Binding of Distinct Substrate Conformations Enables Hydroxylation of Remote Sites in Thaxtomin D by Cytochrome P450 TxtC

Article abstract of DOI:10.1021/jacs.8b08864

Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation, and natural product biosynthesis. Here we report biochemical, structural, and theoretical studies of TxtC, an unusual bifunctional CYP involved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites within the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the α-carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionalization, X-ray crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density corresponding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-intermediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen complex into the reactive ferryl intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the ?€-face of the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second, energetically similar but conformationally distinct binding mode in which the α-hydrogen of the Phe residue is positioned over the heme prosthetic group. Molecular dynamics simulations confirmed that the α-hydrogen is sufficiently close to the ferryl oxygen atom to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and monohydroxylated intermediate.

Full text of DOI:10.1021/jacs.8b08864

Subscriber access provided by Gothenburg University Library
Article
Binding of distinct substrate conformations enables hydroxylation
of remote sites in thaxtomin D by cytochrome P450 TxtC
Lona Mary Alkhalaf, Sarah Barry, Dean Rea, Angelo Gallo, Daniel
Griffiths, Jozef Lewandowski, Vilmos Fülöp, and Gregory L. Challis
J. Am. Chem. Soc., Just Accepted Manuscript • Publication Date (Web): 05 Dec 2018
Downloaded from http://pubs.acs.org on December 5, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 8
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
Binding of distinct substrate conformations enables hydroxylation
of remote sites in thaxtomin D by cytochrome P450 TxtC
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
§‡
§†‡
¥
§
§Ψ
Lona M. Alkhalaf , Sarah M. Barry , Dean Rea , Angelo Gallo , Daniel Griffiths , Józef R.
§
¥
§§#*
Lewandowski , Vilmos Fulop and Gregory L. Challis
§
Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK; ¥ School of Life Sciences, University of
§
Warwick, Coventry, CV4 7AL, UK; Warwick Integrative Synthetic Biology Centre, University of Warwick, Coventry, CV4
7
#
AL, UK; Department of Biochemistry and Molecular Biology, Monash University, Clayton, VIC 3800, Australia
ABSTRACT: Cytochromes P450 (CYPs) catalyze various oxidative transformations in drug metabolism, xenobiotic degradation
and natural product biosynthesis. Here we report biochemical, structural and theoretical studies of TxtC, an unusual bifunctional CYP
involved in the biosynthesis of the EPA-approved herbicide thaxtomin A. TxtC was shown to hydroxylate two remote sites within
the Phe residue of its diketopiperazine substrate thaxtomin D. The reactions follow a preferred order, with hydroxylation of the -
carbon preceding functionalization of the phenyl group. To illuminate the molecular basis for remote site functionalization, X-ray
crystal structures of TxtC in complex with the substrate and monohydroxylated intermediate were determined. Electron density
corresponding to a diatomic molecule (probably dioxygen) was sandwiched between the heme iron atom and Thr237 in the TxtC-
intermediate structure, providing insight into the mechanism for conversion of the ferrous-dioxygen complex into the reactive ferryl
intermediate. The substrate and monohydroxylated intermediate adopted similar conformations in the active site, with the -face of
the phenyl group positioned over the heme iron atom. Docking simulations reproduced this observation and identified a second,
energetically similar but conformationally-distinct binding mode in which the -hydrogen of the Phe residue is positioned over the
heme prosthetic group. Molecular dynamics simulations confirmed that the -hydrogen is sufficiently close to the ferryl oxygen atom
to be extracted by it and indicated that the two substrate conformations cannot readily interconvert in the active site. These results
indicate that TxtC is able to hydroxylate two spatially remote sites by binding distinct conformations of the substrate and
monohydroxylated intermediate.
