the functional significance of the F/G loop, it was necessary to map out
the dynamics of TxtE by sampling timescales 200–2000x longer than previous reports on
cytochrome P450 enzymes,
22
-
35
i.e. a time scale relevant to these motions and in a format
that allows for their repeated observation. To achieve this, we performed Markov state model
(MSM)
36
molecular dynamics (MD) simulations on the 100 μs timescale. The substrate co-
crystal structure of TxtE (PDB ID: 4TPO)
21
served as the structural basis for the simulations
after homology-based reconstruction and equilibration of the missing F/G loop (Fig. 1a,
Supplementary Fig. 1). We carried out several rounds of adaptive sampling
37
and monitored
Dodani et al.
Page 2
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
the progress through hidden Markov models (HMMs).
38
Based on what is known about the
structural plasticity in P450s
8
, we chose to define the conformational space of TxtE in terms
of the F/G and B/C loop motions. Clustering of the dataset revealed that the F/G loop obeys
bimodal dynamics that do not appear to be coupled with the motions of the B/C loop. The
F/G loop of TxtE transitions between an unstructured set of open-lid ensembles similar to
the MD starting structures that feature a solvent-accessible active site and a narrow set of
closed-lid conformations that cut off the active site from bulk water (Fig. 1b). We achieved
convergence within the conformational space of the F/G and B/C loops and observed on the
order of 100 transitions between the open- and closed-lid conformations. Throughout, the
active site was represented as the substrate-bound ferric peroxynitrite intermediate, which
allowed us to investigate the catalytic step just prior to nitration (Supplementary Fig. S2).
While in line with the general picture of P450 F/G loop dynamics, our simulation data
afforded details that had been inaccessible using structural methods alone. The closed-lid
state is characterized by a tightly packed active site in which a small number of water
molecules occupy discrete positions, as previously reported.
39
The active site is sealed off
from bulk solvent by a network of hydrogen bonds that involves Tyr89 of the B’
2
/C loop,
Tyr175 of the F/G loop, the amino acid moiety of the substrate, and a water molecule (Fig.
2a). Strikingly, His176 of the F/G loop is in direct contact with the substrate through a
tightly packed edge-to-face interaction (Supplementary Fig. S3). In contrast, none of the
open-lid structural ensembles shows Tyr89 and Tyr175 in contact with one another, and
His176 points away from the active site (Fig. 2b). The set of open-lid TxtE conformations is
instead characterized by an influx of disordered water molecules into the active site. While
the open-lid F/G loop is necessary for substrate binding and product release, the exclusion of
solvent is necessary for a catalytically productive active site. Its hydration instead frequently
results in the separation of the substrate and peroxynitrite-heme by layers of water
molecules, which allows the substrate to rearrange and adopt a variety of loosely-bound
poses at the opening of the active site (Supplementary Fig. S4).
While the amino acid moiety of the substrate is bound to the protein in a manner consistent
with the co-crystal structure,
21
the substrate indole ring can occupy the active site pocket in
two distinct orientations. One shows the indole-NH in contact with the heme-bound
peroxynitrite oxygen (Supplementary Fig. S5) and resembles the orientation evident in the
co-crystal structure (‘unflipped’). The other has the indole ring rotated by ~180° (‘flipped’),
with its C4 closest to the nitrogen atom of the heme-bound peroxynitrite (Supplementary
Fig. S5). The flipped orientation accounts for the overwhelming majority of observed
closed-lid states (78%, Table 1). However, substrate poses with a flipped indole ring are the
exception in the open-lid state and account for a mere 7%. Instead, the predominant
substrate orientation is that of the unflipped indole ring (81%), with the remaining 12%
accounting for substrate positions that are loosely bound and distant from the heme center.
Decomposition of these two macroscopic states, open- and closed-lid, into their underlying
micro-states allowed us to further subdivide and discretize the free energy landscape. Rather
than being defined by a large-scale conformational change, the geometric differences
between these micro-states are on the order of side chain reorganizations and lend
themselves to a more comprehensive analysis of the closed-to-open dynamics. We
Dodani et al.
Page 3
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
determined the connectivity between these sub-states and employed transition path theory
(TPT)
40
to determine the flux between states and to map out the kinetic rates of transitions
across the network (Fig. 2c). This statistical framework allowed us to outline the steps
separating the closed- from the open-lid conformations and to identify the interactions that
gate their transitions. In particular, we characterized a single intermediate state central to all
observed closed-to-open transitions in our dataset (Fig. 2d). Unsurprisingly, this state shares
attributes of both the open- and the closed-lid conformations, featuring an interrupted
Tyr175-Tyr89 contact, a new His176-Tyr89 interaction, and a partial opening of the F/G
loop. This result suggests a transition whereby His176 substitutes for Tyr175, producing a
significantly weakened interaction between the F/G loop and Tyr89.
