Use of a Tyrosine Analogue to Modulate the Two Activities of a Nonheme Iron Enzyme OvoA in Ovothiol Biosynthesis, Cysteine Oxidation versus Oxidative C-S Bond Formation

Article abstract of DOI:10.1021/jacs.7b13628

Ovothiol is a histidine thiol derivative. The biosynthesis of ovothiol involves an extremely efficient trans-sulfuration strategy. The nonheme iron enzyme OvoA catalyzed oxidative coupling between cysteine and histidine is one of the key steps. Besides catalyzing the oxidative coupling between cysteine and histidine, OvoA also catalyzes the oxidation of cysteine to cysteine sulfinic acid (cysteine dioxygenase activity). Thus far, very little mechanistic information is available for OvoA-catalysis. In this report, we measured the kinetic isotope effect (KIE) in OvoA-catalysis using the isotopically sensitive branching method. In addition, by replacing an active site tyrosine (Tyr417) with 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid (MtTyr) through the amber suppressor mediated unnatural amino acid incorporation method, the two OvoA activities (oxidative coupling between cysteine and histidine, and cysteine dioxygenase activity) can be modulated. These results suggest that the two OvoA activities branch out from a common intermediate and that the active site tyrosine residue plays some key roles in controlling the partitioning between these two pathways.

Full text of DOI:10.1021/jacs.7b13628

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Use of a tyrosine analog to modulate the two activities of
a non-heme iron enzyme OvoA in ovothiol biosynthesis,
cysteine oxidation versus oxidative C-S bond formation
Li Chen, Nathchar Naowarojna, Heng Song, Shu Wang,
Jianyun Wang, Zixin Deng, Changming Zhao, and Pinghua Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13628 • Publication Date (Web): 15 Mar 2018
Downloaded from http://pubs.acs.org on March 16, 2018
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1
Li Chen, Nathchar Naowarojna, Heng Song, Shu Wang, Jiangyun Wang, Zixin Deng,
1,2
2
Changming Zhao, * Pinghua Liu *
1
Key Laboratory of Combinatory Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sci-
ences, Wuhan University, Hubei 430072, People’s Republic of China
2
Department of Chemistry, Boston University, Boston, Massachusetts 02215, United States
3
College of Chemistry and Molecular Sciences, Wuhan University, Hubei 430072, People’s Republic of China
4
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, People’s Republic of China
Supporting Information Placeholder
5
-9
has been an intriguing question for decades. Their
sulfur incorporation strategy differs from other reported
ABSTRACT: Ovothiol is a histidine thiol derivative. The
biosynthesis of ovothiol involves an extremely efficient
trans-sulfuration strategy. The non-heme iron enzyme
OvoA catalyzed oxidative coupling between cysteine
and histidine is one of the key steps. Besides catalyzing
the oxidative coupling between cysteine and histidine,
OvoA also catalyzes the oxidation of cysteine to cysteine
sulfinic acid (cysteine dioxygenase activity). Thus far,
very little mechanistic information is available for
OvoA-catalysis. In this report, we measured the kinetic
isotope effect (KIE) in OvoA-catalysis using the
isotopically sensitive branching method. In addition, by
replacing an active site tyrosine (Tyr417) with 2-amino-
3-(4-hydroxy-3-(methylthio) phenyl) propanoic acid
(MtTyr) through the amber suppressor mediated unnatu-
ral amino acid incorporation method, the two OvoA ac-
tivities (oxidative coupling between cysteine and
histidine, and cysteine dioxygenase activity) can be
modulated. These results suggest that the two OvoA ac-
tivities branch out from a common intermediate and that
the active site tyrosine residue plays some key roles in
controlling the partitioning between these two pathways.
3,10-17
natural product sulfur incorporation pathways.
Three ergothioneine biosynthetic pathways were
1
8
discovered in recent years (Scheme 1). Both the
mycobacterial and the fungal pathways are aerobic
biosynthetic pathways (Scheme 1A), in which the trans-
sulfuration is achieved by non-heme iron enzyme-
catalyzed oxidative C-S bond formation reactions (2 
1
9-20
3 conversion catalyzed by EgtB
conversion catalyzed by Egt1
or 2  4
2
1-23
) and PLP-lyase
2
4
catalyzed C-S cleavage (EgtE, Egt2-catalysis). In some
anaerobic microbes, a rhodanase-like EanB enzyme
incorporates sulfur into hercynine to produce
ergothioneine using an activated “S” from cysteine
2
5
desulfurase IscS (Scheme 1B).
The enzyme responsible for the first step of ovothiol
biosynthesis (OvoA) in Erwinia tasmaniensis has been
2
6
identified (Scheme 1C). Similar to the EgtB and Egt1
enzymes in the aerobic ergothioneine biosynthetic path-
ways (Scheme 1A), OvoA is a mononuclear non-heme
iron enzyme that catalyzes oxidative coupling between
cysteine and histidine. The OvoA enzyme differs from
Egt1 and EgtB-catalysis in both substrate selectivity and
product C-S bond formation regioselectivity (Scheme
.
INTRODUCTION
27-28
1A vs. 1C).