Introduction
herbicide (registration number: 84059-12), although it has yet
to reach the market. Five genes within a mobile pathogenicity
island are responsible for thaxtomin biosynthesis in S. scabies
Cytochromes P450 (CYPs) are versatile monoxygenases that
employ a heme prosthetic group ligated to a conserved active
site Cys residue and an electron transfer protein partner to
11
8
7.22 (Figure 2). The txtA and txtB genes encode a dimodular
1
IV
nonribosomal peptide synthetase (NRPS) that assembles
activate molecular oxygen. The resulting ferryl (Fe =O)
reactive intermediate effects a wide range of oxidative
transformations. These enzymes frequently play key roles in the
biosynthesis of microbial natural products, including the
assembly of non-proteinogenic amino acid precursors, and the
catalysis of diverse on and post-assembly line tailoring
thaxtomin D 2 from L-Phe, L-4-nitrotryptophan and two units of
1
3
S-adenosyl-L-methionine (SAM) (Figure 2). The txtE gene
encodes an unusual CYP that catalyzes regiospecific nitration
2
of L-Trp at the 4-position of the indole using O and NO, which
is generated from L-Arg by the nitric oxide synthase encoded
3
, 14
2
-6
by txtD.
Gene deletion experiments have implicated txtC,
reactions. A handful of CYPs, all of which tailor the products
of polyketide synthase assembly lines, have been shown to be
which is also predicted to encode a CYP, in the conversion of
15
7
thaxtomin D 2 to thaxtomin A 1. However, direct evidence for
multifunctional . These enzymes catalyze the sequential
hydroxylation of both the Phe -carbon and the phenyl group
in thaxtomin D 2 by TxtC is lacking. Moreover, catalysis of
both these reactions by a single CYP
oxidation of two or three proximal carbon centers and in most
cases come from Actinobacteria (Figure 1), although fungal
examples are also known. Structural studies of two such
enzymes have indicated that only small changes in the
positioning of the substrate relative to the heme prosthetic group
are required to achieve the successive oxidation reactions,
because the atoms undergoing functionalization are positioned
O
O
NH
O
O
O
O
OH
O
O
O2N
O
HO
O
Aureothin
8
-10
adjacent to each other.
Tirandamycin
N
O
Several plant-pathogenic
Actinobacteria,
including
O
HO
O
Streptomyces acidiscabies, Streptomyces turgidscabies and
Streptomyces scabies, produce thaxtomins, a family of
phytotoxic diketopiperazines that inhibit cellulose biosynthesis
OH
O
O
O
O
HO
O
O
O
O
1
1
O
O
in expanding plant tissues. S. scabies causes common scab in
OH
OH
1
2
O
potatoes and other economically important crops, and
thaxtomin A 1 has been approved by the EPA for use as an
HO
FD-891
Mycinamycin
Figure 1: Structures of actinobacterial polyketides assembled
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 2 of 8
by multifunctional CYPs. The oxygen atoms introduced by the
CYP in each case are highlighted in red.
MS analysis showed that the molecular formula of the higher
molecular weight compound corresponds to that for thaxtomin
A 1, whereas the lower molecular weight compound has the
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
could be considered somewhat remarkable, given that the
resulting hydroxyl groups are separated by almost 7 Å in the X-
1
same molecular formula as thaxtomin B 3 (Figure S4). H NMR
1
6
spectroscopic comparisons of the purified products with
authentic standards of thaxtomins A and B isolated from S.
acidiscabies 84.104 confirmed these structural assignments
ray crystal structure of thaxtomin A 1.
Here we report the overproduction in Escherichia coli and
purification of TxtC, and show that it catalyzes sequential
hydroxylation of the Phe -carbon and the phenyl group in
thaxtomin D 2. X-ray crystal structures of TxtC in complex with
thaxtomin D 2 and thaxtomin B 3 (the monohydroxylated
intermediate), combined with docking and molecular dynamics
(see supporting information). In addition to thaxtomin A 1,
small amounts of two additional products with molecular
weights 32 Da higher than the substrate were observed in the
TxtC-catalyzed reaction. These compounds could not be
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
purified in sufficient quantities to permit
H NMR
simulations,
suggest
that
the
enzyme
catalyzes
spectroscopic analysis. However, ESI-Q-TOF-MS showed that
they have the same molecular formulae as thaxtomin A 1 (see
supporting information). These are likely isomers of thaxtomin
A 1, in which the aromatic hydroxyl susbtituent is situated ortho
or para to the methylene group, because such compounds are
known to be biosynthesized in small amounts by thaxtomin-
producing Streptomyces species. To substantiate this
hypothesis, we purified an authentic standard of the o-isomer
from S. acidiscabies 84.104, confirmed its identity by HRMS
functionalization of these two spatially remote carbon centers
by binding distinct conformational states of its two substrates.
