Substrate Specificity in Thiol Dioxygenases - ACS Publications

Article Cite This: Biochemistry XXXX, XXX, XXX−XXX

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Substrate Specificity in Thiol Dioxygenases Sekotilani Aloi,† Casey G. Davies,† P. Andrew Karplus,‡ Sigurd M. Wilbanks,§ and Guy N. L. Jameson*,†,∥ †

Department of Chemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand Department of Biochemistry and Biophysics, Oregon State University, 2011 Ag & Life Sciences Building, Corvallis, Oregon 97331, United States § Department of Biochemistry, University of Otago, P.O. Box 56, Dunedin 9054, New Zealand ∥ School of Chemistry, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, 30 Flemington Road, Parkville, VIC 3010, Australia Biochemistry Downloaded from pubs.acs.org by IDAHO STATE UNIV on 05/02/19. For personal use only.

‡

S Supporting Information *

ABSTRACT: Thiol dioxygenases make up a class of ferrous iron-dependent enzymes that oxidize thiols to their corresponding sulfinates. X-ray diffraction structures of cysteinebound cysteine dioxygenase show how cysteine is coordinated via its thiolate and amine to the iron and oriented correctly for O atom transfer. There are currently no structures with 3mercaptopropionic acid or mercaptosuccinic acid bound to their respective enzymes, 3-mercaptopropionate dioxygenase or mercaptosuccinate dioxygenase. Sequence alignments and comparisons of known structures have led us to postulate key structural features that define substrate specificity. Here, we compare the rates and reactivities of variants of Rattus norvegicus cysteine dioxygenase and 3-mercaptopropionate dioxygenases from Pseudomonas aureginosa and Ralstonia eutropha (JMP134) and show how binary variants of three structural features correlate with substrate specificity and reactivity. They are (1) the presence or absence of a cis-peptide bond between residues Ser158 and Pro159, (2) an Arg or Gln at position 60, and (3) a Cys or Arg at position 164 (all RnCDO numbering). Different permutations of these features allow sulfination of L-cysteine, 3-mercaptopropionic acid, and (R)-mercaptosuccinic acid to be promoted or impeded.

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salt bridge with Arg60. Thus, all three functional groups of cysteine are used, ensuring the correct orientation for the oxygenation reaction. In contrast to cysteine, 3-mercaptopropionic acid (3-MPA) possesses only a thiolate and carboxylate group. Structures of Pa3MDO4 and Re3MDO13,14 show that 3MDO enzymes have a Gln in place of Arg60 that is present in CDO enzymes (i.e., are “Gln-type” rather than “Arg-type” CDO homologues13) and that 3MDO active sites have a different conserved Arg (residues 168 in Pa3MDO and 173 in Re3MDO) that replaces Cys164 of rat CDO (RnCDO). Our working model for how a substrate binds to the active site (presented in Figure 1b) is based on the assumptions that the carboxylate of 3-MPA forms a salt bridge with this Arg while allowing the thiolate and the subsequently bound dioxygen to coordinate to the iron in positions similar to those thought to occur in CDO (see Materials and Methods for details of the modeling). While this proposed binding mode differs from a thiolate-only binding

hiol dioxygenases make up a class of enzymes that catalyze the first step in the oxidative breakdown of thiols.1,2 They catalyze the addition of molecular oxygen to the thiol to form the corresponding sulfinate. These enzymes are found in plants,3 bacteria,4−6 fungi,7 and higher organisms.8 The range of compounds that are currently known to be substrates for these enzymes are presented in Scheme 1. All but cysteamine and the ethylene response factor contain at least one carboxylate and thus have the potential to make highly specific salt bridges with the guanidino group of an arginine residue. Nonetheless, the answer to the question of whether and how these residues contribute to differing specificities is not obvious. Mammalian cysteine dioxygenase (CDO) is the most well studied thiol dioxygenase, with spectroscopic and crystallographic studies providing concrete details about its substrate binding.9−11 Ferrous iron is held in octahedral geometry with three histidines coordinating the iron on one face. The other three sites are taken by water molecules or sometimes a chloride. Upon cysteine binding, the solvent molecules are displaced and cysteine coordinates to the iron via its thiolate and amine groups (Figure 1a), with the carboxylate forming a © XXXX American Chemical Society

Received: January 28, 2019 Revised: April 6, 2019

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DOI: 10.1021/acs.biochem.9b00079 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry Scheme 1. Known Substrates of Thiol Dioxygenases