Based on these observations, we hypothesized that mutations at His176 could shift the loop
equilibrium to the catalytically competent closed state when substrate is bound, with the
goal of improving binding and thereby enhancing catalytic efficiency, particularly on non-
natural substrates. We selected two relatively conservative mutations, intended either to
provide a stronger contact to Tyr89 as a preemptive measure against lid opening (His176Tyr)
or raise the free energy barrier of the closed-to-open transition through increased steric
demand (His176Phe), which were tested with L-tryptophan. Consistent with our hypothesis,
the binding affinity of the His176Phe and His176Tyr variants for L-tryptophan increased 15-
and 8-fold, respectively, compared with the wild-type enzyme (Table 1). However, instead of
the anticipated 4-nitro-L-tryptophan, we detected a new product at a shorter retention time
but with a mass spectrum that nevertheless corresponds to a nitrated tryptophan (Fig. 3,
Supplementary Fig. S8–S10). Derivatives of nitrotryptophan have characteristic spectral
signatures that can be used to determine the connectivity of the nitro group to the tryptophan
core.
41
-
43
The wavelength of maximum absorption for the new nitro product (
λ
max
= 330
nm) was consistent with that reported for 5-nitro-L-tryptophan (
λ
max
= 330 nm)
41
and was
further confirmed by HPLC co-injection with an authentic standard (Fig. 3, Supplementary
Fig. S9–S10, Supplementary Fig. S31–S32). No other naturally occurring or engineered
enzymes have been reported to exclusively nitrate L-tryptophan at the C5 position.
Motivated by these results, we performed site-saturation mutagenesis at His176 to explore
the effect any of the remaining amino acid substitutions might have on regioselectivity.
While most mutations significantly reduced or completely abolished activity, several variants
generated detectable levels of nitrated product(s). Mutation from His176 to asparagine,
glycine, serine, cysteine, or methionine resulted in a mixture of 4- and 5-nitro-L-tryptophan
(Supplementary Fig. S12–S16), while substitution of His176 with phenylalanine, tyrosine,
and tryptophan all resulted in nitration exclusively at the C5-position.
To better understand how the substitutions at residue 176 give rise to improved binding of L-
tryptophan and a complete shift in regioselectivity we characterized the His176Phe/Tyr/Trp
variants with MD and the His176Phe/Tyr variants with X-ray crystallography. Compared
with the nearly 50:50 ratio of closed vs. open states observed in the wild-type TxtE
simulations, the computational data of the His176Phe/Tyr/Trp mutants showed a substantial
increase in their relative closed state populations with ratios around 90:10 (Table 1). HMM-
state decomposition and TPT mapping reveal significant kinetic and thermodynamic effects
for each of the C5-selective variants. The larger aromatic side chains of these amino acids
Dodani et al.
Page 4
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
allow for tighter packing of the closed-lid F/G loop and provide thermodynamic stabilization
compared to the open-lid conformation. The added bulk further increases the contact surface
of the F/G loop with neighboring residues, particularly Tyr89, during the closed-to-open
transition, leading to an increased kinetic barrier and slowed conformational exchange.
Thermodynamic stabilization of the closed-lid conformation and slowed closed-to-open
transitions are consistent with the tighter binding constants and lower total turnover numbers
(TTNs) determined for the C5-selective variants relative to wild-type TxtE (Table 1). As
with His176 in the wild-type closed-lid conformation, the MD simulations show the side
chains of the residues at position 176 in the C5-selective variants engaged in edge-to-face
interactions with the substrate indole moiety (Fig. 4b–d) and the substrate adopting one of
two orientations: a minor conformation in which the indole nitrogen faces the iron center
and a major one in which it is flipped. In the former orientation, the substrate alignment does
not change relative to the wild-type enzyme.
21
The slowed closed-to open transitions also
limit the partial substrate release necessary for the flipping of the indole moiety
(Supplementary Fig. S4). Further analysis of the substrate populations suggests that the
Phe/Tyr/Trp variants favor the catalytically relevant flipped orientation of the substrate
indole moiety in the closed-lid conformation to a lesser extent than wild-type TxtE (Table
1). Interestingly, in the flipped scenario, the increased steric demand of Phe/Tyr/Trp at
position 176 shifts the substrate alignment relative to the heme co-factor, placing the indole
C5 closest to the nitrogen of the ferric peroxynitrite (Fig. 4b–d, 5). In contrast, the wild-type
simulations show the C4 position closest to the peroxynitrite, corresponding with the
observed regioselectivity (Fig. 5).