txtDtxtE
txtA
txtB
txtC
OH
L-Arg
TxtD O2, e-
OH
NH2
O
O
O
N
N
TxtA, TxtB
L-Phe
NH2
NO
O
O2N
1
TxtE, O2,
Fd, FdR,
NADPH
ATP
and H NMR spectroscopy, and showed that its retention time
O2N
NH Mg2+, SAM
NH
is identical to one of the minor dihydroxylated products of the
enzymatic reaction (Figure S4). By a process of elimination, we
assigned the other dihydroxylated product of the enzymatic
reaction as the p-isomer. All four hydroxylated products were
absent from control reactions in which the enzyme was
inactivated by boiling, or from which the ferredoxin, ferredoxin
reductase or NADPH were omitted (Figure S5).
NH
2
OH
OH
O
N
N
O
N
N
TxtC
O2
TxtC
O2
O
O
O2N
O2N
Fd, FdR,
NADPH
Fd, FdR,
NADPH
OH
NH
NH
3
1
To investigate whether the TxtC-catalyzed hydroxylation
reactions follow a defined order, the disappearance of the
substrate and the formation of mono- and dihydroxylated
products was monitored over a 60 minute period (Figure 3).
Within 5 minutes, most of the substrate is consumed and
thaxtomin B 3 (along with a small amount of another
monohydroxylated species), thaxtomin A 1 and its o-isomer are
formed. Over the next
Figure 2: Organization of the mobile pathogenicity island in S.
scabies 87.22 responsible for thaxtomin biosynthesis
(biosynthetic genes are shaded blue) and proposed pathway for
assembly of thaxtomin A 1 (Fd = ferredoxin, FdR = Fd
reductase).
Results and discussion
TxtC from Streptomyces acidiscabies 84.104 has previously
been overproduced in E. coli, but UV-Vis spectroscopic
analysis of the purified protein indicated that it exists in the
1
5
catalytically inactive P420 form. We cloned txtC from S.
scabies 87.22 and overexpressed it in E. coli as an N-terminal
His
purified from cell lysate using Ni-NTA chromatography
Figure S1). The identity of the purified protein was confirmed
6
-fusion. The resulting protein was soluble and was readily
(
by peptide mass fingerprinting and gel filtration
chromatography indicated that it is monomeric (Figure S2).
Absorbance maxima at 419 and 450 nm were observed for the
ferric and ferrous-CO forms of the protein, respectively,
confirming that this CYP is produced in a catalytically active
form (Figure S3).
A mutant of S. acidiscabies 84.104 in which txtC has been
disrupted accumulates thaxtomin D 2, suggesting that this
1
5
metabolite could be a substrate for TxtC. To test this
hypothesis, we incubated purified recombinant TxtC with
thaxtomin D 2, isolated from the txtC mutant, NADPH, and
spinach ferredoxin/ferredoxin reductase (required to transfer
electrons form NADPH to the heme prosthetic group of CYPs).
LC-MS analysis of the reaction mixture identified two major
products, giving rise to ions with m/z values 16 and 32 Da
higher than the substrate. Semi-preparative HPLC purification
of these products from a scaled-up reaction and ESI-Q-TOF-
Figure 3: Extracted ion chromatograms (EICs) from LC-MS
analyses of TxtC-catalyzed hydroxylation of thaxtomin D 2
showing that thaxtomin B 3 is an intermediate in its conversion
to thaxtomin A 1. Top: EIC at m/z = 429 corresponding to
+
[
M+Na] for 2. Bottom: EIC at m/z = 445 and 461
+
corresponding to [M+Na] for 1 and 3, respectively. The p- and
ACS Paragon Plus Environment

Page 3 of 8
Journal of the American Chemical Society
o-isomers of 1 have retention times of ~10 and ~14 minutes,
suggesting that the unliganded enzyme has a high degree of
conformational flexibility. The overall structure of TxtC is very
similar to that of other well-characterized bacterial CYPs, such
as P450cam (Figure 4). Both complexes were found to adopt
an essentially identical closed conformation in which the
substrates are sequestered from solvent.