Figure 1. Active site comparison of CDO and 3MDO. (a) Cysteine-bound CDO [Protein Data Bank (PDB) entry 4IEV] showing Arg60 forms a salt bridge with the carboxylate of the substrate cysteine and the presence of a Ser-Pro cis-peptide bond. The Cys93−Tyr157 thioether cross-link has been omitted for the sake of clarity. (b) Pa3MDO (PDB entry 4TLF, chain C with iron-bound waters removed) showing the lack of a cispeptide bond moves the backbone so that Arg168 (the equivalent of Cys164 in RnCDO) is in position to form a salt bridge with the carboxylate of 3-MPA in a binding mode we propose here. 3-MPA has been docked manually as described in the text. Picture produced with Chimera.12

mode proposed for Av3MDO,5 which does not include the substrate carboxylate−Arg168 interaction, in our view the model we propose has the advantage of allowing for the greatest possible mechanistic similarity to CDO. However, it is important to note that neither model is validated, and the question of how substrates bind to 3MDO remains open. Although Arg has a side chain that is longer than that of Cys, due to a change in the loop conformation compared to RnCDO,13 the Arg173 guanidino group does not intrude so far into the active sites of Pa3MDO and Re3MDO as one would expect from the RnCDO structure. Specifically, the removal of a cis-peptide bond (Ser158/Pro159 in RnCDO) is associated with a rotation and displacement of the 159−168 loop in Pa3MDO.4 This rotation of the residues around the active site ensures the hydroxyl of Tyr159 is placed in the correct position relative to iron for catalysis but moves the α-carbon of Arg168 away from the iron by ∼1 Å relative to Cys164 in RnCDO to accommodate the larger arginine residue. A bacterial enzyme from Variovorax paradoxus strain B4 was reported to convert mercaptosuccinate (MSA) to its sulfinate, although no direct confirmation of the sulfinated product was

obtained.2,15 Sequence alignment (Figure 2) shows that the residues equivalent to 62 and 168 in Pa3MDO are both Arg. This would make it possible for both of the carboxylates of mercaptosuccinate to be held in place by salt bridges, but there is no crystal structure to confirm this. Likewise, although partially characterized more than 10 years ago,16 cysteamine dioxygenase (ADO) is also structurally uncharacterized apart from the possible presence of a cysteine-tyrosine cross-link like in CDO.8,17,18 Similar structural information is needed for plant cysteine oxidases (PCO).3 Here, we sought to determine what features allow thiol dioxygenases to specifically recognize the carboxylates variously positioned on their substrates. Variants of Pa3MDO were constructed to investigate whether mercaptosuccinate activity could be introduced, while CDO variants were constructed to observe whether 3-MPA activity could be inserted. Finally, we provide the first characterization of Re3MDO kinetics and compare it with Pa3MDO kinetics. B


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Figure 2. Structure-based sequence alignment showing conserved residues within thiol dioxygenases. The 3MDOs from Pseudomonas aeruginosa (PDB entry 4TLF4) and Ralstonia eutropha (PDB entry 4QMA13) and CDOs from Rattus norvegicus (PDB entry 4IEV9) and Bacillus subtilis (PDB entry 4QM913) are compared with the sequence of MSDO from V. paradoxus (protein sequence AGU51201.1). Legend: *, Fe(II) binding His residues; !, Ser-His-Tyr “catalytic triad”; Y, additional substrate binding Tyr in CDOs; #, active site Arg and Gln residues distinguishing CDOs from 3MDOs and MSDO; F, two conserved active site Phe residues in 3MDOs; c, cis-Pro peptide bonds in CDOs. The alignment was generated using PROMALS19 and manually colored according to secondary structure as defined by DSSP:20 cyan for α-helix, blue for 310-helix, and green for βsheet.

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MATERIALS AND METHODS Pa3MDO Variants. The following variants were produced; R168A, R168C, and Q62R. All mutations were generated using primers (Invitrogen) containing a CGT-to-GCT, CGT-toTGC, or CAG-to-CTG 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 Pa3MDO coding region of the expression constructs (pPRIBA1/Pa3MDO/R168A, pPR-IBA1/Pa3MDO/R168C, and pPR-IBA1/Pa3MDO/Q62R) 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 those for wild-type (WT) Pa3MDO using Strep-tag technology.4 Both R168A and R168C did not fold properly as shown by varying amounts of the molecular chaperone GroEL present as an extra band on a sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−PAGE) gel at ∼66 kDa and its cofactor GroES at ∼21.5 kDa (Figure 3a). The identity of these bands was confirmed by mass spectrometry performed at the University of Otago Centre for Protein Research. The lower yield of these proteins and their instability (they easily precipitated) showed that R168 is important for the folding and stability of the protein and suggesting perhaps a structural role for the positive charge in addition to its role during catalysis. In contrast, Q62R was purified in a manner similar to that of WT Pa3MDO with 0.14 ± 0.06 (n = 4 purifications) endogenously bound iron per protein. Reconstitution with ferrous iron ammonium sulfate followed by treatment with Chelex produced protein with an iron occupancy of 0.6 ± 0.1, slightly lower than the wild-type value. RnCDO Variants. Variants of RnCDO from Rattus norvegicus, including R60Q and the double variant R60Q:C164R, were produced using primers (Invitrogen) containing a CAG-to-CTG and CGT-to-ACG codon sub-

Figure 3. SDS−PAGE gels of purified bacterial and mammalian variants used in this study: (a) WT Pa3MDO (15 μg), R168A (7 μg), R168C (6 μg), Q62R (11 μg), and WT Re3MDO (7.9 μg) and (b) WT RnCDO (15 μg), R60Q (15 μg), and R60Q:C164R (10 μg).

