Substrate and pH-Dependent Kinetic Profile of 3-Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa

Article abstract of DOI:10.1021/acs.biochem.5b01203

Thiol dioxygenases catalyze the synthesis of sulfinic acids in a range of organisms from bacteria to mammals. A thiol dioxygenase from the bacterium Pseudomonas aeruginosa oxidizes both 3-mercaptopropionic acid and cysteine, with a ～70 fold preference for 3-mercaptopropionic acid over all pHs. This substrate reactivity is widened compared to other thiol dioxygenases and was exploited in this investigation of the residues important for activity. A simple model incorporating two protonation events was used to fit profiles of the Michaelis-Menten parameters determined at different pH values for both substrates. The pKs determined using plots of k_cat/K_m differ at low pH, but not in a way easily attributable to protonation of the substrate alone and share a common value at higher pH. Plots of k_cat versus pH are also quite different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs. At higher pH, k_cat decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked, the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs reactivity of the crosslinked form.

Full text of DOI:10.1021/acs.biochem.5b01203

Article
pubs.acs.org/biochemistry
Substrate and pH-Dependent Kinetic Profile of
3‑Mercaptopropionate Dioxygenase from Pseudomonas aeruginosa
Matthias Fellner,† Sekotilani Aloi,† Egor P. Tchesnokov,† Sigurd M. Wilbanks,‡ and Guy N. L. Jameson*,†
‡
†Department of Chemistry and Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand
ABSTRACT: Thiol dioxygenases catalyze the synthesis of
sulfinic acids in a range of organisms from bacteria to
mammals. A thiol dioxygenase from the bacterium Pseudomo-
nas aeruginosa oxidizes both 3-mercaptopropionic acid and
cysteine, with a ∼70 fold preference for 3-mercaptopropionic
acid over all pHs. This substrate reactivity is widened
compared to other thiol dioxygenases and was exploited in
this investigation of the residues important for activity. A
simple model incorporating two protonation events was used
to fit profiles of the Michaelis−Menten parameters determined
at different pH values for both substrates. The pKs determined using plots of kcat/Km differ at low pH, but not in a way easily
attributable to protonation of the substrate alone and share a common value at higher pH. Plots of kcat versus pH are also quite
different at low pH showing the monoprotonated ES complexes with 3-mercaptopropionic acid and cysteine have different pKs.
At higher pH, kcat decreases sigmoidally with a similar pK regardless of substrate. Loss of reactivity at high pH is attributed to
deprotonation of tyrosine 159 and its influence on dioxygen binding. A mechanism is proposed by which deprotonation of
tyrosine 159 both blocks oxygen binding and concomitantly promotes cystine formation. Finally, the role of tyrosine 159 was
further probed by production of a G95C variant that is able to form a cysteine-tyrosine crosslink homologous to that found in
mammalian cysteine dioxygenases. Activity of this variant is severely impaired. Crystallography shows that when un-crosslinked,
the cysteine thiol excludes tyrosine 159 from its native position, while kinetic analysis shows that the thioether bond impairs
reactivity of the crosslinked form.
hiol dioxygenases are non-heme mononuclear iron
Tenzymes that catalyze the addition of both oxygen
atoms of dioxygen to a thiol to form the corresponding
sulfinate.1 Examples of these enzymes have been found in
plants,2 bacteria,3,4 fungi,5 and mammals.6−11 Between them,
these enzymes catalyze the oxidation of a range of different
thiols and even peptides.2 In contrast, individual thiol
dioxygenases show an unusually strong preference for a single
substrate, although some cross reactivity has been observed.
Thiol dioxygenases have been split into two main groups.12
The “arg-type”, including cysteine dioxygenase (CDO), uses an
arginine (residue 60 in CDO) to hold the carboxylate of
cysteine through a salt-bridge interaction. The other group, the
“gln-type”, replaces the arginine described above with a
glutamine and shows different specificity, e.g., 3-mercaptopro-
pionate or possibly mercaptosuccinate. It has been further
suggested that the “gln-type” enzymes use another arginine
(residue 173 in the CDO homologue from Ralstonia eutropha)
to position substrate. This binding mode may be incompatible
with use of cysteine as a substrate in most family members.
Mammalian and some bacterial CDOs contain an unusual
post-translational modification near the active site iron. Initially
observed in crystal structures11,13,14 and later described by mass
spectrometry,15 a thioether bond occurs between a cysteine and
a tyrosine in a fraction of molecules in any particular protein
preparation.16 The crosslink has been shown to increase
reactivity,17 and each isoform has different Michaelis−Menten
parameters.18 By producing the C93G variant of CDO, we
recently showed that removal of the crosslink resulted in a shift
to higher pH for the maximum of kcat.19 This increase of ∼1 pH
unit matched model chemistry20 that suggested that a thioether
bond ortho- to a phenol decreased the pKa of the phenol by
about 1 unit. It thus seems that the tyrosine (residue 157 in
CDO), which is highly conserved in all thiol dioxygenases, acts
as a hydrogen bond donor/acceptor. However, it is not yet
clear whether a requirement for a specific pH optimum or other
factors account for the conservation of this crosslink among
mammalian thiol dioxygenases.
Recently we described21 a 3-mercaptopropionate dioxyge-
nase from Pseudomonas aeruginosa (p3MDO) that is unusual
because in vitro it is able to oxidize both 3-mercaptopropionate
(3-MPA) and cysteine with a 40-fold greater efficiency for 3-
MPA, under the conditions probed, the smallest preference yet
reported for two substrates utilized by the same thiol
dioxygenase. The observation of this reactivity is important
for two reasons. First, it shows that cysteine likely binds in a
different way in this enzyme as compared to cysteine
dioxygenase, and second, it provides us with a system that
allows us to probe substrate specificity in thiol dioxygenases.