More interestingly, when wild-type
Ergothioneine (5) and ovothiol (8 – 10) are histidine
OvoA enzyme was characterized in vitro, besides the
oxidative coupling of cysteine and histidine to produce
the coupling product 7, cysteine sulfinic acid was also
detected as a side-product ( 10% of the product mix-
derivatives with a sulfur substitution at the -carbon or
the -carbon of the histidine imidazole side-chain, re-
spectively. Humans obtain ergothioneine from the diet
2
8
1
ture, Scheme 1C). This is the first discovery of such
through an ergothioneine-specific transporter, and
activity in enzymes other than cysteine dioxygenase
ergothioneine has been suggested to play many benefi-
2
9
2-3
(
CDO). The production of cysteine sulfinic acid is de-
cial roles in human health. Ovothiol is present in sea
pendent on the presence of histidine. In the absence of
histidine, cysteine is oxidized to cystine instead of cyste-
urchin eggs at millimolar concentrations and is assumed
to protect the egg content during oxidative envelope
2
8
4
ine sulfinic acid. It was thus proposed that the for-
maturation. Ergothioneine and ovothiol biosynthesis
mation of the oxidative coupling product 7 and cysteine
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sulfinic acid 11 might be two pathways branched out
analog MtTyr can modulate the partitioning between the
two OvoA-pathways: cysteine oxidation versus oxida-
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from a common intermediate in OvoA-catalysis. A simi-
lar idea has also been proposed for EgtB-catalysis in
ergothioneine biosynthesis based on the study on EgtB
3
3
tive C-S bond formation. EgtB mechanistic models in
literature imply that significant isotope effects might
exist and an active site tyrosine residue might play a key
20, 30
active site mutants.
Thus far, there has been no di-
2
0, 34
rect evidence supporting this mechanistic hypothesis. In
this work, by taking the advantage that the wild-type
OvoA produces two products simultaneously in a ratio
role.
Due to these reasons, we have also repeated the
KIE studies using OvoA MtTyr variant to test these hy-
potheses.
28
of  9:1, we use the isotopically sensitive branching
31-32
.
RESULTS
method (Figure 1) to test this hypothesis.
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0
Figure 1. Use of the isotopically sensitive branching
method in OvoA-mechanistic studies. P1 represents com-
pound 7 and P2 represents cysteine sulfinic acid 11 in this
study.
Use of the isotopically sensitive branching meth-
od in mechanistic studies. In cases where two compet-
ing pathways branch out from a common intermediate,
such as some P-450 oxygenase/oxidase-catalyzed reac-
tions, if the step immediately after the common interme-
diate in one pathway has kinetic isotope effect (KIE),
KIE can be measured using the isotopically sensitive
3
1-32
branching method.
Previous studies in both OvoA
and EgtB-catalysis led to the proposal that cysteine
sulfinic acid 11 and oxidative coupling product 7 are
produced by two pathways branched out from a common
2
0, 28, 30
intermediate.
As represented by a simple kinetic
SCHEME 1. A. Two aerobic ergothioneine biosynthetic
pathways in bacteria and fungi; B. The anaerobic
ergothioneine biosynthetic pathway; C. The proposed
ovothiol biosynthetic pathway. D. MtTyr used in this
study and the Cys-Tyr crosslink in cysteine dioxygenase.
model in Figure 1, from a common intermediate (ES*)
along the OvoA catalytic pathway, P1 is the oxidative
coupling product 7 resulting from oxidative C-S bond
formation branch, and P2 is cysteine sulfinic acid 11
produced from the sulfur oxidation branch. Because the
relative amounts of P1 and P2 are directly proportional
to the rate constants of the two steps at the branching
point (k and k in Equaton 1, Figure 1), the isotope ef-
In addition, in recent structural and biochemical
characterizations of EgtB-catalysis, Tyr377 was pro-
posed to be important for the oxidative C-S bond for-
3
5
fect on k (k /k ) can be measured by determining the
2
0, 30
3
3H 3D
mation.
Thus far, there is no OvoA crystal structure
ratio of the two products (P1 and P2) in two sets of reac-
tions: a reaction with unlabeled substrates and a reaction
with isotopically labeled substrates (Equation 2, Figure
information. We created a homology model of OvoA and
this model suggests that Tyr417 in OvoA is the EgtB
Tyr377 counterpart. By replacing Tyr417 in OvoA with
1).
2
-amino-3-(4-hydroxy-3-(methylthio) phenyl) propanoic
Measurement of the substrate deuterium KIE us-
acid (MtTyr, Scheme 1D) through the amber suppressor
mediated unnatural amino acid incorporation method,
we demonstrated that replacing Tyr417 with a tyrosine
ing the wild-type OvoA. To measure the KIE of the k3
step in the branch producing the oxidative coupling
product 7, two approaches were used, one with
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isotopically labeled histidine (Figure 2A) and the other
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by conducting the reaction in D O buffer (Figure 2B).
2
We first carried out the isotopically sensitive branching
2
experiment using [U- H ]-histidine as the substrate. Two
5
sets of parallel reactions were run under identical condi-
tions (Figure 2). In the first reaction, unlabeled cysteine
and histidine were used as substrates, while in the se-
13
2
cond reaction, [β- C]-cysteine and [U- H ]-histidine
5
were used. After the reactions were complete, the two
reaction mixtures were combined. Cysteine sulfinic acid
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11 and the coupling product 7 were then characterized
by mass spectrometry. We used mass spectrometry ion
intensities to quantify the relative amounts of the prod-
ucts (7 vs. 7b and 11 vs. 11b). KIE was calculated ac-
cording to Equation 2 in Figure 1. For KIE measure-
ments, two batches of protein were used and for each
batch of enzyme, two independent KIE measurements
were performed. Therefore, any KIE value reported in
this research is the average of at least four independent
experiments.