We also obtained high-resolution X-ray crystal structures of the His176Phe and His176Tyr
variants bound to L-tryptophan. In contrast to the wild-type TxtE, both of these structures
show fully-resolved closed-lid conformation F/G loops, lending support to our hypothesis
that these mutations stabilize the closed-lid conformation. The conformation of the F/G loop
in these structures shows remarkable agreement with that predicted by our reconstruction
and simulation procedures, and both Phe176 and Tyr176 are shown to interact directly with
the co-crystallized substrate as predicted (Fig. 6). Other than the F/G loop, no significant
structural deviation was observed from the previous co-crystal structure.
21
As with the wild-
type structure, the co-crystallized tryptophan was found to occupy only the unflipped
orientation, most likely due to inherent differences between the crystalline and solution state,
as well as the absence of the ferric peroxynitrite intermediate included in our simulations.
Given the demonstrated control over regioselectivity of the residue at position 176 in the
S.
scabies
TxtE enzyme, we investigated the natural diversity at this position in related
enzymes. Seven unique homologs of TxtE were identified using the BLAST search
algorithm (sequence identity > 65%), and multiple sequence alignment revealed a natural
subpopulation with a tryptophan (but not phenylalanine or tyrosine) at the position
corresponding to His176 in four annotated P450s of unknown function from:
Streptomyces
virginiae
(Uniprot ID K9MYX3),
Streptomyces sp. Mg1
(B4VER3),
Streptomyces
lavendulae
(I2FGE0), and
Saccharomonospora marina
XMU15 (H5X7S6) (Fig. 7a,
Supplementary Fig. S50–S51). All of the other residues that were identified to bind L-
tryptophan in the wild-type structure (PDB ID: 4TPO) are conserved in these P450s,
suggesting that they would also accept L-tryptophan as a substrate (Supplementary Fig.
Dodani et al.
Page 5
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
S50). We synthesized the genes for these enzymes and expressed them in
E. coli
. LC-MS
analysis and HPLC co-injection demonstrated that, as with the C5-selective His176Trp TxtE
variant, all four of the homologous enzymes with tryptophan at the amino acid
corresponding to His176 exclusively nitrate L-tryptophan at the C5 position (Fig. 7b and c,
Supplementary Fig. S17–S20, Supplementary Fig. S34–S37). It is interesting to note that no
natural products have yet been identified with a 5-nitro-L-tryptophan core and that this
nitrated building block may be a component of undiscovered natural products. For a point of
comparison to
S. scabies
TxtE_His176Trp, we further characterized the most stable and
highly expressed homolog from
S. virginiae
(Virginiae_TxtE). Virginiae_TxtE produces
exclusively 5-nitro-L-tryptophan with a higher turnover and lower affinity than the
corresponding His176Trp mutant of
S. scabies
TxtE (Table 1). Upon mutation of
Virginiae_TxtE Trp176 to His176, the nitration activity is significantly attenuated, but the
product distribution shifts to favor C4 nitration (Supplementary Fig. S21). These data
demonstrate that the relationship between residue 176 and regioselective nitration of
tryptophan is preserved across the range of homologous enzymes despite the subtlety of the
rearrangement that gives rise to it and the modest overall sequence identity (67–77%).
Conclusions
In order to create a catalytically productive microenvironment, P450s undergo open-to-
closed transitions that involve large, dynamic motions of the F/G loop and adjoining F and G
helices.
8
,
11
,
12
Through a collaborative approach between experiment and simulation, we
have identified a single F/G loop residue in the nitrating P450 TxtE that not only plays a key
role in gating these transitions, but also acts to control regioselectivity and can be used as a
predictor of regioselectivity in homologous nitrating P450s. This approach also showcases
how a thorough understanding of structure-function relationships, as obtained through
protein engineering efforts, can be a catalyst for the discovery of previously uncharacterized
enzymes.
44
Taken together, our results demonstrate that TxtE largely controls regioselectivity through
precise positioning of the substrate with respect to the ferric peroxynitrite intermediate. TxtE
chemistry stands in contrast to peroxidase or globin catalyzed tryptophan nitration, wherein
5-nitrotryptophan is typically observed as the minor product or not at all.
41
-
43
,
45
-
48
It has
been suggested that the 5-nitrotryptophan regioisomer is produced only in the presence of an
electrophilic nitronium ion.
42
,
48
Notably, the production of 5-nitrotryptophan by the
His176Phe/Tyr/Trp mutants and the TxtE homologs having tryptophan at the corresponding
position makes an electrophilic substitution mechanism a possibility, though a caged radical
mechanism could also give rise to the observed behavior.
20
The mechanism of TxtE-
catalyzed nitration, how TxtE has evolved from its P450 counterparts, and what natural
products contain 5-nitro-L-tryptophan are still outstanding questions.
Our study showcases a rare example of how a single residue in the cytochrome P450 F/G
loop can interact directly with the substrate to contribute to active site organization.