Received: November 6, 2015
Revised: February 11, 2016
© XXXX American Chemical Society
A
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In common with other bacterial thiol dioxygenases,3 p3MDO
does not contain a crosslink because the position of the
cysteine is occupied by a glycine. Our recent crystal structure
(4TLF)21 shows that the tyrosine lies in the same position
relative to iron as the tyrosine in CDOs, although these features
are shifted relative to the rest of the enzyme, correlating with a
cis-peptide bond observed between positions 159 and 160 in all
mammalian CDOs. The importance of the tyrosine and its
position in bacterial thiol dioxygenases was therefore
investigated by studying the pH-dependent kinetics of WT
p3MDO and a G95C variant where the crosslink was artificially
formed.
G95C) were fully sequenced to confirm the absence of
adventitious mutations (Genetic Analysis Services at the
University of Otago). Expression and purification conditions
were the same as for WT p3MDO with 27
4% (n = 3
purifications) endogenously bound iron per protein and
reconstitution21 with ferrous iron ammonium sulfate followed
by treatment with Chelex to produce protein with >70% iron
occupancy. It therefore appeared that the introduced mutation
did not affect iron binding.
SDS-PAGE analysis (Figure 1) showed two bands for G95C
p3MDO and was thus visually similar to WT mammalian
CDO15−18,23 indicating a mixture of cysteine-tyrosine cross-
linked and un-crosslinked protein. Crosslinked CDO has
slightly greater mobility, presumably because of a less extended
conformation. In previous studies, crosslink formation could be
followed during catalytic turnover using densitometric analysis
of gels run at each time point,17 but G95C was remarkably
unreactive (see below), and changes in fraction crosslinked
were not detectable.
MATERIALS AND METHODS
■
Expression and Purification of WT p3MDO. Expression
and purification of WT p3MDO (NP_251292.1) using Strep-
Tag affinity technology were performed as described
previously.10,21 Purified protein was found to be a least 98%
pure by SDS-PAGE analysis and resulted in a single band which
corresponded to the theoretical molecular mass of 23 477 Da
(Figure 1). Purified protein was extensively dialyzed (dilution
1
NMR Investigation of WT p3MDO Reactivity. H NMR
of substrates (5−10 mM), products (5−10 mM), and reaction
mixtures using varying protein concentrations (10−100 μM)
were measured in phosphate buffer (100 mM, pH 6.5−8.5).
The signal was locked using a capillary containing TSP-d4 in
D2O, while water was suppressed by presaturation methods as
described previously.24
Choice of Buffers for pH-Dependent Studies. Sodium
phosphate buffer (pH 7.5, 100 mM), used previously to obtain
kinetic parameters for WT p3MDO,21 was used as a starting
point for designing pH-dependent studies and various buffers
were tested. At pH 7.5 PIPES and Tris buffers showed similar
activity to phosphate buffer, while HEPES and MOPS resulted
in significantly decreased velocities using 3-MPA as substrate.
At pH 6.5 PIPES and phosphate showed similar velocities while
they were reduced in both MES and MOPS. At pH 8.5 Tris and
AMPSO buffers produced identical rates. Although rates
measured in CHES appeared similar, it was ultimately not
utilized in obtaining kinetic parameters. Below pH 6.1 only
sodium acetate buffer provided consistent data even though
one might expect the carboxylate to interfere with substrate
binding and in particular the proposed salt bridge with arginine
168. Other buffers, including citrate and MES, removed activity
altogether or lowered activity significantly. Interestingly, MES
buffer was also problematic for Bruland et al. in their study of a
3-mercaptopropionate dioxygenase.25 Using this information
the pH kinetic profile for 3-MPA as a substrate covered a pH
range from 4.1 to 9.0 by utilizing sodium acetate, PIPES,
sodium phosphate, Tris and AMPSO (all 100 mM). The pH
profile for cysteine as a substrate covered a pH range from 6.1
to 8.0 by utilizing 100 mM PIPES and sodium phosphate.
Measurement of WT p3MDO Activity at 37 °C. Activity
with 3-MPA as the substrate was measured using a 96-well plate
Ellman’s assay as previously reported.21,22 Activity with cysteine
as the substrate was performed using our HPLC-ELSD
method.24 To obtain kcat and Km values at different pH and
buffers, five to seven different substrate concentrations were
measured (0.2−5.5 mM 3-MPA alone or 5−80 mM L-cysteine
in the presence of 20−400 μM bathocuproinedisulfonic acid).
The thiol stock solution was freshly prepared daily in the
appropriate buffer, and the pH was adjusted with concentrated
sodium hydroxide or hydrochloric acid. The thiol was heated to
37 °C under stirring and mixed with a concentrated enzyme
solution, also in the appropriate buffer, to initiate the reaction.
Figure 1. Purified samples of P. aeruginosa thiol dioxygenase p3MDO
and G95C were loaded on a 15% (w/v) polyacrylamide gel, resolved
by SDS-PAGE, and detected by staining with Coomassie Blue dye. 20-
and 40-fold dilutions are shown to gauge purity. Predicted molecular
masses including Strep-tags are 23 747 Da for p3MDO and 23 793 for
G95C. Molecular masses (kDa) of selected markers are shown to the
left and right. CDO containing a cysteine-tyrosine crosslink travels
slightly faster than un-crosslinked.
factor of >109) against sodium phosphate buffer (pH 7.5, 10
mM). Dialyzed protein was concentrated (∼1 mM) and found
to contain endogenously bound iron, 29 8% iron per protein
(n = 10 purifications). The concentrated protein was
reconstituted with iron as previously described10,22 to >80%
occupancy and stored at 4 °C for no longer than 2 months.