1
2
3
4
5
6
7
8
9
respectively. The compound with a retention time of ~12
minutes that forms transiently in small quantities during the
course of the reaction has an m/z corresponding to an alternative
monohydroxylated intermediate.
1
7
In the structure of the TxtC-thaxtomin B complex, the 4-
nitroindole moiety of the substrate is embedded in a pocket
lined by the side chains of predominantly hydrophobic residues
(Leu74, Pro82, Val84, Phe182, Leu228, Met229 and Val232).
On one side of this pocket, the carboxylate side chain of Glu69
forms a hydrogen bond with the N-H group of the indole (Figure
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
A). Two further polar contacts between the substrate and the
enzyme are mediated by water molecules. One of these forms
hydrogen bonds with the side chain of Thr385, and the carbonyl
group of the Phe residue in the substrate (Figure 5A). The other
sits in close proximity to the carbonyl group of 4-
nitrotryptophan and one of the carboxylate groups of the active
site heme (Figure 5A). Together these two water molecules
anchor the diketopiperazine of thaxtomin B 3 in the active site,
positioning the -face of the phenyl group in this
monohydroxylated intermediate over the heme prosthetic group
(Figures 5A and S10). Electron density corresponding to a
diatomic molecule was also found in close proximity to the
heme iron atom with 100% occupancy (Figure 5A). This is
likely dioxygen, which binds to the active site as a result of
heme photoreduction during X-ray data collection, as reported
previously for P450cam.
molecule (relative to the heme) hydrogen bonds to the side
chain of Thr237, a highly conserved residue in CYPs that has
1
8, 19
The distal end of this diatomic
Figure 4: Overall structure of the TxtC-thaxtomin D 2 complex
highlighting the location of the heme prosthetic group (in
green). The -helix and -sheet secondary structure elements
are labeled according to standard CYP nomenclature.
2
0, 21
been shown to be important for catalysis.
A further water
molecule sits within H-bonding distance of the Thr237
hydroxyl group, the distal end of the diatomic molecule, and the
water molecule that interacts with the Thr385 side chain (Figure
55 minutes, the amount of thaxtomin B 3 in the mixture steadily
decreases and a concomitant increase in the quantities of
thaxtomin A 1 and its o/p-isomers is observed. These data imply
that thaxtomin B 3 is the main intermediate in the TxtC-
catalyzed dihydroxylation of thaxtomin D 2. Incubation of
5
A).
The phenyl group of the substrate is positioned close to the
proximal end of the heme-associated diatomic molecule in the
TxtC-thaxtomin B 3 structure (Figure 5A). To investigate
whether this binding mode permits hydroxylation of the phenyl
group, we replaced the heme and its associated diatomic
molecule with compound I (heme oxyferryl complex) and
carried out molecular dynamics (MD) simulations. The results
of these simulations show that all five unsubstituted carbon
atoms of the phenyl group are able to approach the ferryl
oxygen to within ~3.2 Å (Table 1), explaining why a mixture of
o-, m- and p-hydroxylated products is formed. The average and
minimum distances of the unsubstituted carbon atoms from the
ferryl oxygen are smallest for the o-positions and largest for the
p-positions (Table 1), although the differences are small and
may not be relevant to the observed preference for m-
hydroxylation. Nevertheless, it is possible that thaxtomin A 1
results only partially from direct attack of the ferryl oxygen at
the m-position (Figure 6). The intermediate resulting from
attack of the ferryl oxygen at the o-position could undergo 1,3-
elimination to form an arene oxide that rearranges via an “NIH
shift” to form the m-product (Figure 6). While the involvement
of arene oxides in CYP-catalyzed aromatic hydroxylation
thaxtomin
B
3
with TxtC, NADPH and spinach
ferredoxin/ferrodoxin reductase resulted in the formation of
thaxtomin A 1 as well as small quantities of its o/p-isomers,
consistent with this hypothesis (Figure S4).
Taken together our data unambiguously show that TxtC
catalyzes the final two steps in thaxtomin A 1 biosynthesis.
They further suggest that these reactions occur in a preferred
order, with hydroxylation of the Phe -carbon to give thaxtomin
B 3 occurring first, followed by functionalization of the phenyl
group to give thaxtomin A 1, along with small quantities of its
o and p-isomers. The fact that thaxtomin B 3, but no isomers in
which the aromatic ring of the Phe residue is hydroxylated
instead of its -carbon are produced by thaxtomin-producing
Streptomyces species further supports this conclusion.