14
-
16
We
speculate that direct interactions between the F/G loop and the substrate could exist, albeit
transiently, in other P450s as well. A more rigorous and extensive understanding of the
dynamics and stabilization of flexible loops in P450s and other enzymes could be useful
Dodani et al.
Page 6
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
towards the engineering of new substrate contacts to alter active site chemistry or even
engineer novel enzymatic function.
49
,
50
Methods
Computational details
The high-resolution TxtE co-crystal structure with PDB accession ID 4TPO served as the
structural template. Missing residues of the TxtE F/G loop were reconstructed with
Schrödinger Inc.'s structure prediction tool Prime. The heme-iron(III)-peroxynitrite complex
involved in the TxtE catalyzed aromatic nitration cycle was used to model the active form of
TxtE in the presence of L-Trp substrate. Force field parameters were generated with the
antechamber module of AMBER12, and production MD simulations were carried out
utilizing the GPU-accelerated pmemd.cuda code. One hundred independent production runs
were performed for 0.5 microseconds each, utilizing the GPU nodes of NCSA's Blue Waters
supercomputer. Mixtape v.0.2.2 (zenodo.org/record/12638#.Vjtef7Rx1N0), which is now
part of MSMBuilder 3 (msmbuilder.org), was used to construct a 10 state hidden Markov
model (HMMs) of this dataset. 100 new trajectories (10 from each state) were seeded from
this state decomposition and were run for 0.2 microseconds each. This equal-weighted
adaptive sampling process was performed a total of three times, resulting in an aggregate
sampling of 110 microseconds. The final dataset was used to generate a 9 state HMM and its
connectivity was examined with transition path theory (TPT). A more detailed description of
the computational protocol is available as part of the
Supplementary Information
.
Purified enzyme reactions
A solution of purified enzyme (150 μL, 2.5 μM in 25 mM Tris pH 8) was aliquoted in
quadruplicate into a 96-well plate (Evergreen Scientific). A solution (50 μL) containing the
following was added to each well: L-tryptophan (5 μL, 20 mM in 25 mM Tris pH 8, 500 μM
final concentration), spinach ferredoxin NADP
+
reductase (2 μL, 17 U/mL in 1 M Tris pH 8,
0.17 U/mL final concentration), spinach ferredoxin (2 μL, 1 mg/mL in Tris pH 8, 0.01
mg/mL final concentration), nicotinamide adenine dinucleotide phosphate (NADPH,
Codexis, Inc.) (2 μL, 89 mM in 25 mM Tris pH 8, 890 μM final concentration), and
diethylamine NONOate sodium salt hydrate (DEANO, 1 μL, 100 mM in 10 mM sodium
hydroxide) in 38 μL of 25 mM Tris pH 8. The plate was covered, wrapped in foil, and the
reactions proceeded overnight with shaking. To each well, a solution of the internal standard
p
-toluenesulfonamide (25 μL, 4 mM stock in 0.15 M HCl with 0.4% DMSO, 444 μM final
concentration) was added with shaking for 5 min. Each reaction (200 μL) was applied to a
0.5-mL 3-kDA MWCO centrifugal filter (Millipore) and centrifuged at 20,817g for 1 h at
room temperature. The filtrate was transferred to a 96-well assay plate (Agilent
Technologies) and analyzed by HPLC for total turnover numbers or transferred to a vial
insert (Agilent Technologies) and analyzed by liquid chromatography mass spectrometry
(LC-MS). The wavelength of maximum absorption for the new nitro product (
λ
max
= 330
nm) was consistent with that reported for 5-nitro-L-tryptophan (
λ
max
= 330 nm)
41
and 1-
nitro-
N
-
α
-acetyl-L-tryptophan amide (
λ
max
= 332 nm).
42
,
43
Since the latter compound is
known to be unstable in acidic conditions, treatment of the enzyme-generated products with
Dodani et al.
Page 7
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
hydrochloric acid (10 μL, 6M) allowed us to distinguish between 1- and 5-nitro-L-
tryptophan.
42
,
43
Dissociation constants
A solution of purified enzyme (100 μL, 4.3–5 μM) in 25 mM Tris buffer (pH 8) was
aliquoted in a half area 96 well plate (Greiner Bio One) and L-tryptophan prepared in 25
mM Tris (pH 8) was titrated at varying concentrations. For TxtE_His176Phe and
TxtE_His176Trp the following final L-tryptophan concentrations were used: 0, 1, 3, 6, 12,
24, 47, 94, 189, 377, 566, 755, and 1132 μM. For TxtE_His176Tyr the following final L-
tryptophan concentrations were used: 0, 3, 6, 12, 24, 47, 94, 189, 377, 566, 755, and 1132
μM. For Virginiae_TxtE the following final L-tryptophan concentrations were used: 0, 24,
47, 189, 377, 566, 755, and 1132 μM. The volume change (6 μL) was the same for each
well. After at least 10 min of shaking, spectra were recorded from 350 to 500 nm with a 5-
nm step size on a plate reader (Infinite M200, Tecan) at least in triplicate. For each substrate
concentration, differential UV-visible spectra were determined by subtracting the enzyme
alone control spectrum from the enzyme with substrate. The difference in the absorbance of
each spectrum at the
λ
max
and
λ
min
was calculated in at least 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 as follows ΔΔA = (ΔΔA
max
[L-tryptophan]) /
(
K
d
+ [L-tryptophan]) (Supplementary Fig. S49).