C


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Biochemistry stitution. A polymerase reaction and sequencing of the expression construct (CDO/pPR-IBA1/R60Q and CDO/ pPR-IBA1/R60Q:C164R) were carried out as described above for the Pa3MDO variants. Expression and purification were carried out as previously described,11 and a >98% pure and ∼20% cross-linked protein was confirmed by SDS−PAGE (Figure 3b) Re3MDO. Purified 3MDO from Ralstonia eutropha (15.8 mg/mL) was a kind gift from the Joint Centre for Structural Genomics (JCSG). Product Identification and Distribution. End-point analysis of the products was carried out using 1H nuclear magnetic resonance (NMR) using a Varian 500 MHz AR spectrometer as previously described.21 Reaction mixtures contained 30 μM enzyme and 10 mM substrate and were allowed to react for >10 h. Control experiments in the absence of protein determined the amount of non-enzymatic oxidation over the same time frame, under the same reaction conditions. When the product was unknown, the resultant solutions were then tested by mass spectrometry in negative ion mode on a Bruker microTOFQ instrument connected to a Dionex Ultimate 3000 high-performance liquid chromatography (HPLC) instrument. Kinetic Assays and Fitting. Kinetic assays were carried out using either Ellman’s reagent-based22 or HPLC-based assays21 developed in our laboratories and previously described in detail. As before,23,24 a range of buffers were tried, and by comparing results at overlapping pHs with different buffers, we identified a selection of buffers that produced consistent results over the pH range of 5.1−8.5. The following buffers were used: acetate, PIPES, TRIS, MOPS, and CHES (all at 100 mM, ionic strength of at least 150 mM). The pH-dependent data were modeled whereby the enzyme existed with two ionizable groups with only one active form of the enzyme (see Scheme 2). The pH profiles of kcat and

Km =

kcat/Km were fitted to the parameters given in Scheme 2, using eqs 1 and 2, respectively.25 The parameters from these fits were then used in eq 325 to simulate a fit for the pH profile of Km.

kcat = Km

kcat′ [H+] KES1

+1+

KES2 [H+]

(1)

kcat′ K m′ [H+] KE1

+1+

KE2 [H+]

[H+] KE1

[H+] KES1

+1+

+1+

KE2 [H+]

KES2 [H+]

) (3)

Resolution of Racemic MSA. A modified version of the method published by Shiraiwa et al.26 was used to resolve racemic MSA using (1S,2S)-2-amino-1-phenyl-1,3-propanediol (APP). A 2:1 resolving reagent:MSA molar ratio was mixed in 10−11 mL of methanol (HPLC grade, BDH Chemicals) and stirred for 2−3 h. The precipitate (R)-MSA·(S)-APP was filtered, collected, and washed with 6−8 mL of methanol. The collected filtrate was evaporated at 30 °C using a rotary evaporator to yield the (S)-MSA·(S)-APP salt as a brown syrup. Both the (R)-MSA·(S)-APP salt and the (S)-MSA·(S)APP syrup were dried under vacuum in a desiccator overnight. Purified (S)-MSA·(S)-APP salt was crystallized from 3 mL of methanol and dried under vacuum. To obtain purified (R)MSA and (S)-MSA, the resolving reagent was removed by treatment with Amberlite protonated resin. To both (R)-MSA· (S)-APP and (S)-MSA·(S)-APP was added 5−6 mL of H2O along with 2.5 g of resin per gram of salt. The mixtures were allowed to stand for >16 h at room temperature, while occasionally being stirred. Then, the resin was washed with 10 mL of H2O and the eluate freeze-dried. The freeze-dried samples were then recrystallized from water on ice. Docking Studies. The crystal structures of Pa3MDO (PDB entry 4TLF) and Re3MDO (PDB entry 4QMA) were combined with models of 3-MPA built and optimized in Avogadro27 and used within Chimera12 to conduct docking studies with Autodock vina.28 The standard options were chosen, and very similar structures were observed with the carboxylate of 3-MPA interacting with Arg168/173. These structures are similar to those produced through manual docking below (see below and the Supporting Information). Varying the search volume and/or employing flexible arginine residues within the structures did not greatly affect the binding modes produced. However, the algorithms used in these methods are unable to effectively model the Fe−S bond, so manual docking was applied to generate our final proposed model. Manual docking was informed by the position of substrate cysteine in the structure of rat CDO (PDB entry 4IEV) when overlaid with Pa3MDO. We assumed that dioxygen would coordinate in the same site as CDO, i.e., trans to His89 in Pa3MDO. This is purposefully not the same position (trans to His147) occupied by dioxygen in the crystal structure of Re3MDO (PDB entry 4QMA), because there is good reason to believe that the binding site seen in that crystal structure is not catalytically relevant. The main reason, as was explicitly pointed out in the original publication,13 is that studies on these thiol dioxygenases consistently imply that dioxygen binds only after the thiol substrate is present,5,29 yet in the 4QMA structure, the dioxygen seen is bound in the absence of a thiol substrate. On the basis of our docked model, we consider that the binding site seen for dioxygen in 4QMA can be rationalized as dioxygen binding as a carboxylate mimic in the same way carboxylates have been seen as peroxide mimics.30 Placement of the thiol of 3-MPA so that it occupies the position equivalent to that of cysteine in CDO allows the carboxylate to form a salt bridge with Arg168 by rotation of the CH2−CH2 and CH2−CO2− bonds of 3-MPA. Positioned for this salt-bridge interaction, the carboxylate occludes the space