Shown in Figure 2 are the spectra from one set of
experiments. The m/z of 289.0612 (left panel of Figure
-
2
A) corresponds to coupling product 7 ([M-H] form)
produced from the first reaction, while m/z of 294.0896
(
(
[
left panel of Figure 2A) is the coupling product 7b
-
[M-H] form) produced from the second reaction using
1
3
2
β- C]-labeled cysteine and [U- H ]-histidine as the
5
substrates. The ratio of 7 and 7b ([P1 ]/[P1 ] = 0.88,
H
D
Figure 2A) reflects the relative amounts of coupling
products produced from these two reactions. Using a
similar approach, the ratio of the cysteine sulfinic acids
(11 and 11b) produced from these two reactions was
accurately measured ([P2 ]/[P2 ] = 0.87, Figure 2A
H
D
right panel). Based on the [P1 ]/[P1 ] and [P2 ]/[P2 ]
H
D
H
D
(
Figure 2), the KIE on k was calculated using Equation
3
2
(Figure 1). The average of four repeats was reported
as the KIE here, to be k /k 1.01  0.02. In addition to
H
D
Figure 2. Measurement of the KIE using the
mass spectrometric studies, the same set of reactions
2
isotopically sensitive branching method. A. Using [U- H ]-
5
1
3
were also characterized by C-NMR, which gave a con-
sistent conclusion as that obtained from mass spectrome-
try analysis (Figure S1). There are at least two possible
explanations for the close to unity KIE obtained in this
study. This result implies the lack of a substrate primary
histidine as the substrate. On the top are the conditions of
the two reactions and at the bottom are the mass spectra of
cysteine sulfinic acid 11 and the coupling product 7. B.
Solvent KIE measurement by running the reactions in H O
2
buffer and D O buffer, respectively. Any ion intensity pre-
2
deuterium KIE on the k step, suggesting that the C-H
sented here is the average height of twenty mass spec-
trometry scans.
3
bond cleavage at the histidine δ-position is not the step
at the branching point (k step in Figure 1). Alternatively,
3
substrate isotope labeling affects both pathways (k and
3
Measurement of the solvent deuterium KIE using
wild-type OvoA. We also examined whether there is
k5) at a comparable level, which will be discussed later.
solvent KIE on the k step. Two sets of reactions were
3
run in parallel (Figure 2B). In the first reaction, unla-
beled cysteine and histidine were used as the substrates
and the reaction was run in the H O buffer, while in the
2
13
second reaction, [β- C]-cysteine and unlabeled histidine
were used and the reaction was run in D O buffer. After
2
the two reactions were complete, the products were ana-
lyzed as described in Figure 2A. Based on the ratios of
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4
0-45
compounds 7 and 7c, and 11 and 11b (Figure 2B), the
ty.
Because wild-type OvoA has some level of cyste-
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solvent deuterium KIE on k was calculated using Equa-
tion 2 (Figure 1), to be 1.29  0.01. C-NMR analysis
ine dioxygenase activity, we decided to replace Y417 by
MtTyr (Scheme 1D), a mimic of the Tyr-Cys cross-link
in the cysteine dioxygenase active site. We first synthe-
sized MtTyr enzymatically using an engineered variant
3
13
also gave results consistent to those obtained from mass
spectrometry analysis (Figure S2). Recently, OvoA sol-
vent KIE studies have been conducted using steady state
kinetics and a small solvent KIE (1.2  0.1) was report-
3
3
of tyrosine phenol lyase (TPL). The identity of the re-
sulting MtTyr was further confirmed by HPLC isolation
1
13
35
and detailed characterization using H-NMR, C-NMR
ed.
and high-resolution mass spectrometry (Figure S8-10).
Characterization of the OvoA Y417F mutant. The
detected solvent KIE, although small, is consistent with
the idea that the formation of coupling product 7 and the
oxidation of cysteine to cysteine sulfinic acid 11 are two
branches of a common intermediate, with solvent ex-
Recently, we have developed a Methanococcus
0
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jannaschii
tyrosyl
amber
suppressor
tRNA
Tyr
(MjtRNA CUA)/tyrosyl-tRNA synthetase (MjTyrRS)
3
3
pair to specifically recognize MtTyr.
OvoA
Y417MtTyr protein was overexpressed based on this
system (Figure S11) and the identity was further veri-
fied by tandem mass spectrometry analysis (Figure S12).
The iron content of OvoA Y417MtTyr variant was de-
termined by the previously reported ferrozine assay used
changeable protons being part of the k step of the
3
branch leading to the formation of the coupling product
7
. Alternatively, the solvent may affect the two pathways
to slightly different degrees, leading to the small KIE
observed in this measurement. This observation imme-
diately raises the question as to what could be the factors
that govern the partitioning between the two pathways.
Thus far, there is no reported OvoA crystal structure,
even though the structure of Mycobacterium
thermoresistibile EgtB in ergothioneine biosynthesis
4
6-47
for the wild-type OvoA protein.
The purified OvoA
Y417MtTyr protein had 0.94  0.04 equivalent of Fe,
which is close to that of the wild-type OvoA, suggesting
that the incorporation of MtTyr does not significantly
disturb the metal-center.
20
(
Scheme 1A reaction) is available. The wild-type M.
The OvoA Y417MtTyr variant was then character-
ized by the three assays discussed earlier: oxygen con-
thermoresistibile EgtB does not have sulfur dioxygenase
activity. However, upon mutation of an active tyrosine
1
13
28
sumption, H-NMR, and C-NMR analysis. Using a
NeoFox oxygen electrode, we directly monitored the
oxygen consumption rate. The kinetic parameters deter-
(Y377) to phenylalanine, the oxidation of γ-Glu-Cys to
γ-Glu-Cysteine sulfinic acid becomes the dominant reac-
2
0, 30
tion,
very low level of oxidative C-S bond formation activity
 0.1% of the total activity). These results implied that
while the EgtB Y377F mutant does maintain a
mined for OvoA Y417MtTyr variant were: k 1.14 
cat
-1
0.02 s , K of cysteine 253  25 M and K of histidine
m
m
(
27
5
06  35 M. Compared with wild-type OvoA, the k
cat
an active site tyrosine residue might play a key role in
modulating the partitioning between these two activities:
oxidative coupling vs. cysteine dioxygenase activity.
of OvoA Y417MtTyr variant is 10-fold less (Figure
1
S13). In the H-NMR assay, the peak at 7.75 ppm (Fig-
ure S14) correlates to the formation of compound 7.