We note that we are characterizing the recombinant TxtE outside its native context and
without its native reductase or nitric oxide source. Instead a surrogate spinach reductase
system and an artificial nitric oxide donor with a short half-life are used as previously
reported.
20
Under these reaction conditions, TxtE activity is low and measurements of
k
cat
,
K
M
, and specific activity are not possible. Instead, as is common for cytochrome P450s, we
have reported total turnover numbers and dissociation constants, which are useful for
assessing relative activities.
Additional materials and procedures are described in the
Supplementary Information.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Dr. Jens Kaiser and Pavle Nikolovski 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. This work was funded by the Gordon and Betty Moore Foundation through grant
GBMF2809 to the Caltech Programmable Molecular Technology Initiative (to F.H.A). S.C.D is supported by Ruth
L. Kirschstein NRSA postdoctoral fellowship from the National Institutes of Health (5F32GM106618). G.K.
acknowledges support from the Lawrence Scholars Program, the NIH Simbios Program (U54 GM072970), and the
Center for Molecular Analysis and Design (Stanford). J.K.B.C. acknowledges the support of the Resnick
Sustainability Institute (Caltech). 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. Sabine Brinkmann-Chen, Dr. Todd K. Hyster, Dr. John A. McIntosh, Dr. Christopher K. Prier,
Robert T. McGibbon, and Mohammad M. Sultan for helpful discussions. This research is part of the Blue Waters
sustained-petascale computing project, which is supported by the National Science Foundation (awards
OCI-0725070 and ACI-1238993) and the state of Illinois. Correspondence and material requests should be
Dodani et al.
Page 8
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
addressed to F.H.A. for experimental work and V.S.P. for computational work. 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.
References
1. Meunier B, de Visser SP, Shaik S. Mechanism of oxidation reactions catalyzed by cytochrome P450
enzymes. Chem. Rev. 2004; 104:3947–3980. [PubMed: 15352783]
2. Shaik, S.; de Visser, SP. Cytochrome P450: Structure, Mechanism, and Biochemistry. Ortiz de
Montellano, PR., editor. Vol. 2. Plenum; New York: 2005. p. 45-85.
3. Whitehouse CJ, Bell SG, Wong LL. P450(BM3) (CYP102A1): connecting the dots. Chem. Soc.
Rev. 2012; 41:1218–1260. [PubMed: 22008827]
4. Podust LM, Sherman DH. Diversity of P450 enzymes in the biosynthesis of natural products. Nat.
Prod. Rep. 2012; 29:1251–1266. [PubMed: 22820933]
5. Li S, et al. Substrate recognition by the multifunctional cytochrome P450 MycG in mycinamicin
hydroxylation and epoxidation reactions. J. Biol. Chem. 2012; 287:37880–37890. [PubMed:
22952225]
6. Denisov, IG.; Sligar, SG. Cytochrome P450: Structure, Mechanism, and Biochemistry. Ortiz de
Montellano, PR., editor. Springer; Cham: 2015. p. 69-109.Ch. 3
7. Ortiz de Montellano, PR. Cytochrome P450 Structure, Mechanism, and Biochemistry. Ortiz de
Montellano, PR., editor. Springer; Cham: 2015. p. 111-176.Ch. 4
8. Pochapsky TC, Kazanis S, Dang M. Conformational plasticity and structure/function relationships
in cytochromes P450. Antiox. Redox Signal. 2010; 13:1273–1296.
9. Poulos, TL. Fifty years of Cytochrome P450 Research. Yamazaki, H., editor. Springer; 2014. p.
75-94.
10. Poulos, TL. Fifty years of Cytochrome P450 Research. Yamazaki, H., editor. Springer; Tokyo:
2014. p. 75-94.Ch. 4
11. Hasemann CA, Kurumbali RG, Boddupalli SS, Peterson JA, Diesenhofer J. Structure and function
of cytochromes P450: a comparitive analysis of three crystal structures. Structure. 1995; 2:41–62.
[PubMed: 7743131]
12. Poulos TL. Cytochrome P450 flexibility. Proc. Natl. Acad. Sci. U.S.A. 2003; 100:13121–13122.
[PubMed: 14597705]
13. Fasan R, Chen MM, Crook NC, Arnold FH. Engineered alkane-hydroxylating cytochrome
P450(BM3) exhibiting nativelike catalytic properties. Angew. Chem. 2007; 46:8414–8418.