Scheme 2. Enzyme Reaction Used To Model the pH Profiles

kcat =

(

K m′

(2) D


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Biochemistry needed for a water molecule to bind to iron trans to His142. This is consistent with the previous observation that Arg168 would clash with the amine of cysteine showing that 3MDOs cannot bind substrate cysteine in the canonical bidentate orientation.13 The Mössbauer spectrum of 3-MPA-bound Pa3MDO4 has parameters (δ = 1.11 mm/s; ΔEQ = 2.5 mm/ s) consistent with a five/six-coordinate resting state.31,32 If the carboxylate occupies the site opposite His142 and if a water molecule can occupy the dioxygen binding site trans to His89, as it does in CDO, the model is consistent with the Mössbauer data. However, if the iron coordination site opposite His142 is left completely empty, then the geometry at the iron will be a mixture of four/five-coordinate and inconsistent with the experimental Mössbauer parameters. Nevertheless, because the primary interaction is with Arg168, we expect the interaction of the carboxylate of 3-MPA with iron is likely to be weak. Thus, the carboxylate was placed so one oxygen would also weakly coordinate the Fe(II) atom. This was achieved by overlaying the C−O with the CH−NH2 of cysteine in rat CDO and the putative O2 bound to Re3MDO,13 which also occupies a similar position. The 3-MPA was then moved or rotated to ensure interaction distances and angles were of reasonable magnitudes: Fe−S and Fe−O bond distances (∼2.2−2.6 Å), salt-bridge N···O distances (∼2.6−3.0 Å), and NH···O angles (∼140−180°). Finally, the structure was visually optimized using the surfaces of the bound 3-MPA and binding pocket (Figure S1). It can be seen that Phe78 makes van der Waals contacts with both substrate methylenes and provides excellent complementarity to this substrate binding mode. This structure therefore provides us with an excellent model for exploring substrate binding but, in the absence of a crystal structure, still needs to be validated. This proposed substrate binding mode differs from that put forward by Pierce et al.5 for the 3MDO of Azotobacter vinelandii that posited a thiolate-only coordination to the iron with the carboxylate of 3-MPA hydrogen bonded to the conserved active site Tyr (equivalent to Tyr159 in Pa3MDO) rather than formation of a salt bridge with an Arg. However, AvMDO is able to oxidize cysteamine to hypotaurine, while Pa3MDO4 cannot, which implies that there are differences across the family that could reflect differences in the modes of substrate binding.

Figure 4. 1H NMR spectra showing the product distribution at pH 7.5 of the aerobic reactions of Q62R Pa3MDO with L-cysteine (Renantiomer) and 3-MPA to form 3-SPA, CSA, and respective disulfides (green). Reference spectra of 3-SPA (red) and CSA (blue) standards are provided for comparison.