1
3
We generated an OvoA structural model using the I-
Interestingly, C-NMR results clearly indicated that
OvoA Y417MtTyr variant behaves differently from
wild-type OvoA (Figure 3A). In wild-type OvoA-
catalysis, less than 10% of the products is cysteine
sulfinic acid 11 (Figure S15). However, in OvoA
Y417MtTyr catalysis, up to 30% of the products is cys-
teine sulfinic acid (Figure 3A and Figure S15). By
changing Tyr417 to MtTyr, we can modulate the parti-
tioning between the two branches of OvoA-catalysis:
oxidative C-S bond formation and cysteine oxidation.
36
TASSER program (Figure S3). This analysis suggests
that Y417 in OvoA might be the EgtB Y377 counterpart.
To test this prediction, we generated the OvoA Y417F
mutant (Figure S4) and characterized it by the same
three assays reported in our earlier studies, oxygen con-
1
13
sumption, H-NMR and C-NMR analysis (Figure S5-
). Similar to the case of EgtB studies, there was barely
7
any coupling product formation in the OvoA Y417F re-
action. This means that for this mutant, it would be chal-
lenging to obtain an accurate measurement of the KIE of
the oxidative-coupling branch of Figure 1 by the
isotopically sensitive branching method.
In previous studies, it has been demonstrated that
OvoA has flexible substrate selectivity. In addition, the
product C-S bond formation regioselectivity can be
modulated by the methylation state of the histidine
Replacing Y417 in OvoA with 2-amino-3-(4-
hydroxy-3-(methylthio) phenyl) propanoic acid
2
7
amine group or the stereochemistry of the histidine
3
5
(MtTyr). To resolve the above issue, we decided to re-
stereocenter. When hercynine (2) or D-histidine re-
placed L-histidine (1) as the substrate, C-S bond for-
mation regioselectivity changes from the histidine side-
chain δ-position to -position. Since there is no OvoA
crystal structure information to date, we cannot make
concise conclusions on how the addition of methylthiol
group from MtTyr affects the OvoA active site (e.g.,
place Y417 with a tyrosine analog. In the cysteine
dioxygenase structure, one of the important feature3s7-i3s9
the presence of a Cys-Tyr crosslink (Scheme 1D).
Such cross-link is not essential for CDO activity since
the mutation of C93 to alanine or serine significantly
reduces its activity, but does not abolish the CDO activi-
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hydrogen bonding network, active site waters, or even
product 7 (left panel) and cysteine sulfinic acid 11 (right
panel) by running the reactions in H O buffer or D O buff-
er, respectively. Any ion intensity presented here is the
average height of twenty mass spectrometry scans.
1
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5
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7
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9
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positions of active site residues). However, the disturb-
ance is probably minimal since the reaction catalyzed by
OvoA Y417 MtTyr variant still produces 7 and 11 as the
products, and the C-S bond formation regioselectivity is
2
2
2
7, 35
unchanged.
Interestingly, the ratio between 7 and 11
Measurement of the solvent deuterium KIE using
OvoA Y417MtTyr variant. We also conducted solvent
deuterium KIE experiments using OvoA Y417MtTyr
variant by following the protocol outlined in Figure 2B.
The results are shown in Figure 3C. Based on the ratio
of compounds 7 and 7c (1.95), and 11 and 11b (0.93),
changes from 9:1 with the wild-type OvoA to 7:3 with
the OvoA Y417MtTyr variant. Because the amounts of
these two products (7 & 11) in OvoA Y417MtTyr reac-
tion are at comparable levels, we decided to further
characterize it by the isotopically sensitive branching
method.
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
the solvent deuterium KIE on k was calculated using
3
Measurement of the substrate deuterium KIE us-
ing OvoA Y417MtTyr variant. Similar to the wild-type
OvoA studies (Figure 2), unlabeled histidine and unla-
beled cysteine were used as substrates for one reaction,
Equation 2 (Figure 1), to be 2.09  0.02. Again, shown
in Figure 3 is one of four sets of data used to calculate
KIE. This solvent KIE provides another set of evidence
supporting the hypothesis that cysteine sulfinic acid (11)
and the oxidative coupling product (7) are produced by
two pathways branched out from a common intermedi-
2
13
and [U- H ]-histidine and [β- C]-cysteine were used as
5
substrates for the other reaction. Shown in Figure 3 are
the results from one set of experiments. The ratio of 7
and 7b ([P1 ]/[P1 ], Figure 3B) was 0.68, which re-
13
ate and that their ratio can be modulated. The C-NMR
assays were also consistent with product ratio changes
observed from mass spectrometry analysis (Figure S17).
In the reaction catalyzed by the OvoA Y417MtTyr vari-
ant, the ratio of 11 to 7 increases from 3:7 in the reaction
H
D
flects the amounts of cysteine sulfinic acid produced
from these two reactions. The ratio of 11 and 11b
(
[P2 ]/[P2 ], Figure 3B) was 0.63, which reflects the
H D
amounts of the coupling products formed from these two
conducted in H O buffer to approximately 1:1 when
2
reactions. From this ratio, KIE for k on the oxidative C-
conducted in D O buffer.