Expression and Purification of the G95C p3MDO
Variant. The glycine 95 to cysteine (G95C) mutation was
generated using primers (Invitrogen) containing a GGC-to-
TGC codon substitution. A polymerase chain reaction
catalyzed by PfuUltra High-Fidelity DNA polymerase (Stra-
tagene, Agilent Technologies) was conducted according to the
manufacturer’s protocol. Both strands of the CDO coding
region of the expression construct (pPR-IBA1/p3MDO/
B
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The experimental setup for 3-MPA consisted of 12 time points
measured every minute. For each time point an aliquot of the
reaction mixture (15 or 20 μL) was quenched by injection into
Ellman’s reagent (185 or 180 μL). The final enzyme
concentration was different depending on pH and optimized
for reactivity, 9 μM from pH 4.5 to 8.0 and 18 μM at pH 4.1
and 8.5−9.0. When cysteine was used as a substrate, six time
points were measured every 10 min starting after 20 min. An
aliquot of the reaction mixture (40 μL) was quenched by
injection into ice cold acetone (120 μL). Because of the lower
reactivity with cysteine, a higher enzyme concentration (20
μM) was used. In all cases, an enzyme titration ensured that
under the conditions used, the velocity was linearly dependent
on enzyme concentration. Furthermore, control experiments
showed that neither substrate was oxidized in the absence of
enzyme during the time period measured under all conditions.
Velocities of the different substrate concentrations at a given
setup (buffer, pH, enzyme concentration) were normalized to
the active site iron concentration and plotted in Graph Pad
Prism (version 6.0) to determine kcat and Km.
Measurement of G95C p3MDO Variant Activity at 37
°C. Preliminary experiments showed no measurable activity
when using the system described for WT p3MDO. The enzyme
concentration had to be increased to 100 μM for both
substrates to observe activity. For 3-MPA the same
experimental setup as described for WT p3MDO was used,
and for cysteine the time points were altered to six time points
every 20 min after addition of enzyme.
Crystallization and Structural Determination of the
G95C pTDO Variant. Crystals were grown at 18 °C using the
hanging drop vapor diffusion method. Protein that had been
reconstituted with iron (2.5 μL, 18 mg/mL) in phosphate
buffer (pH 7.5, 10 mM) was mixed with a precipitant solution
(1 μL) containing sodium acetate buffer (pH 5.3, 140 mM) and
PEG 4000 (8% w/v). The reservoir contained 500 μL of the
precipitant solution. Crystals were cryo-protected with a
mixture of precipitant solution (75% v/v) and ethylene-glycol
(25% v/v) before freezing. Data were collected at the MX2
beamline of the Australian Synchrotron. Using 0.5° oscillations,
100° of data were collected on an ADSC Quantum 315r
detector with a detector distance of 360 mm at 13 000 eV, 90%
attenuation, and 2 s exposure time. Data were indexed in space
group P41212, with unit cell dimensions of 66.54, 66.54, and
376.85 Å, and integrated in an automated fashion with XDS.27
Scaling used Aimless in CCP4,28 and 5% of reflections were
reserved for the calculation of Rfree (Table 1). The resolution
Table 1. X-ray Data Collection, Reduction, and
Crystallographic Refinement Statistics of G95C p3MDO
Resting State (PDB 4WVZ)
a
resolution range (Å)
unique reflections
multiplicity
39.91−2.09 (2.16−2.09)
a
51609 (4897)
a
7.0 (3.8)
a
completeness (%)
mean I/σ(I)
99.60 (97.07)
a
28.13 (4.35)
R-meas
0.050
Modeling the pH-Dependent Kinetic Data. The enzyme
was treated as having two dissociable groups with only the
singly protonated form proceeding through catalysis illustrated
in Scheme 1 and described in-depth elsewhere.26 The data
a
CC1/2
1 (0.92)
a
R-work
0.177 (0.241)
a
R-free
0.227 (0.300)
number of non-hydrogen atoms
macromolecules
ligands
6930
6436
4
Scheme 1. Enzyme Reaction Used To Model the pH Profiles
water
560
794
0.006
1.00
96
protein residues
RMS bonds (Å)
RMS angles (deg)
Ramachandran favored (%)
Ramachandran outliers (%)
Clashscore
0
3.79
36
average B-factor (Å2)
a
Values in parentheses indicate the highest-resolution shell.
was cut at 2.09 Å based on the I/σ and CC1/2 values (0.5) in
the hk plane; diffraction extended significantly further parallel
to the l axis. All four chains of PDB entry 4TLF21 were used for
molecular replacement in Phenix Phaser29 with four chains in
the asymmetric unit. The active site iron was placed using
Phaser-EP29 (MR-SAD). Parameters for the iron atoms were
created with Phenix ReadySet, and each active site iron atom in
all four chains was modeled and refined with an occupancy of
one. Phenix Refine,29 MolProbity,30 and COOT31 were further
used for refinement. Coordinates and structure factors were
deposited in the Protein Data Base as entry 4WVZ.
clearly indicated a bell shape curve, and therefore the pH
profiles of kcat and kcat/Km were fitted to the parameters given in
Scheme 1, using eqs 1 and 2 respectively.26 The parameters
from these fits were then used in eq 326 to simulate a fit for the
pH profile of Km.
k′cat
kcat
=
[H+]
KES1
KE+S2
+ 1 +
(
)
[H ]
(1)
(2)
k ′cat
K ′m
kcat
Km
=
[H+]
KE1
KE2
+ 1 +
+
(
)
RESULTS
[H ]
■
Kinetic Studies of the pH Dependence of 3-MPA
Dioxygenation by WT p3MDO. 1H NMR shows that 3-MPA
is converted into 3-sulfinopropionic acid (3-SPA) with high
fidelity (negligible amounts of disulfide are formed regardless of
pH, see Figure 2). Reaction velocities measured by
[H+]
KE1
KE2
K′m
+ 1 +
+
(
)
[H ]
Km
=
[H+]
KE+S2
+ 1 +
(
)
KES1
[H ]
(3)
C
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1
Figure 2. H NMR showing product distribution after 16 h of reaction of p3MDO (30 μM) with either 10 mM 3-MPA (top) or 10 mM cysteine
(bottom) at various pHs. Peaks are differentiated by color: 3-SPA (light green), 3MPA (black), cysteine (blue), CSA (dark green), and cystine (red).
Peak assignments are indicated by α and β. All spectra have been normalized.