Table 1. Sulfinate/Thiol Coupling Efficiencies at pH 6.5 Using 1H NMR coupling efficiency (%) substrate Pa3MDO

RnCDO

wild type

3-MPA L-Cys R-MSA

Q62R

100 31 0 coupling efficiency (%)

98 31 99

substrate

wild type

R60Q

R60Q:C164R

3-MPA L-Cys

0 95

0 91

0 38

fitted to a simple model with two ionizable groups and only one active form (see Scheme 2). The parameters determined alongside those of wild-type Pa3MDO as a comparison are listed in Table 2. Oxidation of cysteine to cysteine sulfinate is possible in these enzymes, but as shown previously, cysteine is not their main substrate.4,23 For this reason, cysteine oxidation was measured at only a few pH values to compare with wild-type Pa3MDO. The plots of kcat and kcat/Km versus pH are presented in Figure S2 and show that kcat is increased compared to that of the wild type as it is when 3-MPA is the substrate (see Table 2). Pa3MDO Q62R Has (R)-Mercaptosuccinate Dioxygenase Activity. Our rationale for substituting glutamine 62 with arginine was to see if it was able to react with mercaptosuccinic acid. Initial 1H NMR studies of Q62R with racemic MSA showed that it did react but that it appeared to specifically oxygenate one of the enantiomers. For this reason, we resolved racemic MSA into its (R)- and (S)-enantiomers and showed that indeed (R)-MSA is selectively oxidized to the sulfinic acid while (S)-MSA forms the disulfide (Figure 6). The 1 H NMR results were confirmed by mass spectrometry (m/z 180.9828). Kinetic analysis showed that the reaction was very slow, but on the basis of the low Km of ∼0.5 mM (Figure 5), we infer that the (R)-MSA substrate binds relatively tightly in the active site, which is consistent with its two carboxyl groups interacting with Arg62 and Arg168. This was confirmed by competition experiments that showed (R)-MSA was preferred

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RESULTS Pa3MDO Q62R Retains 3-MPA and Cysteine Dioxygenase Activity. Initial experiments with 1H NMR at pH 7.5 in phosphate buffer showed that the Q62R variant could form the sulfinate of both 3-MPA and cysteine (Figure 4). 3-MPA was converted almost stoichiometrically to 3-sulfinopropionic acid (3-SPA), while cysteine was converted to both cystine and cysteinesulfinic acid (CSA). Thus, the variant behaves very much like wild-type Pa3MDO.23 The coupling efficiencies at pH 6.5 [amount of sulfinate formed/amount of thiol depleted (see the Supporting Information)] were 98% for 3-MPA and 31% for cysteine, which are indistinguishable from the efficiencies of wild-type (WT) Pa3MDO4 (Table 1). Then, pH-dependent kinetic studies using both 3-MPA and cysteine as substrate were carried out. Both the kcat and the kcat/Km of 3-MPA oxidation by Q62R Pa3MDO showed a bellshaped dependence on pH and are compared with previously published wild-type data (Figure 5). The values of kcat and Km for Q62R are similar but higher, with the maximum value of kcat shifted to a higher pH (∼0.5 pH unit). These data could be E


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Figure 5. (a−c) pH-dependent kinetic parameters of 3-MPA oxidation by WT Pa3MDO (red, previously published) and Q62R Pa3MDO (black). (d−f) (R)-MSA oxidation by Q62R Pa3MDO. (g−i) 3-MPA oxidation by Re3MDO. The following buffers were used: (●) acetate, (◇) piperazine, (▲) MES, (○) PIPES, (mostly filled circles) MOPS, (◆) potassium phosphate, (□) TRIS, (△) AMPSO, and (■) CHES.

Table 2. pH-Independent Parameters Determined by Fitting of pH-Dependent Values of kcat and kcat/Km According to Scheme 2 −1

kcat′ (s ) Km′ (mM) kcat′/Km′ (M−1 s−1) pKE1 pKE2 pKES1 pKES2

WT Pa3MDO (3-MPA)a

Q62R Pa3MDO (3-MPA)

Q62R Pa3MDO (R-MSA)

Re3MDO (3-MPA)

0.28 ± 0.03 0.8 ± 0.2 350 ± 30 5.8 ± 0.2 7.2 ± 0.2 5.0 ± 0.3 7.0 ± 0.2

0.67 ± 0.03 0.9 ± 0.2 740 ± 40 6.5 ± 0.3 7.0 ± 0.2 5.8 ± 0.2 7.3 ± 0.2

0.020 ± 0.002 0.5 ± 0.2 40 ± 10 6.5 ± 0.2 8.5 ± 0.2 6.5 ± 0.2 8.3 ± 0.2

0.35 ± 0.03 0.7 ± 0.2 500 ± 50 5.8 ± 0.2 6.9 ± 0.2 5.5 ± 0.2 7.0 ± 0.2

a

Previously published.23

MPA into 3-SPA with only negligible amounts of disulfide formed. A high coupling efficiency of ∼100% was determined for this reaction (Table 1). The kinetics of Re3MDO were measured using our Ellman’s assay over a pH range similar to that of Pa3MDO and produced similar results (Figure 5g−i and Table 2).23 Enzyme activity with L-cysteine analyzed by our HPLC− ELSD method showed no CSA formation at concentrations of substrate and enzyme previously used for Pa3MDO.23 Therefore, even higher concentrations of Re3MDO (120− 400 μM) were tried, but even under these extreme conditions and extended reaction times (>10 h), no CSA peak was

over cysteine (Supporting Information). However, both competition experiments and the kinetic parameters (Table 2) show that 3-MPA remains the preferred substrate. Re3MDO Is a 3-Mercaptopropionate Dioxygenase with Only Minor Cysteine Dioxygenase Activity. Enzyme activity was determined using both 3-MPA and L-cysteine as substrates. Enzyme activity with 3-MPA analyzed by mass spectrometry showed formation of the dioxygenated product 3-SPA (m/z 136.9914), thereby confirming that Re3MDO has 3MDO activity as previously predicted.13 1H NMR analysis was performed to determine the product distribution of the reaction. Figure 7 shows that Re3MDO efficiently converts 3F