3
2
S bond formation branch was calculated using Equation
2
(Figure 1). The average of four repeats were reported
13
.
DISCUSSION
as the KIE here, to be 1.08  0.01. The C-NMR assay
results were also consistent with the mass spectrometry
results (Figure S16).
Mechanistic models. When Seebeck and co-
workers first reported their discovery of the OvoA en-
zyme, a few mechanistic options were proposed, similar
2
6
to those outlined in Scheme 2A. Later on, when it was
discovered that OvoA exhibits cysteine dioxygenase
2
8
activity, the question was raised immediately on which
one is the first-half reaction: Scheme 2A vs. Scheme 2B.
To form product 7, in Scheme 2A, oxidation of thiolate
to sulfenic acid occurs first, while in Scheme 2B, the
oxidative C-S bond formation is the first-half reaction.
Thus far, there is no reported mechanistic or structural
20
work on OvoA. However, EgtB structural information
and mechanistic investigations using density function
theory or quantum mechanics/molecular mechanics
3
4, 48
(QM/MM) method are both available.
OvoA and
EgtB/Egt1 differ in both substrate selectivity and prod-
1
9, 21, 26-28
uct C-S bond regioselectivity.
However, the
chemistries involved in these two biosynthetic pathways
are similar (Scheme 1). For this reason, information
from EgtB mechanistic studies serves as the guide when
we discuss OvoA mechanistic models.
In Scheme 2A, upon substrate binding, two ex-
-
Figure 3. KIE measurement using OvoA Y417MtTyr
changeable ligands (H O or OH ) are replaced by the
2
1
3
variant as the enzyme. A. C-NMR spectra of the reaction
mixture. B. Mass spectra of the coupling product 7 (left
panel) and cysteine sulfinic acid 11 (right panel) by running
the reactions with unlabeled substrates (histidine and cyste-
substrates. At the same time, a vacant site is created for
oxygen binding and activation. After oxygen is activated,
the Fe(III)-superoxo intermediate reacts with the thiolate
IV
to form a cysteine sulfenic acid and an Fe =O species
2
13
ine) or labeled substrates ([U- H ]-histidine and [β- C]-
5
(12, Scheme 2A). Several different ways might account
for the subsequent oxidative C-S bond formation (two
cysteine), respectively. C. Mass spectra of the coupling
ACS Paragon Plus Environment

Journal of the American Chemical Society
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3
5
electron chemistry vs. one-electron chemistry, pathways
kinetic analysis. In pathway II of Scheme 2A, oxida-
tion of imidazole goes through a step-wise process. Re-
cently, when fluoro-histidine was used in OvoA-
catalysis, it was recognized as an OvoA substrate and its
26
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
I, II and III in Scheme 2A). These models were modi-
fied based on Seebeck’s initially mechanistic proposal
29, 49-50
and EgtB structural information.
In pathway I, the
IV
35
Fe =O species abstracts a hydrogen atom from the
histidine imidazole ring -carbon to produce an imidaz-
ole-based radical (13). Subsequent recombination be-
tween this radical and a thiol radical will lead to the
formation of the oxidative coupling product 7. In path-
activity was comparable to that of histidine. Because
the introduction of a fluoro-substitute affects the
histidine reduction potential, the lack of obvious effects
on OvoA activity suggests that OvoA does not follow
this mechanistic model or this step is not the rate-
IV
35
way II, the Fe =O species oxidizes the imidazole ring to
limiting step if this mechanistic model is followed.
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
produce an imidazole cation radical (14), followed by C-
S bond formation through a radical recombination. Re-
cent computational work based on density function theo-
ry by Wei et al. suggests that Y377 participates in EgtB-
catalysis by deprotonating the hercynine -position, and
Y417 might function as an acid to provide the proton in
the 12  14 conversion step, while serves as a base in
the 15  7 conversion. Recent calculation studies from
Wei et al. based on density function theory led to a pro-
posal close to the pathway II model (Scheme 2A). In the
EgtB-model proposed by Wei et al., they predicted that
the deprotonation step (e.g., 15  7) is the rate-limiting
step and a substrate deuterium KIE as high as 5.7 was
48
this step is the rate-limiting step in EgtB-catalysis.
Therefore, in pathway II mechanistic model (Scheme
2A), Y417 may function as a Lewis acid/base (15  7
48
conversion, Scheme 2A). In pathway III, the reaction
follows a two-electron chemistry. Nucleophilic attack
by the imidazole ring on the sulfenic acid functional
group leads to C-S bond formation (12  15). Subse-
quent deprotonation of the imidazole ring (15) by a base
from the protein active site produces the final coupling
product (7).
predicted. The deprotonation step is also part of path-
way III in Scheme 2A. Such a large substrate deuterium
4
8
KIE predicted from Wei et al. was not observed in our
OvoA studies.
Faponle et al. reported their EgtB mechanistic stud-
ies using
a
combination of quantum mechan-
3
4
ics/molecular mechanics (QM/MM) approach. Results
from this study led to a model very similar to the one in
Scheme 2B, in which formation of a thioether is the first
half of the reaction. In this model, after substrate binding,
2+
oxygen binds to the Fe center leading to the first in-
3+
termediate, an Fe -superoxo species (16). From Y417
and through the active site water network, a proton-
coupled electron transfer process then reduces the
3+
superoxo species (16) to the Fe -hydroperoxo species
(20) and at the same time, producing a Y417-based radi-
cal. A radical attack from the cysteine sulfur atom to the
imidazole side-chain of histidine links the two substrates
together through a thioether bond. Simultaneous to the
C-S bond formation, the hydrogen atom of the
hydroperoxo species relays back to Y417-based radical
2+
to regenerate tyrosine and produces an Fe -superoxo
2+
species (21). In the next step, Fe -superoxo (21) ab-
stracts a hydrogen atom from the imidazole ring to re-
2+
generate the aromaticity and produces Fe -hydroperoxo
intermediate (22). In this mechanistic model, Y417 is
involved in redox-chemistries. In another recent report,
Seebeck et al. suggested that Y377 in EgtB may func-
30
tion as Lewis acid/base.