Table 2. Michaelis−Menten Parameters for 3-MPA and Cysteine Dioxygenation by WT p3MDO. Determined at Various pH
Values at 37°C
WT p3MDO with 3-MPA
WT p3MDO with cysteine
condition
kcat (s−1
)
KM (mM)
kcat/Km (M−1 s−1
)
kcat (s−1
)
KM (mM)
kcat/Km (M−1 s−1
)
a
acetate 4.7
acetate 5.1
acetate 5.5
PIPES 6.1
PIPES 6.5
phosphate 6.5
PIPES 7.0
phosphate 7.0
PIPES 7.5
phosphate 7.5
Tris 7.5
0.11 0.01
0.14 0.02
0.22 0.02
0.23 0.01
0.19 0.01
0.20 0.01
0.13 0.01
0.11 0.01
0.10 0.01
0.10 0.01
0.10 0.01
0.03 0.01
0.05 0.01
0.03 0.01
0.02 0.01
4
4
5
1
2
2
30 10
40 20
50 30
0.9 0.1
1.0 0.1
1.5 0.2
0.5 0.1
0.9 0.1
0.8 0.1
0.7 0.1
0.6 0.1
0.5 0.1
0.7 0.1
1.4 0.3
1.1 0.2
260 50
200 40
130 20
270 50
130 20
120 20
130 20
180 30
60 10
0.011 0.001
0.022 0.002
16
16
2
4
0.7 0.1
1.4 0.4
0.033 0.001
15
2
2.2 0.4
0.019 0.001
0.021 0.001
13
13
2
2
1.5 0.3
1.6 0.3
phosphate 8.0
Tris 8.0
0.013 0.001
12
2
1.1 0.3
70 20
Tris 8.5
18
18
5
4
AMPSO 8.5
a
Standard errors associated with the Fit are reported.
discontinuous HPLC, and chromogenic methods were used to
compute Michaelis−Menten parameters over a pH range from
4.1 to 9.0. Enzymatic activity was only detectable between pH
4.7 and 8.5 (Table 2). As described above, multiple buffers were
tried, and only those with similar activity at overlapping pHs
were used for analysis. This gives confidence in the resulting
bell-shaped curve of the kcat pH profile (Figure 3A). A
maximum at pH 6.1 is observed, and the profile could be fitted
to an enzyme with two dissociable groups according to eq 126
(parameters in Table 3). Fitting kcat/Km versus pH according to
eq 2 was not as straightforward. Small changes in Km have a
large impact on the resulting kcat/Km values. To fit kcat/Km it was
therefore decided to place emphasis on the higher pH range as
data points of complementary buffers were available. A
reasonable bell-shaped fit to the data points was obtained
(Figure 3B and Table 3). Constraining all parameters in eq 3 to
the values obtained from fitting kcat and kcat/Km resulted in a
simulation of Km versus pH, which followed the overall trend of
the data points, although residuals are significant at certain pHs
(Figure 3C).
Kinetic Studies of the pH Dependence of Cysteine
Dioxygenation by WT p3MDO. Initial experiments by
HPLC-ELSD showed that the pH range of cysteine
dioxygenase activity was narrower compared to 3-MPA
dioxygenase activity. Indeed, cysteine sulfinic acid (CSA)
production was barely detectable at pH 5.5 or above pH 8.0.
The pH profile of kcat indicated a bell-shaped curve (Figure 3A)
with a maximum at pH 7.1. The pH profile for kcat/Km appeared
to be very similar to the profile for kcat (Figure 3B), and it was
therefore fitted with pKE1 and pKE2 equal to pKES1 and pKES2
D
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each other out in eq 3. The simulation shows that K′m is
probably overestimated but overall the fit to eq 3 does not
cause us to question the fits to eqs 1 and 2 (Figure 3B). Our
HPLC-ELSD method24 allows simultaneous monitoring of
cysteine depletion and CSA formation. At low pH (6.1) and
low cysteine concentrations, cysteine depletion matched CSA
formation, and only at very high cysteine concentrations was
cystine formation observable (Figure 4A). However, at pH 6.5
cysteine depletion was about 1.5 times faster than CSA
formation. This trend continued with increasing pH, and by pH
8.0, cysteine depletion was about 30 times faster than CSA
formation (Figure 4B). As discussed previously,21 this denotes
uncoupling of the enzyme, presumably due to the substrate
thiol not being oriented properly in the active site. Note that
higher enzyme concentrations results in higher coupling, and
thus coupling is a difficult concept to quantify.
1H NMR experiments show that the difference between
cysteine depletion and CSA formation corresponds to cysteine
disulfide (cystine) formation (Figure 2). As pH increases, the
proportion of cystine formed increases, until cystine is almost
the sole product at pH 8.5. Interestingly, the formation of CSA
remained linear and appeared to be unaffected by cystine
formation as long as sufficient cysteine remained, strongly
suggesting that cystine formation occurs in parallel. The rate of
cystine formation was found to depend linearly on the total
enzyme concentration ([p3MDO]T), and thus initial rates were
normalized with respect to the enzyme concentration and
plotted against initial cysteine concentration ([cysteine]T). A
linear trend was observed rather than hyperbolic showing the
reaction was first order in cysteine (Figure 4C). The slope of
these lines provided the second order rate constant kcystine. This
rate constant was then measured as a function of pH, and the
resultant sigmoidal curve could be fitted to a single pK of 7.2
(Figure 4D).
The overall rate equation for cystine formation was therefore
given by
k
cystine[p3MDO]T [cysteine]T
rate of cystine formation =
[H+]
1 +
{
}
K
(4)
Figure 3. Kinetic pH profile of 3-MPA (black) and cysteine (blue)
dioxygenation reaction catalyzed by WT p3MDO. (A) kcat. The solid
lines represent the best fit according to eq 1. (B) kcat/Km. The solid
line represent the best fit according to eq 2. (C) Km. The dashed lines
represent simulations using eq 3 and the parameters obtained from
fitting kcat and kcat/Km. All parameters are listed in Table 2. Sodium
acetate buffer, full circle; PIPES, open circle; sodium phosphate, full
square; Tris, open square; AMPSO, full triangle.