[PubMed: 17886313]
14. Yano JK, et al. Crystal structure of a thermophilic cytochrome P450 from the archaeon Sulfolobus
solfataricus. J. Biol. Chem. 2000; 275:31086–31092. [PubMed: 10859321]
15. Kells PM, Ouellet H, Santos-Aberturas J, Aparicio JF, Podust LM. Structure of cytochrome P450
PimD suggests epoxidation of the polyene macrolide pimaricin occurs via a hydroperoxoferric
intermediate. Chem. Biol. 2010; 17:841–851. [PubMed: 20797613]
16. Zhang H, et al. Structural analysis of HmtT and HmtN involved in the tailoring steps of himastatin
biosynthesis. FEBS Lett. 2013; 587:1675–1680. [PubMed: 23611984]
17. Buddha MR, Tao T, Parry RJ, Crane BR. Regioselective nitration of tryptophan by a complex
between bacterial nitric-oxide synthase and tryptophanyl-tRNA synthetase. J. Biol. Chem. 2004;
279:49567–49570. [PubMed: 15466862]
18. Winkler R, Hertweck C. Biosynthesis of nitro compounds. Chembiochem. 2007; 8:973–977.
[PubMed: 17477464]
19. Ju KS, Parales RE. Nitroaromatic compounds, from synthesis to biodegradation. Microbiol. Mol.
Bio. Rev. 2010; 74:250–272. [PubMed: 20508249]
20. Barry SM, et al. Cytochrome P450-catalyzed L-tryptophan nitration in thaxtomin phytotoxin
biosynthesis. Nat. Chem. Biol. 2012; 8:814–816. [PubMed: 22941045]
21. Dodani SC, et al. Structural, functional, and spectroscopic characterization of the substrate scope
of the novel nitrating cytochrome P450 TxtE. Chembiochem. 2014; 15:2259–2267. [PubMed:
25182183]
Dodani et al.
Page 9
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
22. Zhang H, Kenaan C, Hamdane D, Hoa GH, Hollenberg PF. Effect of conformational dynamics on
substrate recognition and specificity as probed by the introduction of a de novo disulfide bond into
cytochrome P450 2B1. J. Biol. Chem. 2009; 284:25678–25686. [PubMed: 19605359]
23. Sano E, et al. Mechanism of the decrease in catalytic activity of human cytochrome P450 2C9
polymorphic variants investigated by computational analysis. J Comput. Chem. 2010; 31:2746–
2758. [PubMed: 20839301]
24. Hendrychova T, et al. Flexibility of human cytochrome P450 enzymes: molecular dynamics and
spectroscopy reveal important function-related variations. Biochim. Biophys. Acta. 2011;
1814:58–68. [PubMed: 20656072]
25. Hendrychova T, Berka K, Navratilova V, Anzenbacher P, Otyepka M. Dynamics and hydration of
the active sites of mammalian cytochromes P450 probed by molecular dynamics simulations. Curr.
Drug Metab. 2012; 13:177–189. [PubMed: 22208532]
26. Miao Y, et al. Coupled flexibility change in cytochrome P450cam substrate binding determined by
neutron scattering, NMR, and molecular dynamics simulation. Biophys. J. 2012; 103:2167–2176.
[PubMed: 23200050]
27. Pulawski W, et al. Low-temperature molecular dynamics simulations of horse heart cytochrome c
and comparison with inelastic neutron scattering data. Eur. Biophys. J. 2013; 42:291–300.
[PubMed: 23224355]
28. Cong S, Ma XT, Li YX, Wang JF. Structural basis for the mutation-induced dysfunction of human
CYP2J2: a computational study. J. Chem. Inf. Model. 2013; 53:1350–1357. [PubMed: 23647230]
29. Cui YL, et al. Molecular dynamic investigations of the mutational effects on structural
characteristics and tunnel geometry in CYP17A1. J. Chem. Inf. Model. 2013; 53:3308–3317.
[PubMed: 24205838]
30. Kobayashi K, et al. Evaluation of influence of single nucleotide polymorphisms in cytochrome
P450 2B6 on substrate recognition using computational docking and molecular dynamics
simulation. PLoS One. 2014; 9:e96789. [PubMed: 24796891]
31. Hollingsworth SA, Poulos TL. Molecular dynamics of the P450cam-Pdx complex reveals complex
stability and novel interface contacts. Protein Sci. 2015; 24:49–57. [PubMed: 25307478]
32. Fan JR, Zheng QC, Cui YL, Li WK, Zhang HX. Investigation of ligand selectivity in CYP3A7 by
molecular dynamics simulations. J. Biomol. Struct. Dyn. 2015:1–8.