Currently available data from investigations on
testing these mechanistic models. Pathway I in
Scheme 2A is inconsistent with the current results be-
2
cause no primary KIE was observed when [U- H ]-
5
histidine was used as the substrate in this study. Similar
conclusions were reached from previous steady-state
Scheme 2. Proposed OvoA mechanistic models.
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Journal of the American Chemical Society
Wei et al. commented that it is possible that, mecha-
If the conclusions reached by Faponle et al. in their
QM/MM EgtB study are also true for OvoA -catalysis,
the 20  21 conversion step in Scheme 2B is rate-
4
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
nistically, OvoA and EgtB might behave differently.
Obtaining OvoA structural information in the future will
be important. Theoretical calculation and experimental
characterizations could then be conducted on the same
system for comparative studies. For the fluoro-histidine
analog, there are no KIE studies yet, so it will be worth-
while to re-examine this substrate analog quantitatively
by these KIE studies.
34
limiting. We did not observe substrate deuterium KIE
(Figure 2). There might be at least three possible rea-
sons. First, as Wei et al. suggested, OvoA may indeed be
48
slightly different from EgtB-catalysis. Second, there
may be a similar level of deuterium KIE for the pro-
posed rate-limiting steps of the two branches (the 20 
21 conversion and the 16  17 conversion). Third, it
may also be possible that, in OvoA-catalysis, some key
species (16, 17, and 20) are actually in equilibrium and
what is observed in our kinetic isotopically sensitive
branching studies reflects the change of their relative
distribution upon isotope labeling.
Our current results are partially consistent with the
Scheme 2B model. In cysteine dioxygenase studies, it
was suggested that the creation of a partial cysteine sul-
fur radical (17, Scheme 2B) is a key factor in directing
the formation of the first S−O bond in the CDO catalytic
cycle. It has also been suggested that this is the rate-
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
2
9, 49-
limiting step along the cysteine oxidation pathway.
Our studies using OvoA Y417MtTyr variant provid-
ed new insights into this novel conversion. Similar to the
wild-type OvoA, OvoA Y417MtTyr variant takes
histidine and cysteine as the substrates. In addition, cys-
teine dioxygenase activity and oxidative C-S coupling
are still the only two observed activities. More im-
portantly, the oxidative C-S bond formation
regioselectivity of OvoA Y417MtTyr variant is also the
same as that of the wild-type OvoA. Upon replacing
Y417 in OvoA with MtTyr, the ratio between 7 and 11
changes from 9:1 in wild-type enzyme reaction to 7:3 in
OvoA Y417MtTyr variant reaction. Unnatural tyrosine
incorporation method clearly provided us with an effi-
cient way to fine-tune the OvoA activities. In addition,
for OvoA Y417MtTyr variant, the ratio between 7 and 11
5
2
If the mechanistic model proposed by Faponle et al.
on EgtB-catalysis is followed by OvoA-catalysis
Scheme 2B), the proton-coupled electron transfer from
(
3
+
Y417 to Fe -superoxo species (16) serves as an effi-
cient mechanism to divert the superoxo intermediate (16)
3
+
from the cysteine oxidation pathway to the Fe -
hydroperoxo intermediate (20) for the oxidative C-S
bond formation. Therefore, the 16  20 conversion will
be the critical step in controlling the partitioning be-
tween the cysteine dioxygenase and the oxidative C-S
bond formation pathways. Indeed, in our OvoA studies
reported here, the dominant activity of the OvoA Y417F
mutant is the cysteine dioxygenase activity.
For EgtB Y377F mutant, a small solvent deuterium
KIE was observed for the oxidative C-S bonding for-
further changes from 7:3 in H
O buffer to approximately
2
3
0
mation activity. Steady-state kinetic studies of wild-
type OvoA indicated that there is a small solvent deuter-
1:1 in D O buffer. These changes were also reflected by
2
the solvent deuterium KIE value, which is 2.09  0.02
for the OvoA Y417MtTyr variant and 1.29  0.01 for the
wild-type OvoA. The other intriguing result is the ob-
servation of a small, yet reproducible isotope effect
35
ium KIE (1.2  0.1). This is very close to our solvent
deuterium KIE (1.29  0.01) measured using the
isotopically sensitive branching method. Although there
is a close match between the two KIEs, we should be
cautious in making a direct link between these two
measurements because our KIE studies measure the par-
titioning between the two pathways and solvent may
affect both pathways. There were reports of solvent KIE
studies for cysteine dioxygenase. However, the cysteine
oxidation branch of OvoA-catalysis is different from the
cysteine dioxygenase-catalysis characterized previous-
(1.08  0.01) in OvoA Y417MtTyr variant when [U-
2
H
]-histidine was used as the substrate.
5
Mutating Tyr417 to MtTyr changes both the pK and
a
33, 53-55
reduction potential.
In a small molecular model
system that mimics the Tyr-Cys crosslink, the pK of 2-
a
(
methylthio)-p-cresol was lowered by  0.8 units relative
to p-cresol, and the reduction potential of 2-
methylthio)-p-cresol was lower than that of p-cresol by
(

2
9,49-52
ly.