X-ray Crystallography Characterization of the G95C
p3MDO Variant. To probe the effect of a mammalian CDO-
like thioether crosslink and to assess the role of protonation of
tyrosine 159, we mutated glycine to cysteine at position 95 and
assessed structure and activity. The overall structure and the
organization of the active site of G95C p3MDO (4WVZ)
appear to be unchanged when compared to WT p3MDO
(4TLF) (Figure 5A,B). Using Cα-atom alignment resulted in an
average RMSD of only 0.2 Å when the four chains in the
asymmetric units of WT and G95C pTDO were compared
(alignment and calculation by UCSF Chimera32). The
differences between the chains were uninfluenced by the single
point mutation and have already been discussed for WT
p3MDO.21 The only major difference can be seen at the
mutated residue 95 and its influence on the nearby tyrosine
159. If modeled without a crosslink, the introduced side chain
of cysteine 95 clashed with the tyrosine 159 phenyl ring,
suggestive of a similar thioether bond to that observed in WT
mammalian CDO crystal structures.9,13,14 However, in contrast
to WT mammalian CDO which is entirely crosslinked in single
crystals, G95C p3MDO could not be modeled this way. Two
conformations for residues 95 and 159 representing a
Table 3. pH-Dependent Parameters for WT p3MDO
Modeled As an Enzyme with Two Dissociable Groups
3-MPA
cysteine
k′cat (s−1
K′m (mM)
pKE1
)
0.28
0.8
5.8
7.2
5.0
7.0
0.08
15
6.9
7.2
6.9
7.2
pKE2
pKES1
pKES2
respectively. k′cat was constrained to 0.08 s−1 and the resulting
K′m equaled 15 mM (Table 3). Constraining all parameters in
eq 3 to simulate a pH profile for Km resulted in a horizontal line
at the value of K′m, because the constrained pK values cancel
E
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Figure 4. Kinetics of cystine formation observed via HPLC ELSD-based assay. (A) At low pH (6.1), cysteine depletion (blue triangles) equals CSA
formation (green circles) except at high cysteine levels (e.g., 40 mM, as in this panel) where cystine formation can be calculated to equal CSA
formation (red dashed line in inset, showing detail at [CSA] < 1 mM). (B) At high pH (8.0), cysteine depletion is faster than CSA formation (NMR
confirms cystine formation), and significant cystine formation (red dashed line) can be calculated. (C) Initial rate of cystine formation normalized to
p3MDO concentration (red squares) can be plotted against initial cysteine concentration and shown to be linearly dependent (e.g., pH 8.0).
Formation of CSA (green circles) shows Michaelis−Menten kinetics under the same conditions. (D) The overall second order rate constant of
cystine formation determined from plots like C is plotted against pH and fitted to eq 4. A pK of 7.2 was derived (denoted as a vertical dashed line).
crosslinked and un-crosslinked version of the crystal structure
were modeled at varying occupancies. In the four chains an
average of 70 5% crosslinked and 30 5% un-crosslinked
(Figure 5C) was used to fit the data. The orientation of
tyrosine 159 in crosslinked G95C protein was the same as in
WT p3MDO with the tyrosine hydroxyl groups in the four
chains 3.9 0.1 Å away from the active site iron compared to
4.1 0.1 Å in WT (4TLF) (Figure 5D). However, cysteine 95
remains in a similar orientation in both versions. In the un-
crosslinked orientation, the thiol forces tyrosine 159 further
away from the active site iron: (4.6 0.1 Å).
The water molecules bound to the iron varied between the
four chains in the asymmetric unit. The position opposite
histidine 142 showed the most density and as in WT p3MDO21
was modeled conservatively as a single water molecule.
Dioxygen or acetate molecules were also tried as they were in
the related R. eutropha structure,12 but these models were not
supported by density in all chains. The other water molecules,
opposite histidine 89 and 91, showed diverging amounts of
electron density when comparing the four chains. Only in chain
A were both positions modeled as water molecules, resulting in
the octahedral coordination of iron exhibited by WT p3MDO.
The water molecule between glutamate residues 104 and 132 is
also observed in all chains in G95C; however the water
molecule observed in some chains of the WT p3MDO
structure, linking 104 to the active site, is not observed in
G95C (Figure 5B).
MPA and cysteine. The formation of the products 3-SPA and
CSA were confirmed by detection of these products by mass
spectrometry, as previously described for WT p3MDO.21 It was
possible to obtain Michaelis−Menten parameters when the
enzyme concentration was increased significantly as described
above in Materials and Methods. A simple screening of activity
across a similar pH range as described for WT p3MDO
revealed no striking differences and low overall activity. Thus,
kcat and Km were only obtained at pH 6.5 in PIPES. This pH
was chosen as it lies between the WT pMDO kcat optimum for
both substrates 3-MPA and cysteine. Measurements at pH 6.5
G95C p3MDO resulted in a kcat of 0.007 0.001 s−1 and Km
0.9 0.1 mM for 3-MPA and a kcat of 0.003 0.001 s−1 and Km
22
3 mM for cysteine. Cystine formation by the G95C
variant was similar to WT p3MDO but was not investigated in
detail.
