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 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 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.
T
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 dioxygenase 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 3MPA, 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.
hiol dioxygenases are non-heme mononuclear iron enzymes 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-mercaptopropionate 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 © XXXX American Chemical Society
Received: November 6, 2015 Revised: February 11, 2016
A
DOI: 10.1021/acs.biochem.5b01203 Biochemistry XXXX, XXX, XXX−XXX
Article
Biochemistry 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 crosslinked 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. NMR Investigation of WT p3MDO Reactivity. 1H 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.
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MATERIALS AND METHODS Expression and Purification of WT p3MDO. Expression and purification of WT p3MDO (NP_251292.1) using StrepTag 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
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. 20and 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-toTGC codon substitution. A polymerase chain reaction catalyzed by PfuUltra High-Fidelity DNA polymerase (Stratagene, 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
DOI: 10.1021/acs.biochem.5b01203 Biochemistry XXXX, XXX, XXX−XXX
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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
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. 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
Table 1. X-ray Data Collection, Reduction, and Crystallographic Refinement Statistics of G95C p3MDO Resting State (PDB 4WVZ) resolution range (Å) unique reflections multiplicity completeness (%) mean I/σ(I) R-meas CC1/2 R-work R-free number of non-hydrogen atoms macromolecules ligands water protein residues RMS bonds (Å) RMS angles (deg) Ramachandran favored (%) Ramachandran outliers (%) Clashscore average B-factor (Å2)
Scheme 1. Enzyme Reaction Used To Model the pH Profiles
a
kcat = Km
k′cat
(
+
[H ] KES1
KES2 [H+]
)
(1)
k ′cat K ′m
(
[H+] KE1
K ′m Km =
+1+
(
(
+1+
[H+] KE1
KE2 [H+]
+1+
+
[H ] KES1
+1+
)
KE2 [H+]
KES2 [H+]
)
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. kcat =
39.91−2.09 (2.16−2.09)a 51609 (4897)a 7.0 (3.8)a 99.60 (97.07)a 28.13 (4.35)a 0.050 1 (0.92)a 0.177 (0.241)a 0.227 (0.300)a 6930 6436 4 560 794 0.006 1.00 96 0 3.79 36
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(2)
RESULTS
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
) (3) C
DOI: 10.1021/acs.biochem.5b01203 Biochemistry XXXX, XXX, XXX−XXX
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Figure 2. 1H 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
a
condition
kcat (s−1)
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 phosphate 8.0 Tris 8.0 Tris 8.5 AMPSO 8.5
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.11 0.14 0.22 0.23 0.19 0.20 0.13 0.11 0.10 0.10 0.10 0.03 0.05 0.03 0.02
KM (mM) a
0.01 0.02 0.02 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01
4 4 5 0.9 1.0 1.5 0.5 0.9 0.8 0.7 0.6 0.5 0.7 1.4 1.1
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
1 2 2 0.1 0.1 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.3 0.2
WT p3MDO with cysteine
kcat/Km (M−1 s−1) 30 40 50 260 200 130 270 130 120 130 180 60 70 18 18
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
10 20 30 50 40 20 50 20 20 20 30 10 20 5 4
kcat (s−1)
KM (mM)
kcat/Km (M−1 s−1)
0.011 ± 0.001 0.022 ± 0.002
16 ± 2 16 ± 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 ± 2 13 ± 2
1.5 ± 0.3 1.6 ± 0.3
0.013 ± 0.001
12 ± 2
1.1 ± 0.3
Standard errors associated with the Fit are reported.
(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
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 D
DOI: 10.1021/acs.biochem.5b01203 Biochemistry XXXX, XXX, XXX−XXX
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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. 1 H 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 rate of cystine formation =
X-ray Crystallography Characterization of the G95C p3MDO Variant. To probe the effect of a mammalian CDOlike 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
0.28 0.8 5.8 7.2 5.0 7.0
0.08 15 6.9 7.2 6.9 7.2
[H+] K
{1 + } (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.
k′cat (s−1) K′m (mM) pKE1 pKE2 pKES1 pKES2
kcystine[p3MDO]T [cysteine]T
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
DOI: 10.1021/acs.biochem.5b01203 Biochemistry XXXX, XXX, XXX−XXX
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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).
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.
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 uncrosslinked 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). Kinetic Studies of the G95C p3MDO Variant. The G95C p3MDO variant showed reduced activity toward both 3-
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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 F
DOI: 10.1021/acs.biochem.5b01203 Biochemistry XXXX, XXX, XXX−XXX
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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
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 (30-fold excess over 3-MPA (data not shown). In contrast, pKES1 for cysteine cannot reflect the pKa of the cysteine carboxylate because pKES1 for cysteine is almost 2 pK
dioxygenase, and through crystallography, spectroscopy and kinetics we propose how differences in the active sites of CDO and p3MDO control reactivity. 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). Mössbauer 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. G
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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 Michaelis−Menten kinetics. 1H 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 intermediate,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 uncrosslinked 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. One would expect G95C would have a different pH maximum caused by a decrease in the pKa of the phenol
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 computational 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. 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 H
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Cysteine Dioxygenases: a New Route of Cysteine Degradation for Eubacteria. J. Bacteriol. 188, 5561−5569. (4) Wenning, L., Stoveken, N., Wubbeler, J. H., and Steinbuchel, A. (2016) Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16. Appl. Environ. Microbiol. 82, 910−921. (5) Kasperova, A., Kunert, J., Horynova, M., Weigl, E., Sebela, M., Lenobel, R., and Raska, M. (2011) Isolation of recombinant cysteine dioxygenase protein from Trichophyton mentagrophytes. Mycoses 54, e456−462. (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, purification, and characterization of recombinant rat cysteine dioxygenase. J. Biol. Chem. 280, 9865−9869. (7) Gardner, J. D., Pierce, B. S., Fox, B. G., and Brunold, T. C. (2010) Spectroscopic and Computational Characterization of SubstrateBound 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 implications of persulfenate and persulfide binding in the active site of cysteine dioxygenase. Biochemistry 52, 7606−7617. (10) Tchesnokov, E. P., Wilbanks, S. M., and Jameson, G. N. L. (2012) A Strongly Bound High-Spin Iron(II) Coordinates Cysteine and Homo-cysteine in Cysteine Dioxygenase. Biochemistry 51, 257− 264. (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. (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 CrossLink and the Substrate-Bound Fe Site Influence the O2 Coupling Efficiency in Mouse Cysteine Dioxygenase. Biochemistry 52, 9104− 9119. (19) Davies, C. G., Fellner, M., Tchesnokov, E. P., Wilbanks, S. M., and Jameson, G. N. L. (2014) The Cys-Tyr Cross-Link of Cysteine Dioxygenase Changes the Optimal pH of the Reaction without a Structural Change. Biochemistry 53, 7961−7968.
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.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +64 3 479 8028. E-mail:
[email protected]. 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.
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ABBREVIATIONS CDO, cysteine dioxygenase; p3MDO, 3-mercaptopropionate dioxygenase from Pseudomonas aeruginosa; 3-MPA, 3-mercaptoprionic 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-(Nmorpholino)ethanesulfonic acid; AMPSO, 3-([1,1-dimethyl-2hydroxyethyl]amino)-2-hydroxypropanesulfonic acid; CHES, N-cyclohexyl-2-aminoethanesulfonic acid
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REFERENCES
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