In cysteine dioxygenase, cysteine serves as a
56
370 mV. In OvoA MtTyr variant, both the pK and
a
bidendate ligand. In OvoA and EgtB, cysteine is
monodendate and the histidine imidazole side-chain is
the other ligand. In the absence of histidine, OvoA does
reduction potential of MtTyr are less than that of tyro-
sine. If the Scheme 2B model is followed, it predicts an
outcome favoring more oxidative-coupling product for-
mation. Surprisingly, the oxidative coupling product
decreases from 90% with wild-type OvoA to roughly 70%
with OvoA Y417MtTyr variant. Thus, if OvoA-catalysis
follows the mechanistic model showed in Scheme 2B, a
likely explanation to account for the above results is that
MtTyr substitution also affects the cysteine dioxygenase
activity. This is possible because in cysteine
dioxygenase, although the active tyrosine is not directly
28
not have any cysteine dioxygenase activity, which sug-
gests that histidine ligation to the iron-center plays a
crucial role in the cysteine oxidation branch of the
OvoA-catalysis. Therefore, literature information related
to cysteine dioxygenase-catalysis may not be directly
applied to the cysteine oxidation branch of the OvoA-
catalysis.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 11
involved in redox chemistry, the formation of the Tyr-
Cys does improve cysteine dioxygenase activity by near-
were purchased from Sigma-Aldrich (St. Louis, MO) or
1
3
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
Fisher Scientific (Pittsburgh, PA). L-Cysteine (3- C, 99%
2
9
2
ly 10-fold. In OvoA Y417MtTyr variant reaction, the
amount of oxidative coupling product further decreases
from 70% in H O buffer to 50% in D O buffer. If
purity) and [U- H ]-histidine (ring-2,4-D ; α, β, β-D ,
5
2
3
98% purity) were purchased from Cambridge Isotope
Laboratories, Inc. Q5®Site-Directed Mutagenesis Kit
was purchased from New England BioLabs. Strep-
Tactin resins were purchased from IBA life science.
Measurement of the substrate deuterium KIE us-
ing wild-type OvoA. For the KIE measurement, three
OvoA enzymatic reactions were conducted:
2
2
MtTyr does affect both pathways, the observed solvent
KIE suggests that MtTyr modulates these two pathways
to different degrees at the critical steps of the two
branches (k vs. k , Figure 1).
3
5
Our studies clearly indicate that MtTyr incorpora-
tion can be used to modulate OvoA-catalysis. However,
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
Reaction 1: OvoA reaction using unlabeled histidine
and unlabeled cysteine. The reaction mixture contained
because both pK and reduction of MtTyr are different
a
33, 53-55
20 mM air-saturated KPi buffer in H O at pH 8.0, 10
2
from that of tyrosine,
it is challenging to differen-
mM histidine, 10 mM cysteine, 3 mM DTT, 1 mM sodi-
um ascorbate, and 8 μM iron-reconstituted OvoA. The
reaction was run aerobically at 25℃ for 2 h. After the
reaction was complete, the protein was removed by ul-
trafiltration.
tiate between the contributions of these two factors from
just the current sets of studies. By using another tyrosine
analog, 3-methoxytyrosine, we can differentiate the con-
tributions of pK or reduction potential in cytochrome c
a
5
7
oxidase studies. 3-methoxytyrosine has almost the
Reaction 2: OvoA reaction using unlabeled histidine
same pK as tyrosine. However, its reduction potential is
a
13
5
7
and [β- C]-cysteine. The reaction mixture contained 20
different than that of tyrosine by almost 200 mV. In the
future, further characterization using 3-methoxytyrosine
may provide additional mechanistic information related
to OvoA- and EgtB-catalysis. All of the mechanistic
models discussed in Scheme 2 were proposed based on
related studies in EgtB-catalysis. As commented by Wei
et al. recently, it is possible that, mechanistically, OvoA
mM air-saturated KPi buffer in H O at pH 8.0, 10 mM
2
13
histidine, 10 mM [β- C]-cysteine, 3 mM DTT, 1 mM
sodium ascorbate, and 8 μM iron-reconstituted OvoA.
The reaction was run aerobically at 25℃ for 2 h. After
the reaction was complete, the protein was removed by
ultrafiltration.
2
4
8
Reaction 3: OvoA reaction using [U- H ]-histidine
5
and EgtB might behave differently. Therefore, in the
near future, given by the recently reported EgtB struc-
13
and [β- C]-cysteine. The reaction mixture contained 20
2
0
34
mM air saturated KPi H O buffer at pH 8.0, 10 mM [U-
2
tural and theoretical calculation studies, similar types
of KIE studies about EgtB-catalysis will be worthwhile.
2
13
H ]-histidine, 10 mM [β- C]-cysteine, 3 mM DTT, 1
5
mM sodium ascorbate, and 8 μM iron-reconstituted
OvoA. The reaction was run aerobically at 25℃ for 2 h.
After the reaction was complete, the protein was re-
moved by ultrafiltration.
The reaction mixtures of reaction 1 and reaction 3
were combined and then analyzed by LC-MS (negative
ion mode). A HYPERCARB column (Thermo scientific)
was employed and the gradient was: 0-3 min, 95% B; 3-
In conclusion, our results presented here clearly in-
dicate that we can modulate the two activities in OvoA-
catalysis by replacing the active site tyrosine using a
tyrosine analog. Our current results partially align with
the Scheme 2B mechanistic model, while more charac-
terizations in the future will be needed in order to accu-
rately interpret the mechanistic implications of these
KIE results.