DISCUSSION
■
Although some cross reactivity has been observed,33 thiol
dioxygenases each appear to be quite specific for one substrate,
and even are unreactive with seleno-derivatives.34 Intriguingly,
we recently found that a bacterial 3-mercaptopropionate
dioxygenase from P. aeruginosa has the ability to also oxidize
cysteine in reasonable amounts.21 This is exciting because it
allows us to compare the pH-dependent kinetic parameters for
two different substrates and thus gain insight into the important
residues/moieties involved in catalysis. To test the role of
tyrosine 159, we also introduced the thioether crosslink
observed in mammalian CDO into this 3-mercaptopropionate
Kinetic Studies of the G95C p3MDO Variant. The
G95C p3MDO variant showed reduced activity toward both 3-
F
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Figure 5. Comparison of chain C of WT (4TLF) and G95C (4WVZ) p3MDO crystal structures. (A) The WT protein is shown in blue, selected side
chains are shown with oxygen, nitrogen, sulfur, and iron atoms highlighted in red, blue, yellow, and orange (octahedral iron coordination black lines,
hydrogen bonds red lines). (B) The G95C variant is shown in the same orientation in red. (C) Refined model of G95C p3MDO superimposed on
maps after initial phase determination by molecular replacement with G95/Y159A model; mFo − DFc electron density in green mesh (RMS = 1.12,
contoured at level 2.5) and the anomalous map in solid yellow (RMS = 1.01, contoured at level 2.5). (D) Superposition of residue 95, 159 and the
active site iron of WT in blue and G95C in red with average tyrosine-hydroxyl to iron distances of all four chains indicated. Figures created by UCSF
Chimera.32
dioxygenase, and through crystallography, spectroscopy and
kinetics we propose how differences in the active sites of CDO
and p3MDO control reactivity.
Greater differences between pKES1 for 3-MPA and cysteine
are observable. This pK refers to deprotonation of the ES
complex. Unfortunately, substrate bound crystal structures are
not yet available, but the data presented here provide insight
into substrate binding and allows some pKs to be assigned. As
mentioned above, both substrates appear to bind by their thiol
to the ferrous atom, and in the case of 3-MPA, the carboxylate
is most likely held in place by a salt bridge with arginine 168.
The pKa of the carboxylate of 3-MPA is significantly higher
(∼4.5) than cysteine (<2) due to the absence of an adjacent
amine group.39 pKES1 therefore refers to deprotonation of the
carboxylate of 3-MPA. At lower pH the carboxylate is
protonated, breaking the salt bridge to arginine 168 and thus
disrupting both binding of the substrate and its correct
orientation. This exhibits itself as a low kcat and high Km in
this pH regime (Figure 3). It is important to note that data in
this region were collected with acetate buffer (100 mM), which
has a similar pKa to 3-MPA. We believe that acetate is acting as
a weak competitive inhibitor at higher pH. This is shown by a
slight decrease in Km between pH 5.5 and 4.7 as more of the
acetate is protonated and loses its inhibitory potential.
However, the overall increase in Km at lower pH is attributable
to protonation of the ES complex rather than the buffer. We
believe this for two reasons. First, as more acetate is protonated
at pH 4.7, one would expect Km to return to a value of ∼1 that
it shows at all other pHs tested. Second, experiments with 3-
hydroxypropionic acid (an alcohol derivative of 3-MPA) only
shows slight inhibition at concentrations >30-fold excess over
3-MPA (data not shown).
pH-Dependent Kinetic Studies. Studies of the pH
dependence of p3MDO show bell-shaped kinetics using either
3-MPA or cysteine and can be described by a simple model that
involves two ionizable groups, with only the singly protonated
ES complex able to react. The form of the equations means that
careful fitting of the pH dependence of both kcat and kcat/Km
allow all four pK values (two ionizable groups in both ES and E
+ S) to be determined for the reaction with each substrate,
while simulation of the pH dependence of Km using the
parameters previously determined provides an internal check of
the fitting.
Because of proton ambiguity, pKE1 and pKE2 refer to either
deprotonation of a residue in the enzyme (or indeed a weighted
average of several residues) or the substrate alone (Scheme 1).
Mossbauer spectra21 of the ES complexes with 3-MPA and
̈
cysteine support thiolate binding as is known to occur in CDO
and thus one might expect deprotonation of the substrate thiol
to be important. The thiol of 3-MPA and cysteine in solution
have pKas that are quite different, approximately 10 and 8
respectively.35−38 Although pKE2 values are the same for both
substrates (7.2, Table 3), the values of pKE1 obtained here using
3-MPA or cysteine as substrate are quite different (5.8 vs 6.9).
pKE1 therefore does not correlate well with the free pKa of the
respective thiols; indeed although 3-MPA has the highest pKa it
has the lowest pKE1. These pKs therefore suggest a more
complicated process but deprotonation of substrate thiol does
not seem to be rate limiting.
In contrast, pKES1 for cysteine cannot reflect the pKa of the
cysteine carboxylate because pKES1 for cysteine is almost 2 pK
G
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units higher than for 3-MPA. Indeed, cysteine almost certainly
cannot bind in the same way as in CDO. In CDO, cysteine
binds via thiol and amine to the iron while the carboxylate is
held in place by an arginine (60, rat CDO numbering).40
Rotation of the active site (correlating to the occurrence of a
trans-peptide in place of a cis-peptide that is conserved in
mammalian CDOs) means the equivalent residue to arginine
60, glutamine 62 is further away from the iron as well as lacking
charge. It is possible that cysteine coordinates via just the thiol
with the carboxylate held in place by arginine 168 as described
for 3-MPA above. Certainly, this model is supported by
crystallographic studies of a 3-mercaptopropionate dioxygenase
from R. eutropha that suggest that arginine 168 sterically
hinders amine coordination that is observed in CDO.12 In this
different orientation, the protonated, positively charged amine
of cysteine would point away from the iron. This would reduce
the positive charge near the iron but deprotonation may still
stabilize the ES complex. Although a pKa of 6.9 for an amine
appears unlikely, when cysteine is bound and within the active
site, this cannot be ruled out.
pKES2 is approximately the same for both 3-MPA and
cysteine. In both cases, deprotonation of the ES complex
reduces dioxygenase activity. The most likely source of this
effect is tyrosine 159 and its influence on the water molecule
that is bound between it and the iron atom. Comparison with
crystal structures of CDO9,40,42 and R. eutropha 3MDO12
suggests that this water molecule is displaced to open a site for
dioxygen binding. We propose that the protonation state of
tyrosine 159 affects binding of both water and dioxygen at this
site and thus enzymatic activity. Support for this proposal
comes from four sources. First, spectroscopic and computa-
tional analysis by Blaesi et al.43 showed that the inactive
iron(III) form of the enzyme binds cysteine but retains a
hydroxide in the sixth coordination site. Second, mutation of
histidine 155 to alanine in mouse CDO eliminates a hydrogen
bond to hydroxyl of tyrosine 157 and stabilizes a water
molecule in this same site in the cysteine-bound complex as
assessed by magnetic circular dichroism.44 Third, Driggers et
al.40 showed that at higher pH (>8) structural changes of
unliganded CDO strongly suggest this water is likely
deprotonated to form a hydroxide, even though oxidation of
the iron is minimal. Indeed, their crystallographic work
supports dioxygen being unable to bind in this pH range
even though cysteine is bound and they concluded with limited
data points that the pK was ∼7.5, consistent with our results.