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Figure 6. 1H NMR spectra showing the product distribution at pH 7.5 of the aerobic reaction of Q62R Pa3MDO with (R)- and (S)MSA. The spectra of the standard solutions before reaction are also provided for comparison.

Figure 8. 1H NMR spectra showing the product distribution at pH 7.5 of the aerobic reaction of R60Q and R60Q:C164R RnCDO with L-cysteine (R-enantiomer) and D-cysteine to form L- and D-CSA (blue) and L,L- and D,D-cystine (green).

increasing level of cystine formation as the wild type is changed by mutation of first one and then two residues involved in the active site. Over a range of conditions and pH values, no 3-SPA product was observed by either 1H NMR or Ellman’s assay for any of the rat CDO variants. It is therefore not possible to engineer 3MDO activity by simply swapping the arginine from position 60 to 164.

Figure 7. 1H NMR spectra showing the product distribution at pH 6.5 of the aerobic reaction of Re3MDO with L-cysteine (Renantiomer) and 3-MPA to form 3-SPA (red), CSA (blue), and cystine (green).

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DISCUSSION This study relates specific structural features to reactivity. Detailed crystallographic studies of cysteine dioxygenase9,33 and related enzymes4,13 suggested the hypothesis that saltbridge interactions are important for correct substrate orientation in all thiol dioxygenases where the substrate contains a carboxylate. Our previous pH-dependent kinetic studies of RnCDO24 and Pa3MDO23 support the importance of Tyr157 (RnCDO numbering) and made us further hypothesize that the relative orientation of the thiolate− iron−dioxygen and tyrosine hydroxyl4 is crucial to reactivity in a range of substrates. To test the contribution of salt bridges to substrate specificity in thiol dioxygenases, we compared bacterial and mammalian enzymes in combination with selected variants. By Converting Gln62 into an Arg, We Have Added Reactivity to Pa3MDO. The Q62R variant is able to stereospecifically oxidize (R)-MSA, while (S)-MSA is converted to the disulfide. We used NMR and mass spectrometry to directly observe the product (R)-MSS for the first time. A recent study of a true MSDO15 showed depletion of dioxygen and total thiol through an Ellman’s assay using a racemic mixture of MSA. Our results indicate that MSDO should be stereospecific for the (R)-isomer, and this will be an interesting hypothesis to test. Furthermore, because (S)-MSA is also oxidized by our variant but to the disulfide, it will be necessary to confirm the kinetics and product distribution by measuring sulfinate formation directly as we have in this study. Despite the expanded reactivity for Q62R Pa3MDO, 3-MPA is still its preferred substrate with a kcat/Km (Figure 5, Table 2,

observed in the chromatogram. To confirm these results, 1H NMR analysis was performed. The measured spectra showed minimal CSA formation but considerable cystine formation. RnCDO R60Q and R60Q:C164R Retain Cysteine Dioxygenase Activity but Are Unable To Oxidize 3MPA. 1H NMR showed that CDO activity was retained by both variants, but in the case of the double variant, the CDO activity was reduced ∼10-fold and the major product of the reaction was cystine (Figure 8). These results show the importance of Arg60 in orienting substrate cysteine correctly in the active site. Comparison of the kinetic parameters of cysteine depletion and CSA formation for WT, R60Q, and R60Q:C164R at pH 8.1 (Table 3) sheds further light on their reactivity. Replacement of Arg60 with Gln does not remove CDO activity, and the kinetic parameters for CSA formation are similar to those of WT; however, oxidation of cysteine to cystine now becomes more prominent. While productive binding for CDO activity has not been compromised, the increase in the level of incorrect products suggests that the enzyme is less able to reject unproductive binding as we described recently when using the non-native substrate aminobenzene thiol.32 The double variant significantly decreases CDO activity, however, with a lower kcat and a higher Km. We suspect that the presence of the Arg164 side chain in the active site pocket sterically hinders the productive mode of Cys binding. This decrease in the level of productive binding relative to unproductive continues the trend of an G


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Biochemistry Table 3. Kinetic Parameters Measured at pH 8.1 in TRIS Buffer at 22 °C cysteine depletion