To develop an understanding of a how a single CYP is able to
catalyze the successive hydroxylation of two spatially remote
sites in a single molecule, TxtC was co-crystallized with its
substrate thaxtomin
D
2
and the monohydroxylated
intermediate thaxtomin B 3. The X-ray structures of these
complexes were solved to 2.8 and 1.7 Å, respectively, by
molecular replacement using MoxA from Nonomuraea
recticatnina (PDB ID: 2Z36) as a template. Attempts to
crystallize TxtC in the absence of substrate were unsuccessful,
2
2
reactions has been questioned on theoretical grounds, such a
species has recently been implicated as an intermediate in the
P450cam-catalysed oxidation of t-butyl-benzene.
2
3
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 8
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5: (A) Active site in the TxtC-thaxtomin B 3 complex (PDB ID: 6F0C), highlighting key hydrogen bonding interactions
between the substrate, protein and active site water molecules as dashed lines (top) and showing the electron density for the substrate,
heme prosthetic group, diatomic molecule and three ordered water molecules (bottom). (B) Active site in the TxtC-thaxtomin D (2)
complex (PDB ID: 6F0B) highlighting hydrogen bonds as dashed lines (top) and showing the electron density for the substrate, heme
prosthetic group, and single ordered water molecule (bottom). Both 2F
ligand atoms omitted from the density calculations.
0 c
-F electron density maps were contoured at 1 with the bound
Table 1: Minimum, maximum and average distances (Å)
between the ferryl oxygen and the unsubstituted carbon atoms
of the thaxtomin B 3 phenyl group observed in MD simulations.
of the heme-associated diatomic molecule to the carboxylate
side chain of Asp236 (Figure 5A) and are thus proposed to
function as a proton relay (Figure 7). A similar mechanism, in
which the corresponding aspartate residue (Asp251) and a
single active site water molecule participate in proton delivery
during the rate limiting step of O-O bond cleavage, has been
position
o/o'
minimum
2.81/2.83
3.19/3.13
3.23
maximum
4.58/4.91
5.35/5.95
5.94
average
3.61/3.75
4.28/4.64
4.67
2
4
m/m'
p
proposed for P450cam. Consistent with this hypothesis, a
D251N mutant was reported to be much less active than the wild
2
4
type enzyme. More recently, the binding of the electron donor
putidaredoxin to P450cam has been shown to break a salt bridge
between Lys178/Arg186 and Asp251, allowing the side chain
of the latter to pick up a proton from the bulk solvent and deliver
The structure of the TxtC-thaxtomin B complex also suggests a
mechanism for the transfer of protons from bulk solvent to the
ferrous dioxygen complex via Asp236, which forms an
interface between the active site and a water filled exterior
pocket (Figure 7). Two water molecules connect the distal end
2
5, 26
it to the active site.
This indicates that reduction of the
ferrous-dioxygen intermediate and protonation of the resulting
ACS Paragon Plus Environment

Page 5 of 8
Journal of the American Chemical Society
ferric peroxide are tightly coupled. TxtC contains an analogous
phenyl group of the substrate is positioned above the heme iron
atom, accurately recapitulating the experimentally-observed
substrate binding orientation in the TxtC-thaxtomin D structure
(Figure S12). In another state, arising from rotation about the β-
γ bond of the 4-nitrotryptophan residue, the diketopiperazine is
flipped such that the phenyl group is pointing towards the roof
of the active site (Figures 8 and S12). This positions the -
hydrogen atom of the Phe residue in close proximity to the heme
iron atom and it was found to adopt a near attack conformation
in 6% of the frames in MD simulations (Figures 8 and Table
S9). No interconversion between the different substrate binding
orientations was observed in the MD simulations. Close
examination of the substrate binding site in TxtC indicates that
a steric clash between the side chain of the Phe residue in the
substrate and the heme / residues 231-236 in the I helix prevents
this. The switch in conformation of the monohydroxylated
intermediate that enables hydroxylation of its phenyl group thus
appears to require release from the active site of TxtC.