Lastly, the pK is remarkably similar to that of un-crosslinked
CDO and a C93G variant we determined via a pH-dependent
kinetic profile.19 This pK shifted down by ∼1 unit when the
tyrosine was crosslinked strongly supporting assignment of this
pK to tyrosine.
publication. That enzyme has sequence similarity to the
P. aeruginosa enzyme presented here, so it is surprising that
kinetic parameters differ considerably. However, they reach the
same conclusion that 3-MPA is the preferred substrate over a
broad pH range and also postulate that deprotonation of
tyrosine 159 leads to reduced activity at high pH.
Cystine formation likely occurs in the active site as has been
suggested for other nonheme mononuclear iron enzymes.46
The rate of cystine formation in the presence of p3MDO,
which is measured indirectly through loss of cysteine not
involved in CSA production, is linear and does not obey simple
1
Michaelis−Menten kinetics. H NMR confirmed the fact that
CSA and cystine are the only two products (Figure 2). The rate
of cystine formation is first order in both total p3MDO and
cysteine concentrations (Figure 4). It is thus reminiscent of
glutathione peroxidases, which also show such a rate equation
where formation of the sulfenate is rate determining.47 The
proposed mechanism of the related enzyme cysteine
dioxygenase involves production of a sulfenate intermedi-
ate,48,49 and so it is appealing to suggest that this is the branch
point for formation of either CSA by addition of a second
oxygen atom or cystine by rapid reaction of this intermediate
with another cysteine molecule. Of course, other mechanisms
that are consistent with the kinetic data cannot yet be ruled out.
The pH dependence of the second order rate constant for
cystine formation is sigmoidal and can be fitted to a pK of 7.2
(Figure 4D, eq 4). This pH dependence is reminiscent of iron
catalyzed cysteine autoxidation,50 although there are conflicting
studies showing this reaction is complicated and subject to an
array of different influences.51 However, the value of the fitted
pK is the same as pKES2 and thus an increase in cystine
formation correlates well with loss of CSA production. As
discussed above, pKES2 most likely refers to tyrosine 159.
Deprotonation of this residue will enable deprotonation of the
water bound to the iron and thus slow dioxygen binding and
stabilize the oxidized form of the enzyme. This could then
provide the conditions for sulfenate production and subsequent
cystine formation through nucleophilic attack by a second
cysteine molecule.
Artificial Thioether Crosslink Insertion and Its Effect
on Activity. The thioether crosslink between cysteine 93 and
tyrosine 157 in CDO (rat numbering) is known to increase
reactivity of the enzyme and the kinetic parameters for each
form appear to be quite different.17−19 However, this crosslink
is not present in bacterial CDOs and in other thiol
dioxygenases.1,3,25 Exchange of glycine 95 for a cysteine residue
allowed the crosslink to be engineered into p3MDO. The fact
that it so easily formed shows that spatial proximity of the
cysteine and tyrosine contributes substantially to crosslink
formation as has been suggested previously.52 The crystal
structure of G95C p3MDO variant shows some important
features that aid our understanding of the crosslink. Like rat
CDO, not all protein is crosslinked, but unlike rat CDO, both
isoforms are visible in the crystal structure. The crosslinked
form of G95C p3MDO shows tyrosine 159 lies in the same
position as WT p3MDO. In contrast, tyrosine 159 in the un-
crosslinked isoform has been pushed away by the side chain of
cysteine 95 that is significantly larger than the naturally
occurring glycine. This appears to result in the water opposite
histidine 89, the proposed site of dioxygen binding, to be less
well-defined.
Cystine Formation. Cysteine is not the principal substrate
of p3MDO. Previous investigations21 showed that 3-MPA is
oxidized exclusively when in competition with cysteine at pH
7.5. The studies presented here show that 3-MPA is a preferred
substrate at all pHs, and indeed the pH independent specificity
constants that can be derived from Table 3 (350 vs 5.3 M−1
s−1) are ∼70 times higher for 3-MPA. The slower reaction rate
with cysteine, the higher susceptibility of cysteine to disulfide
formation and the fact the enzyme active site is not optimized
for cysteine binding, means that it is not surprising that cystine
is also a significant product depending on conditions. While our
paper was in revision following review, a study of Azotobacter
vinlandii MDO by Pierce et al.45 became available ahead of
One would expect G95C would have a different pH
maximum caused by a decrease in the pKa of the phenol
H
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Article
Cysteine Dioxygenases: a New Route of Cysteine Degradation for
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(2016) Substrate and Cofactor Range Differences of Two Cysteine
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82, 910−921.
(5) Kasperova, A., Kunert, J., Horynova, M., Weigl, E., Sebela, M.,
Lenobel, R., and Raska, M. (2011) Isolation of recombinant cysteine
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(6) Chai, S. C., Jerkins, A. A., Banik, J. J., Shalev, I., Pinkham, J. L.,
Uden, P. C., and Maroney, M. J. (2005) Heterologous expression,
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Spectroscopic and Computational Characterization of Substrate-
Bound Mouse Cysteine Dioxygenase: Nature of the Ferrous and
Ferric Cysteine Adducts and Mechanistic Implications. Biochemistry
49, 6033−6041.