CSA formation

enzyme

kcat (s−1)

Km (mM)

kcat (s−1)

Km (mM)

WT RnCDO R60Q R60Q:C164R

0.26 ± 0.01 0.46 ± 0.06 0.37 ± 0.04

6.1 ± 0.9 17 ± 6 29 ± 7

0.26 ± 0.01 0.33 ± 0.02 0.024 ± 0.005

6.1 ± 0.9 5±2 9±5

Scheme 3. Important Active Site Features of the Three Thiol Dioxygenases Studied Here and Our Proposal for How Their Respective Substrate Can Bind and Conserve the Geometry of Catalysis

water molecule that is bound to the iron atom and must be displaced by dioxygen and also interacts with tyrosine 159. The increase observed in pKES2 might also be a result of the buildup of negative charge caused by bound (R)-MSA. Changes at Positions 60 and 164 (rat numbering) Do Not Convert the Mammalian Enzyme RnCDO into a 3MDO. Indeed, the R60Q and R60Q:C164R RnCDO variants gain no additional reactivity, while losing specificity for L-Cys, with the coupling efficiency dropping markedly. We infer that the cis-peptide bond found in many CDO enzymes (Ser158/ Phe159 in RnCDO) but not 3MDO enzymes is vital for the correct spatial orientation of the active site residues involved in substrate binding. This inference is supported by the only bacterial CDO that has both been proven to have CDO activity and had its structure resolved (Bacillus subtilis, PDB entry 4QM9).34 The structure has a cis-peptide bond and overlays remarkably well with that of rat CDO. This bond occurs in the sequence as Ser-Pro (highlighted in Figure 2 as c), although many other sequences of the form “Xaa-Pro” can adopt a cis conformation.35 Structural characterization of the CDO from Bacillus cereus34 would provide a stringent test of this correlation: it has CDO activity but lacks the proline usually associated with a cis-peptide bond. We have previously described the structural implications of this cis-peptide bond.4 Comparison of the structures of Pa3MDO and RnCDO showed that the combination of the cis-peptide bond and a covalent linkage between tyrosine 157 and cysteine 93 positions the RnCDO tyrosine hydroxyl identically to that of tyrosine 159 in Pa3MDO that lacks both of those structural features. These absences also provide more room for arginine 168 (Pa3MDO) by moving its α-carbon away from the iron and allowing it, in our model (Figure 1), to interact favorably with the carboxylate of 3-MPA. Altogether, the Data Support the Importance of Three Structural Features in Defining Substrate Specificity and Reactivity. They are (1) the presence or absence of a cis-peptide bond between residues Ser158 and

and Figure S2) that is approximately 20 times larger for 3-MPA than for the other substrates, cysteine and (R)-MSA. Thus, this variant of Pa3MDO remains a 3MDO even with such a substantial change to the active site. This indicates that although the enzyme has been named a “Gln-type” thiol dioxygenase,13 Gln62 is not required for catalysis. The lack of an important role for Gln62 in binding or catalysis is fully consistent with our proposal for how 3-MPA binds to the active site (Figure 1b) yet leaves the reason why Gln62 is so well conserved uncertain. It may be that there is natural selection for the narrow substrate specificity supported by glutamine at this position. The introduced guanidino group aids (R)-MSA binding through salt bridges to Arg62 and Arg168 (Pa3MDO numbering). This shows itself by a low Km (0.5 mM) that is independent of pH. In contrast, 3-MPA shows a slightly increased Km (0.9 mM) compared to that of the wild type (0.8 mM) and cysteine shows an even greater increase (20 mM vs 15 mM). This is consistent with the extra arginine group destabilizing cysteine binding, presumably through interactions with the amine, but the coupling efficiency remains similar between Q62R and the wild type, showing that the substrates are not being drawn into unproductive conformations. Proton ambiguity makes assignment of pKE1 and pKE2 difficult if not impossible from our data (Table 2). pKE1 increases upon mutation of Gln62 to Arg but is invariant with substrate, suggesting it is protein-based. In contrast, pKE2 increases only in the presence of (R)-MSA, implying it might be related to deprotonation of the substrate. In contrast, the values of pKES1 and pKES2 refer to deprotonation of the ES complex and show an increase in both values as the substrate is changed from 3-MPA to (R)-MSA (Table 2). (R)-MSA shows increased pK values compared to those of 3-MPA, consistent with a buildup of greater charge. The increase in pKES1 may reflect the requirement that a higher pH be reached before both carboxylates are deprotonated and both salt bridges formed. Previously,23 we have suggested pKES2 refers to the H