33. Roccatano D. Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from
computer simulations studies. J. Phys. Condens. Matter. 2015; 27:273102. [PubMed: 26061496]
34. Narayan AR, et al. Enzymatic hydroxylation of an unactivated methylene C-H bond guided by
molecular dynamics simulations. Nat. Chem. 2015; 7:653–660. [PubMed: 26201742]
35. Cui YL, et al. Structural features and dynamic investigations of the membrane-bound cytochrome
P450 17A1. Biochim. Biophys. Acta. 2015; 1848:2013–2021. [PubMed: 26025587]
36. Noé, F.; Prinz, JH. An Introduction to Markov State Models and Their Application to Long
Timescale Molecular Simulation Vol. 797 Advances In Experimental Medicine and Biology.
Pande, Vijay S.; Bowman, Gregory R.; Noé, Frank, editors. Springer; Dordrecht: 2014. p.
75-90.Ch. 6
37. Bowman GR, Ensign DL, Pande VS. Enhanced modeling via network theory: Adaptive sampling
of Markov state models. J. Chem. Theory Comput. 2010; 6:787–794. [PubMed: 23626502]
38. McGibbon RT, Ramsundar B, Sultan MM, Kiss G, Pande VS. Understanding protein dynamics
with L1-regularzied reversible hiddne markov models. arXiv. 2014
39. Yu F, et al. Structural insights into the mechanism for recognizing substrate of the cytochrome
P450 enzyme TxtE. PLoS One. 2013; 8:e81526. [PubMed: 24282603]
40. Vanden-Eijnden, E. Computer Simulations in Condensed Matter Systems: From Materials to
Chemical Biology Vol. 703 Lecture Notes in Physics. Ferrario, M.; Ciccotti, G.; Binder, K.,
editors. Springer; Verlag: 2006. p. 453-493.
41. Herold S, Shivashankar K, Mehl M. Myoglobin scavenges peroxynitrite without being significantly
nitrated. Biochemistry. 2002; 41:13460–13472. [PubMed: 12416992]
42. Sala A, Nicolis S, Roncone R, Casella L, Monzani E. Peroxidase catalyzed nitration of tryptophan
derivatives. Mechanism, products and comparison with chemical nitrating agents. Eur. J. Biochem.
2004; 271:2841–2852. [PubMed: 15206949]
Dodani et al.
Page 10
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
43. Suzuki T, et al. Nitration and nitrosation of N-acetyl-L-tryptophan and tryptophan residues in
proteins by various reactive nitrogen species. Free Radical Biol. Med. 2004; 37:671–681.
[PubMed: 15288124]
44. Brinkmann-Chen S, Cahn JK, Arnold FH. Uncovering rare NADH-preferring ketol-acid
reductoisomerases. Metab. Eng. 2014; 26c:17–22. [PubMed: 25172159]
45. Padmaja S, Ramazenian MS, Bounds PL, Koppenol WH. Reaction of peroxynitrite with L-
tryptophan. Redox Rep. 1996; 2:173–177.
46. Herold S. Nitrotyrosine, dityrosine, and nitrotryptophan formation from metmyoglobin, hydrogen
peroxide, and nitrite. Free Radical Biol. Med. 2004; 36:565–579. [PubMed: 14980701]
47. Roncone R, Barbieri M, Monzani E, Casella L. Reactive nitrogen species generated by heme
proteins: mechanism of formation and targets. Coord. Chem. Rev. 2006; 250:1286–1293.
48. Nuriel T, Hansler A, Gross SS. Protein nitrotryptophan: formation, significance and identification.
J. Proteomics. 2011; 74:2300–2312. [PubMed: 21679780]
49. Tokuriki N, Tawfik DS. Protein dynamism and evolvability. Science. 2009; 324:203–207.
[PubMed: 19359577]
50. Nestl BM, Hauer B. Engineering of flexible loops in enzymes. ACS Catal. 2014; 4:3201–3211.
Dodani et al.
Page 11
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 1. Dynamic range of the TxtE F/G loop
a
, The missing density between residues 175 and 184 of the wild-type crystal structure (PDB
ID: 4TPO) indicates a disordered F/G loop (left panel), which was rebuilt through homology
modeling (right panel).
b
, Subsequent large-scale MD simulations show that the F/G loop
transitions between a set of disordered open-lid (left panel) and a set structured closed-lid
conformations in which the structurally unresolved His176 engages in a direct interaction
with the substrate
L
-Trp (right panel). Hydrogen atoms are omitted for the sake of clarity.
The substrate and heme are in black stick rendition and the F/G loop is shown in orange. A
PyMOL session file of (b) is available as part of the
Supplementary Information
.
Dodani et al.