(8) Simmons, C. R., Hirschberger, L. L., Machi, M. S., and Stipanuk,
M. H. (2006) Expression, purification, and kinetic characterization of
recombinant rat cysteine dioxygenase, a non-heme metalloenzyme
necessary for regulation of cellular cysteine levels. Protein Expression
Purif. 47, 74−81.
(9) Souness, R. J., Kleffmann, T., Tchesnokov, E. P., Wilbanks, S. M.,
Jameson, G. B., and Jameson, G. N. L. (2013) Mechanistic
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through ortho-substitution of cysteine 95. Unfortunately, this
substitution severely decreases enzymatic activity so much that
we were unable to get a full pH profile, even with increased
enzyme concentration. The specificity constant for 3-MPA and
cysteine decreases ∼70- and ∼40-fold at pH 6.5. The protein
exists as a ∼1:1 mixture of crosslinked and un-crosslinked form,
but the observed decrease in activity is so great that it cannot be
due purely to formation of an inactive isoform. Thus, not just
the crosslink but cysteine 95, when not bound within the
thioether bond itself, affects reactivity. We previously observed
the effect of this thiol/ate when we studied the pH dependent
kinetic parameters of CDO and its C93G variant.19 Removal of
cysteine in C93G CDO led to lower activity at higher pHs.
Altogether, this study provides important information about
substrate specificity in p3MDO. Detailed kinetic studies across
a broad pH range allow us to determine residues that are
important in binding and reactivity. In particular, the water
hydrogen bonded to tyrosine 159 in the absence of substrate
appears to be crucial for reactivity. We have further investigated
the role of tyrosine 159 by forming the thioether crosslink that
characterizes mammalian CDOs. The almost complete removal
of activity can be traced to cysteine 95 pushing tyrosine 159
aside when not part of the crosslink but that does not alone
explain the reduction in activity of the crosslinked isoform. In
addition to the role of tyrosine 159, specific pKs imply roles for
the substrate carboxylate and residues arginine 60 and 168 in
substrate binding.
AUTHOR INFORMATION
■
(11) Ye, S., Wu, X., Wei, L., Tang, D., Sun, P., Bartlam, M., and Rao,
Z. (2007) An Insight into the Mechanism of Human Cysteine
Dioxygenase. Key Roles of the Thioether-Bonded Tyrosine-Cysteine
Cofactor. J. Biol. Chem. 282, 3391−3402.
(12) Driggers, C. M., Hartman, S. J., and Karplus, P. A. (2015)
Structures of Arg- and Gln-type bacterial cysteine dioxygenase
homologs. Protein Sci. 24, 154−161.
(13) McCoy, J. G., Bailey, L. J., Bitto, E., Bingman, C. A., Aceti, D. J.,
Fox, B. G., and Phillips, G. N., Jr. (2006) Structure and mechanism of
mouse cysteine dioxygenase. Proc. Natl. Acad. Sci. U. S. A. 103, 3084−
3089.
Corresponding Author
*Phone: +64 3 479 8028. E-mail: guy.jameson@otago.ac.nz.
Funding
This work was supported by the Marsden Fund of the Royal
Society of New Zealand (G.N.L.J., PI; S.M.W., AI). E.P.T. was
supported by a Canadian Institutes of Health Research
Postdoctoral Fellowship.
Notes
The authors declare no competing financial interest.
(14) Simmons, C. R., Liu, Q., Huang, Q., Hao, Q., Begley, T. P.,
Karplus, P. A., and Stipanuk, M. H. (2006) Crystal Structure of
Mammalian Cysteine Dioxygenase: a novel mononuclear iron center
for cysteine thiol oxidation. J. Biol. Chem. 281, 18723−18733.
(15) Kleffmann, T., Jongkees, S. A. K., Fairweather, G., Wilbanks, S.
M., and Jameson, G. N. L. (2009) Mass-spectrometric characterization
of two posttranslational modifications of cysteine dioxygenase. J. Biol.
Inorg. Chem. 14, 913−921.
(16) Dominy, J. E., Jr., Hwang, J., Guo, S., Hirschberger, L. L., Zhang,
S., and Stipanuk, M. H. (2008) Synthesis of Amino Acid Cofactor in
Cysteine Dioxygenase Is Regulated by Substrate and Represents a
Novel Post-translational Regulation of Activity. J. Biol. Chem. 283,
12188−12201.
(17) Siakkou, E., Rutledge, M. T., Wilbanks, S. M., and Jameson, G.
N. L. (2011) Correlating crosslink formation with enzymatic activity in
cysteine dioxygenase. Biochim. Biophys. Acta, Proteins Proteomics 1814,
2003−2009.
(18) Li, W., Blaesi, E. J., Pecore, M. D., Crowell, J. K., and Pierce, B.
S. (2013) Second-Sphere Interactions between the C93−Y157 Cross-
Link and the Substrate-Bound Fe Site Influence the O2 Coupling
Efficiency in Mouse Cysteine Dioxygenase. Biochemistry 52, 9104−
9119.
ABBREVIATIONS
■
CDO, cysteine dioxygenase; p3MDO, 3-mercaptopropionate
dioxygenase from Pseudomonas aeruginosa; 3-MPA, 3-mercap-
toprionic acid; 3-SPA, 3-sulfinopropionic acid; CSA, cysteine
sulfinic acid; PIPES, piperazine-N,N′-bis(2-ethanesulfonic
acid); Tris, Tris(hydroxymethyl)aminomethane; HEPES, (4-
(2-hydroxyethyl)-1-piperazineethanesulfonic acid); MOPS, 3-
(N-morpholino)propanesulfonic acid; MES, 2-(N-
morpholino)ethanesulfonic acid; AMPSO, 3-([1,1-dimethyl-2-
hydroxyethyl]amino)-2-hydroxypropanesulfonic acid; CHES,
N-cyclohexyl-2-aminoethanesulfonic acid
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