Article

Biochemistry

Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase. J. Biol. Chem. 290 (40), 24424−24437. (5) Pierce, B. S., Subedi, B. P., Sardar, S., and Crowell, J. K. (2015) The “Gln-Type” Thiol Dioxygenase from Azotobacter vinelandii Is a 3-Mercaptopropionic Acid Dioxygenase. Biochemistry 54 (51), 7477− 7490. (6) Bruland, N., Wübbeler, J. H., and Steinbüchel, A. (2009) 3mercaptopropionate dioxygenase, a cysteine dioxygenase homologue, catalyzes the initial step of 3-mercaptopropionate catabolism in the 3,3-thiodipropionic acid-degrading bacterium variovorax paradoxus. J. Biol. Chem. 284 (1), 660−72. (7) 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 (5), e456−62. (8) Wang, Y., Griffith, W. P., Li, J., Koto, T., Wherritt, D. J., Fritz, E., and Liu, A. (2018) Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine. Angew. Chem., Int. Ed. 57 (27), 8149−8153. (9) Driggers, C. M., Cooley, R. B., Sankaran, B., Hirschberger, L. L., Stipanuk, M. H., and Karplus, P. A. (2013) Cysteine Dioxygenase Structures from pH 4 to 9: Consistent Cys-Persulfenate Formation at Intermediate pH and a Cys-Bound Enzyme at Higher pH. J. Mol. Biol. 425 (17), 3121−3136. (10) Gardner, J. D., Pierce, B. S., Fox, B. G., and Brunold, T. C. (2010) Spectroscopic and Computational Characterization of Substrate-Bound Mouse Cysteine Dioxygenase: Nature of the Ferrous and Ferric Cysteine Adducts and Mechanistic Implications. Biochemistry 49 (29), 6033−6041. (11) 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 (1), 257−264. (12) Pettersen, E. F., Goddard, T. D., Huang, C. C., Couch, G. S., Greenblatt, D. M., Meng, E. C., and Ferrin, T. E. (2004) UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25 (13), 1605−12. (13) Driggers, C. M., Hartman, S. J., and Karplus, P. A. (2015) Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs. Protein Sci. 24 (1), 154−61. (14) 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 (3), 910−21. (15) Brandt, U., Galant, G., Meinert-Berning, C., and Steinbüchel, A. (2019) Functional analysis of active amino acid residues of the mercaptosuccinate dioxygenase of Variovorax paradoxus B4. Enzyme Microb. Technol. 120, 61−68. (16) Dominy, J. E., Jr., Simmons, C. R., Hirschberger, L. L., Hwang, J., Coloso, R. M., and Stipanuk, M. H. (2007) Discovery and characterization of a second mammalian thiol dioxygenase, cysteamine dioxygenase. J. Biol. Chem. 282 (35), 25189−98. (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 (12), 2003−2009. (18) 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 (18), 12188−12201. (19) Pei, J., Kim, B.-H., Tang, M., and Grishin, N. V. (2007) PROMALS web server for accurate multiple protein sequence alignments. Nucleic Acids Res. 35 (Suppl. 2), W649−W652. (20) Joosten, R. P., te Beek, T. A. H., Krieger, E., Hekkelman, M. L., Hooft, R. W. W., Schneider, R., Sander, C., and Vriend, G. (2011) A series of PDB related databases for everyday needs. Nucleic Acids Res. 39 (Suppl.1), D411−D419.

Pro159, (2) an Arg or a Gln at position 60, and (3) a Cys or an Arg at position 164 (all RnCDO numbering). Different permutations of these features allow sulfination of L-cysteine versus 3-MPA versus R-MSA to be promoted. These three features are emphasized in Scheme 3, which also shows our proposal for the three carboxylate-containing thiol substrates in the active sites.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.9b00079. 3-MPA docking, coupling efficiency, cysteine oxidation by Q62R Pa3MDO, and competition experiments (PDF) Accession Codes

Pa3MDO, Q9I0N5; Re3MDO, Q46R41; VpMSDO, T1XFB1; RnCDO, P21816; BsCDO, O32085; BcCDO, Q81CX4.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Guy N. L. Jameson: 0000-0001-9416-699X Author Contributions

S.A. carried out the majority of the work, including all NMR and kinetic investigations, resolving R- and S-MSA, and expression and purification of protein. C.G.D. carried out the pH-dependent kinetic investigations of Q62R Pa3MDO with R-MSA and R60Q:C164R RnCDO with L-cysteine. P.A.K. helped analyze the data. S.M.W. helped design the experiments and provided expert supervision. G.N.L.J. provided overall leadership and wrote the manuscript with input from all coauthors. Funding

The authors thank the University of Otago for a scholarship that allowed S.A. to study for an M.Sc. and the National University of Samoa for leave to carry out further experiments. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Dr. Egor P. Tchesnokov is thanked for initial design and characterization of some of the variants described here. REFERENCES

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Article

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Substrate Specificity in Thiol Dioxygenases - ACS Publications

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