1
2
3
4
5
6
7
8
9
salt bridge
Fe
O
Fe
O
O
OH
3
1
Fe
O
Fe
O
O
O
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
Figure 6: Possible mechanisms for TxtC-catalyzed conversion
of thaxtomin B 3 to thaxtomin A 1.
between Arg380 and Asp236, suggesting that it may employ a
similar mechanism.
Surprisingly, the substrate in the TxtC-thaxtomin D structure is
bound in a very similar conformation to the monohydroxylated
intermediate in the TxtC-thaxtomin B structure (Figures 5B and
S10). In this conformation, the -hydrogen atom of the Phe
residue points away from the heme and appears to be too far
away from the iron center to be extracted by the ferryl
intermediate. Indeed, we could find no frames in MD
simulations of this structure in which this hydrogen atom adopts
a near attack conformation (a distance of >3.5 Å between the
hydrogen atom and the ferryl oxygen and a C-H-O bond angle
of 180 ± 45º; see Table S9). Moreover, no electron density
corresponding to either the diatomic molecule, or the two water
molecules that connect its distal end to Asp236 and Thr385 is
observed in the TxtC-thaxtomin D structure (Figure 5B). As a
consequence, the distance between the substrate and the I-helix
Conclusion
Our work demonstrates that TxtC is an unusual CYP capable of
oxidizing two spatially remote sites in thaxtomin D 2. These
hydroxylation reactions proceed in a preferred order with
functionalization of the Phe -carbon preceding oxidation of
the phenyl group. X-ray crystallographic analysis of TxtC with
the mono-hydroxylated intermediate thaxtomin B 3 bound in its
active site positions the -face of the phenyl group in this
substrate over the iron atom of the heme prosthetic group, such
that hydroxylation can occur. MD simulations of the TxtC-
compound I-thaxtomin B complex confirmed that all five of the
unsubstituted carbon atoms in the phenyl group are able to
approach the ferryl oxygen atom sufficiently close to react. It
appears that the predominant m-substituted product of the
aromatic hydroxylation reaction may be formed by a
combination of direct attack of the ferryl oxygen at the m-
position and attack at the o-position, followed by rearrangement
to the m-product via an arene oxide intermediate. The TxtC-
thaxtomin B structure also revealed that a network of hydrogen
bonds connects a heme iron-bound diatomic molecule
2
7
(containing Asp236 and Thr237) decreases relative to the TxtC-
thaxtomin B structure (Figure S11). In contrast, the water
molecule sandwiched between the 4-nitrotryptophan carbonyl
and heme carboxylate groups adopts a very similar position in
both structures. The substrate binding mode observed in the
TxtC-thaxtomin D structure thus appears to disfavor formation
of a ferrous-dioxygen complex, and the substrate must bind in
a different conformation to permit Phe -carbon hydroxylation.
(probably dioxygen), via a pair of active site water molecules,
To investigate whether thaxtomin D 2 can bind to TxtC in
different conformational states, docking simulations were
to a conserved aspartate residue, which is proposed to shuttle
protons from the bulk solvent into the active site. This enables
protonation of the ferric-peroxide intermediate leading to
formation of the reactive ferryl species. Surprisingly, in the X-
ray crystal structure of the TxtC-thaxtomin D complex
thaxtomin D 2 was bound in the same orientation as thaxtomin
B 3. MD simulations confirmed that the -H of the Phe residue
cannot be extracted by the ferryl
2
8
conducted using SwissDock. The results of these simulations
predict that the active site of the enzyme can accommodate
three distinct conformational states of thaxtomin D, which have
predicted Gibbs free energies of binding within 0.2 kcal of each
other (Figure S12). In all three of these states, the 4-nitroindole
moiety is bound in the same position as that observed in the
TxtC-thaxtomin D structure. Moreover, in one of the states the
bulk
water
D236
bulk
water
D236
D236
D236
O
O
R380
NH2
O
O
O
bulk
water
O
O
H
O
O
O
O
O
D236
H
H2N
H
H
H
O
H
H
O
H
N
H
T385
T385
T385
O
H
T385
O
H
H
H
O
H
e
H
H
H
H
O
O
O
R380
H
H
H
H
H
H
O
H
O
H
O
O
O
O
O
H
H
O
H
O
FeIII
H
O
O
FeIII
O
FeIV
FeII
T237
T237
T237
T237
Figure 7. Proposed mechanism for transfer of protons from bulk solvent to the ferric peroxide intermediate in TxtC, via two active
site water molecules and Asp236. The grey arrows indicate that a 180° rotation of the C -C bond in Asp236 is required to deliver the
 
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 8
protons picked up from the bulk solvent into the active site. The juxtaposition of Asp236 and Arg380 between the substrate binding
pocket and a water-filled exterior pocket is illustrated on the left.