1
3
2 min, 95% B - 30% B; 12-14 min, 30% B; 14-17 min,
0% B - 95% B; 17-20 min, 95% B at a flow rate of 0.2
.
EXPERIMENTAL SECTION
mL/min. Solvent A: H O, adjusted to pH 9.2 by ammo-
2
nia; Solvent B: Acetonitrile, adjusted to pH 9.2 by am-
monia. High resolution mass spectra of compounds 7
and 11 were recorded. The ion intensity of each com-
pound was an average of twenty mass spectrometry
scans. The ratios of 7:7b, 7:7c and 11: 11b were calcu-
lated based on the ion intensity of corresponding com-
pounds obtained from the same LC-MS run. Then it was
used to calculate the isotope effect with the equations
showed in Figure 1. This set of experiments were re-
peated four times.
General. The codon optimized OvoA gene (DNA
sequences are shown in Supporting Information) was
inserted into the KpnI/XhoI multiple cloning site of
pASK-IBA5plus to give recombinant plasmid pPL-
OvoA wild-type. OvoA wild-type protein was overex-
28 1
pressed and prepared as reported previously. H-NMR
1
3
and C-NMR spectra were recorded on a Varian 500
MHz VNMRS. High-performance liquid chromatog-
raphy (HPLC) was performed using an Agilent 1200
Series separations module. The enzyme reaction samples
were analyzed by LC-MS on a LTQ-FT-ICR mass spec-
trometer (Thermo Scientific). The OvoA protein se-
quencing was conducted on a QExactive Plus Hybrid
Quadrupole-Orbitrap mass spectrometer (Thermo Scien-
tific). Oxygen consumption kinetic assay was carried out
by a NeoFox oxygen electrode. Chemicals and reagents
Reaction 2 and reaction 3 were also analyzed by
C-NMR spectroscopy by mixing 400 μL reaction mix-
13
ture with 200 μL D O to prepare the NMR samples.
2
Measurement of the solvent deuterium KIE using
wild-type OvoA. For the OvoA solvent deuterium KIE,
ACS Paragon Plus Environment

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Journal of the American Chemical Society
we have also conducted three OvoA enzymatic reac-
tions:
added to induce pPL-OvoA Y417MtTyr and pBK-
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
MtTyrRS, respectively. Cells were incubated at 25℃ for
an additional 14 h and harvested by centrifugation. The
OvoA Y417MtTyr variant protein was then purified an-
Reaction 4: OvoA reaction in H O using unlabeled
2
histidine and unlabeled cysteine. The reaction mixture
28
contained 20 mM air-saturated KPi H O buffer at pH
aerobically using the protocols for wild-type OvoA.
2
8.0, 10 mM histidine, 10 mM cysteine, 3 mM DTT, 1
For enzymatic characterization and KIE measure-
ment, OvoA Y417MtTyr variant reactions were conduct-
ed by following the same procedures used for wild-type
OvoA enzyme.
mM sodium ascorbate, and 8 μM iron-reconstituted
OvoA. The reaction was run aerobically at 25℃ for 2 h.
After the reaction was complete, the protein was re-
moved by ultrafiltration.
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
Reaction 5: OvoA reaction in H O using unlabeled
2
13
histidine and [β- C]-cysteine. The reaction mixture con-
tained 20 mM air-saturated KPi H O buffer at pH 8.0, 10
2
Supporting Information. Protein production, purification
and characterization details, product isolation, characteriza-
tion (NMR, MS results), EgtB/OvoA sequence alignment
and OvoA structural model created by I-TASSER are in-
cluded as supporting information. This material is available
free of charge via the Internet at http://pubs.acs.org.
13
mM histidine, 10 mM [β- C]-cysteine, 3 mM DTT, 1
mM sodium ascorbate, and 8 μM iron-reconstituted
OvoA. The reaction was run aerobically at 25℃ for 2 h.
After the reaction was complete, the protein was re-
moved by ultrafiltration.
Reaction 6: OvoA reaction in D O using unlabeled
2
13
histidine and [β- C]-cysteine. The reaction mixture con-
tained 20 mM air-saturated KPi D O buffer at pH 8.0, 10
2
13
mM histidine, 10 mM of [β- C]-cysteine, 3 mM DTT, 1
Pinghua Liu: pinghua@bu.edu, Changming Zhao:
cmzhao03@whu.edu.cn
mM sodium ascorbate, and 8 μM iron-reconstituted
OvoA. The reagents were dissolved in KPi D O buffer
2
and the enzyme was also prepared in D O buffer. The
2
reaction was run aerobically at 25℃ for 2 h. After the
reaction was complete, the protein was removed by ul-
trafiltration.
This work is supported in part by grants from the National
Institutes of Health (R01 GM093903) and the National
Science Foundation (CHE-1309148) to P.L., and grant from
National Natural Science Foundation of China (31670030,
31628004) to C.Z. and P.L. L.C. and C.Z. are supported by
fellowship from China Scholarship Council. We thank Prof.
Sean Elliott for constructive comments.
Reaction 4 and Reaction 6 mixture were combined
and the ratios (7:7c and 11: 11b) were measured directly
using LC-MS as described in the previous section to
calculate the solvent KIE using the equations showed in
Figure 1. This set of experiments were repeated four
times as well. Reaction 5 and reaction 6 were also ana-
13
lyzed by C-NMR spectroscopy by mixing 400 μL reac-
(
1) Grundemann, D.; Harlfinger, S.; Golz, S.; Geerts, A.;
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Supporting Information). For the pPL-OvoA Y417MtTyr
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