Page 12
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 2. Productive and non-productive active site arrangements are separated by a single
conformational transition state and depend on whether the F/G loop is closed or open
a
, In the closed-lid state, the F/G loop (orange) promotes a set of productive and tightly-
packed active site conformations that support a small number of enclosed structural water
molecules (W1–W3). These form a hydrogen-bond network with the substrate's amino acid
moiety whereas the hydrophobic substrate indole ring is ‘de-wetted’ and rests proximal to
the ferric peroxynitrite-species.
b
, The open-lid state enables substrate binding and product
release. It also renders the active site accessible to bulk solvent in the presence of substrate,
which can interfere with its productive alignment in the active site.
c
, The productive (C1–
C4) and non-productive (O1–O4) active site states are connected by a single conformational
transition state (TS). The
Supplementary Information
contains additional detail on the
diagram.
d
, Analysis of the TS reveals that the Gly58-Tyr175-Tyr89 contacts in
(a)
are
replaced by a set of His176-Tyr89 interactions, which identifies His176 as a mutational
target. The substrate and heme are in black and represent the geometric mean of the
corresponding state. Water molecules (red spheres) were drawn from 20 random structures
across the corresponding state.
Dodani et al.
Page 13
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 3. Discovery of a nitration regioselectivity switch at His176
a
, Schematic of the regiospecific nitration catalyzed by wild-type TxtE and the
TxtE_His176Phe/Tyr/Trp variants.
b
, LC-MS chromatograms of the nitrated products
synthesized by wild-type TxtE and the TxtE_His176Phe/Tyr/Trp variants.
c
, Diode array
detector (DAD) UV-visible absorption spectra to detect the 5-nitrotryptophan (5NT)
standard and the nitrated products produced by the TxtE_His176Phe/Tyr/Trp variants.
Abbreviation: 4-nitrotryptophan (4NT).
Dodani et al.
Page 14
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 4. MD simulations show the 5-selective variants in similar edge-to-face interactions with
the
L
-Trp substrate indole-moiety than what is observed with wild-type TxtE
Compared to the wild-type His176
(a)
, the increased steric demand of Phe176
(b)
, Tyr176
(c)
, and Trp176
(d)
causes the substrate indole moiety to adopt a shifted binding orientation
that is packed more tightly against the back of the active site, which further determines the
orientation of the heme-Fe-bound peroxynitrite (
Fig. 5
). The substrate indole responds first
by adopting a retreated conformation (compare
(a)
with
(b)
) and then by assuming an
increasingly parallel orientation relative to the plane of the heme cofactor (compare
(b)
with
(c)
and
(d)
). The F/G loop is shown in orange.
Dodani et al.
Page 15
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 5. Mutation of His176 to Phe fine-tunes substrate-to-peroxynitrite alignment and active
site water network
TxtE wild-type simulations predominantly show the C4-carbon of
L
-Trp proximal to the
peroxynitrite-nitrogen, indicated by yellow distance marker
(a)
, while simulations of the
His176Phe mutant predominantly show the C5-carbon closest to the peroxynitrite-N
(b)
.
The two active sites support a distinct set of structured water molecules (highlighted by
yellow circles).
Dodani et al.
Page 16
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 6. Crystal structures of His176Phe/Tyr variants have a resolved closed-lid F/G loop that
aligns with the predicted closed-lid MD geometries
a
, His176Phe (crystal structure in white and orange, MD geometries in blue and green).
b
,
His176Tyr (crystal structure in white and orange, MD geometries in blue and green). The
crystal structures in (a) and (b) (white) show the F/G loop (orange) in the closed-lid state and
further show an active site arrangement that supports the predicted closed-lid MD
geometries - for both, the flipped (blue) and unflipped (green) indole-group ensembles. The
range of dynamics is indicated through the overlaid hair-lines, corresponding to 20 structures
randomly drawn from each of the MD states.
Dodani et al.
Page 17
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript
Figure 7. Identification of naturally-occurring TxtE homologs that catalyze the production of 5-
nitrotryptophan
a
, Sequence sub-alignment of the F/G loops of TxtE and seven homologs (sequence identity:
67–77%) reveals natural occurrence of Trp176 in several homologs. Colors in the sequence
logo refer to the hydrophobic (black), basic (blue), acidic (red), polar (green), and other
(purple) residues.
b
, LC-MS chromatograms of the nitrated products synthesized by the
Trp176-containing TxtE homologs.
c
, DAD UV-visible absorption spectra to detect the 5-
nitrotryptophan (5NT) standard and the nitrated products produced by the Trp176-containing
TxtE homologs from
S. virginiae
,
S. sp. Mg1
,
S. lavendulae
, and
S. marina
XMU15.
Dodani et al.
Page 18
Nat Chem
. Author manuscript; available in PMC 2016 September 21.
Author Manuscript
Author Manuscript
Author Manuscript
Author Manuscript