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
‡
These authors contributed equally.
ACKNOWLEDGMENT
This work was supported by grants from the UK BBSRC (Grant
Ref. BB/H006281/1). We thank Rose Loria (Cornell University)
for providing the txtC mutant and wild type S. acidiscabies. The
Bruker MaXis mass spectrometer used in this research was
obtained, through Birmingham Science City: Innovative Uses for
Advanced Materials in the Modern World (West Midlands Centre
for Advanced Materials Project 2), with support from Advantage
West Midlands (AWM) and part funded by the European Regional
Development Fund (ERDF). Crystallographic data were collected
on beam line I24 at the Diamond Light Source, UK and we
acknowledge the support of the beam line scientists.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
REFERENCES
1
) Denisov, I. J.; Makris,T. M.; Sligar, S. G.; Schlichting, I. Chem.
Rev. 2005, 105, 2253-2277.
) Podust, L. M.; Sherman D. H. Nat. Prod. Rep. 2012, 10, 1251-
1266.
3) Barry, S. M.; Kers, J. A.; Johnson, E. R.; Song, L.; Aston, P. R.;
Figure 8: Example of a near attack conformation observed in
MD simulations of TxtC containing the compound I complex
and thaxtomin D 2 bound in the “flipped” orientation with the
phenyl group pointing towards the roof of the active site. This
positions the -hydrogen atom of the Phe residue sufficiently
close to the ferryl oxygen to be extracted by it.
2
Patel, B.; Krasnoff, S. B.; Gibson, D. M.; Loria, R.; Challis, G. L. Nat
Chem Biol. 2012, 8, 814-816.
4
) Song, L.; Laureti L.; Corre, C.; Leblond, P.; Aigle, B.; Challis, G.
L; J. Antibiot. 2014, 67, 71-76.
5) Zabala, D.; Cartwright, J.W.; Roberts, D.M.; Law, B.J.C.; Song,
L.; Samborskyy M.; Leadlay, P.F.; Micklefield J.; Challis, G.L. J. Am.
Chem. Soc. 2016, 137, 4342-4345.
intermediate when thaxtomin D is bound in this orientation.
Docking simulations recapitulated this binding mode, but also
revealed an energetically similar yet conformationally-distinct
substrate binding orientation that places the Phe -hydrogen
atom close to heme iron atom. MD simulations confirmed that
this hydrogen atom can get close enough to the ferryl oxygen to
be abstracted by it, when the substrate is bound in this
orientation. Overall, our data indicate that the main reaction
path of TxtC involves hydroxylation of first the Phe -carbon
and then the phenyl group of thaxtomin D 2 by binding distinct
conformational states of the substrate and mono-hydroxylated
intermediate, contributing new insights into the mechanisms
employed by multifunctional CYPs for the functionalization of
spatially remote carbon atoms.
6
) Haslinger, K.; Peschke, M.; Brieke, C.; Maximowitch, E.; Cryle,
M. J. Nature, 2015, 521, 105-109.
) Cochrane, R. V. K.; Vederas, J. C. Acc. Chem. Res. 2014, 47,
7
3148–3161.
8) Li, S.; Tietz, D. R.; Rutaganira, F. U.; Kells, P. M.; Anzai, Y.;
Kato, F.; Pochapsky, T. C.; Sherman, D. H.; Podust, L. M. J. Biol.
Chem. 2012, 287, 37880−37890.
9
) Tietz, D. R.; Podust, L. M.; Sherman, D. H.; Pochapsky, T. C.
Biochemistry, 2017, 56, 2701-2714.
0) Zocher, G.; Richter, M. E. A.; Mueller, U.; Hertweck, C. J. Am.
Chem. Soc., 2011, 133, 2292−2302.
1) King, R. R..; Calhoun, L.A. Phytochemistry, 2009, 70, 833-841.
12) Lambert, D.H., Loria, R. Int. J. Syst. Bacteriol. 1989, 39, 387–
392.
1
1
ASSOCIATED CONTENT
13) Johnson, E. G.; Krasnoff, S. G.; Bignell, D. R. D.; Chung, W.
C.; Tao, T.; Parry, R. J.; Loria, R.; Gibson, D. M.; Mol. Microbiol.
Supporting Information: Experimental procedures, SDS-PAGE
analysis of His6-TxtC expression and purification, the results of the
enzyme activity assays, spectroscopic characterization data, and
details of the docking and MD simulations are included in the
supporting information. This material is available free of charge via
the Internet at http://pubs.acs.org. The coordinates of the TxtC-
thaxtomin D complex (Accession No. 6F0B) and the TxtC-
thaxtomin B complex (Accession No. 6F0C) have been deposited
in the Protein Data Bank.
2
009, 73, 409-418.
4) Kers, J.A.; Wach, M. J.; Krasnoff, S. B.; Widom, J.; Cameron,
1
K. D.; Bukkhalid, R. A.; Gibson, D. M.; Crane, B. R.; Loria, R. Nature,
2004, 429, 79-82
1
5) Healy, F. G.; Krasnoff, S. B.; Wach, M.; Gibson, D. M.; Loria,
R. J. Bacteriol. 2002, 184, 2019-2029.
16) Bourgault, J. P.; Maddirala, A. R.; Andreana, P. R. Org. Biomol.
Chem. 2014, 12, 8125-8127
17) Poulos, T.; Finzel, B.C.; Howard, A.J. J. Mol. Biol. 1987, 195,
87-700.
6
AUTHOR INFORMATION
Corresponding Author
18) Beitlich, T.; Kuhnell, K.; Schluze-Briese, C.; Shoeman, R.L.;
Schlichting, I., J. Synchrotron Rad. 2007, 14, 11–23.
19) Schlichting, I.; Berendzen, J.; Chu, K.; Stock, A.M.; Maves,
S.A.; Benson, D.E.; Sweet, R.M.; Ringe, D.; Petsko, G.A.; Sligar, S.G.
Science, 2000, 287, 1615-1622.
*
G.L.Challis@warwick.ac.uk
Present Addresses
20) Martinis, S. A.; Atkins, W. M.; Stayton, P.S.; Sligar, S. G. J. Am.
Chem. Soc. 1989, 30, 11420-11429.
†Department of Chemistry, Kings College London, Strand,
London, WC2R 2LS
Ψ Monash University, Clayton VIC 3800, Australia
2
1) Raag, R.; Martinis, S. A.; Sligar, S. G.; Poulos, T. L.
Biochemistry, 1991, 30, 11420-11429.
22) deVisser, S.P.; Shaik, S. J. Am. Chem. Soc. 2003, 125, 7413-
7
424.
Author Contributions
ACS Paragon Plus Environment

Page 7 of 8
Journal of the American Chemical Society
2
3) Stok, J. E.; Chow, S.; Kresenke, E. H.; Soto, C. F.; Matyas, C.;
Poirier, R. A.; Williams, C. M.; de Voss, J. J. Chem. Eur. J. 2016, 22,
408-4412.
27) Eichler, A.; Gricman, L.; Herter, S.; Kelly, P.; Turner, N.; Pleiss,
J.; Flitsch, S. ChemBioChem, 2015, 5, 426–432.
28) Grosdidier, A.; Zoete, V.; Michielin, O. Nucleic Acids Res.
2011, 39, 270-277.
1
2
3
4
5
6
7
8
9
4
2
2
2
4) Gerber, N.C.; Sligar, S.G., J. Biol. Chem., 1994, 269, 4260-4266.
5) Tripathi, S.; Li, H.; Poulos, T. Science, 2013, 340, 1227-1230.
6) Batabyal, D.; Richards, L.S.; Poulos, T.L. J. Am. Chem. Soc.
2017, 139, 13193-13199.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 8
Table of contents graphic
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
O
NO2
N
NO2
N
OH
NO2
N
OH
TxtC
TxtC
N
N
N
O2
O2
O
O
O
N
NADPH
N
NADPH
N
H
H
H
HO
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
ACS Paragon Plus Environment