Oxidized Forms of Ergothioneine Are Substrates for Mammalian Thioredoxin Reductase
"> Figure 1
<p>Structure of EGT, Asc and their oxidized forms. (<b>Left</b>) Asc• and dehydroascorbate are known substrates for Sec-TrxR [<a href="#B26-antioxidants-11-00185" class="html-bibr">26</a>,<a href="#B27-antioxidants-11-00185" class="html-bibr">27</a>]. (<b>Right</b>) EGT has two tautomeric forms: thione (EGT) and thiol (ESH), with the thione being highly favored in neutral aqueous solution. This study investigated whether oxidized forms of EGT are also substrates for Sec-TrxR. The oxidized forms of EGT and Asc have clear structural similarities, as highlighted in blue and red. Sec-TrxR, a selenoenzyme, is also able to reduce the number of other small molecule substrates, including <span class="html-italic">S</span>-nitrosoglutathione, lipoic acid/lipoamide, lipid hydroperoxides, and ubiquinone [<a href="#B28-antioxidants-11-00185" class="html-bibr">28</a>,<a href="#B29-antioxidants-11-00185" class="html-bibr">29</a>,<a href="#B30-antioxidants-11-00185" class="html-bibr">30</a>,<a href="#B31-antioxidants-11-00185" class="html-bibr">31</a>].</p> "> Figure 2
<p>Mass spectra of EGT and 2-thioHis before and after oxidation with H<sub>2</sub>O<sub>2</sub>. (<b>a</b>) Mass spectrum of EGT freshly prepared in deionized water. (<b>b</b>) Mass spectrum of EGT/H<sub>2</sub>O<sub>2</sub> (2:1) in deionized water following a 10 min incubation with H<sub>2</sub>O<sub>2</sub>. The inset is a close-up of <span class="html-italic">m</span>/<span class="html-italic">z</span> 229 and <span class="html-italic">m</span>/<span class="html-italic">z</span> 230. (<b>c</b>) Mass spectrum of 2-thioHis freshly prepared in deionized water. (<b>d</b>) Mass spectrum of 2-thioHis/H<sub>2</sub>O<sub>2</sub> (2:1) in deionized water following a 10 min incubation with H<sub>2</sub>O<sub>2</sub>.</p> "> Figure 3
<p>Consumption of NADPH by Sec-TrxR is stimulated by the presence of 2-thioHis disulfide, but not in its absence. The blue line shows that NADPH is only consumed when 2-thioHis disulfide is added to the assay. Control conditions are described in the legend at the right of the figure.</p> "> Figure 4
<p>Unoxidized EGT and 2-thioHis stimulate consumption of NADPH by Sec-TrxR. Absorbance versus time plot showing consumption of NADPH by Sec-TrxR over a 10 min time period in the presence of EGT or 2-thioHis, but in the absence of oxidant.</p> "> Figure 5
<p>Consumption of NADPH by GR is stimulated by the presence of 2-thioHis disulfide and GSH. The green line shows that NADPH is only consumed when 2-thioHis disulfide and GSH are added to the assay. Control conditions are described in the legend at the right of the figure. As shown by the orange line, GR shows very poor activity toward the disulfide form of 2-thioHis when GSH is not present.</p> "> Figure 6
<p>Equations that describe the mechanism of reduction of ESSE by GR. The figure and equations are adapted from the work of Eyer and Prodhradský, who elucidated the mechanism of reduction of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) by GR [<a href="#B47-antioxidants-11-00185" class="html-bibr">47</a>].</p> "> Figure 7
<p>Michaelis–Menten curves for 2-thioHis-disulfide and ESSE with GR/GSH. (<b>A</b>) Activity of GR/GSH with 2-thioHis oxidized by H<sub>2</sub>O<sub>2</sub>. (<b>B</b>) Activity of GR/GSH with EGT oxidized by H<sub>2</sub>O<sub>2</sub>.</p> "> Figure 8
<p>Mass spectra of EGT before and after oxidation with <sup>1</sup>O<sub>2</sub>. (<b>A</b>) MS of EGT in deionized water in the absence of <sup>1</sup>O<sub>2</sub>. (<b>B</b>) MS of EGT oxidized with <sup>1</sup>O<sub>2</sub> (rose bengal + light).</p> "> Figure 9
<p>Pathway for the formation of ESSE from 5-oxo-EGT and ESH. The <span class="html-italic">m</span>/<span class="html-italic">z</span> values for each species are provided. This figure was adapted from the GSH/EGT cycle proposed by Gründemann and coworkers [<a href="#B20-antioxidants-11-00185" class="html-bibr">20</a>]. Note that R = −CH<sub>2</sub>CH(COO<sup>−</sup>)N<sup>+</sup>(CH<sub>3</sub>)<sub>3</sub>.</p> "> Figure 10
<p>Absorbance versus time curves for the reaction of TrxR with EGT or 2-thioHis oxidized by <sup>1</sup>O<sub>2</sub>. (<b>A</b>) Change in absorbance over time plot for oxidized 2-thioHis and controls. (<b>B</b>) Change in absorbance over time plot for oxidized EGT and controls. All samples have NADPH added to them. All samples have Sec-TrxR added to them except the blue lines.</p> "> Figure 11
<p>Mass spectra of <sup>1</sup>O<sub>2</sub>-oxidized EGT followed by immediate addition of Sec-TrxR and NADPH. Comparison of the data above with the data in <a href="#antioxidants-11-00185-f008" class="html-fig">Figure 8</a>B shows the disappearance of the peaks corresponding to 5-oxo-EGT and 5-hydroxy-EGT. Our interpretation is that Sec-TrxR can directly reduce the 5-oxo and 5-hydroxy forms back to EGT.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Synthesis of L-2-Thiohistidine
2.3. Enzyme Production
2.4. MS Analysis of EGT Oxidized by H2O2
2.5. Sec-TrxR Activity Assay with ESSE
2.6. GR/GSH Activity Assay with ESSE
2.7. NMR Experiments with EGT and H2O2
2.8. p-Nitrosodimethylaniline (RNO) Bleaching
2.9. MS Analysis of EGT Oxidized by 1O2
2.10. Sec-TrxR Activity Assay with EGT Oxidized by 1O2
2.11. MS Analysis of 1O2-Oxidized EGT following Reduction with Sec-TrxR
2.12. Selenium Dependency of ESSE Recycling by TrxR Experiments
3. Results and Discussion
3.1. Enzymatic Reduction of ESSE with TrxR
3.2. Enzymatic Reduction of ESSE with GR/GSH
3.3. Activity of Sec-TrxR toward 2-ThioHis and EGT Oxidized with 1O2
3.4. Selenium Dependence of the Reactions
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Enzyme | Substrate | KM (μM) | kcat (min−1) | kcat/KM (min−1 M−1) |
---|---|---|---|---|
Sec-TrxR | 2-thioHis disulfide | 930 ± 120 | 4470 ± 300 | 4.81 × 106 |
ESSE | 430 ± 105 | 2900 ± 360 | 6.74 × 106 | |
selenoneine | 2335 ± 615 | 6270 ± 790 | 2.69 × 106 | |
Sec-TrxR/Trx | ESSE | 143 ± 6 | 1925 ± 85 | 1.34 × 107 |
GR/GSH | 2-thioHis disulfide | 2815 ± 500 | 16,425 ± 1690 | 5.84 × 106 |
ESSE | 130 ± 6 | 20,035 ± 1210 | 1.53 × 108 |
Enzyme | Substrate | KM (μM) | kcat (min−1) | kcat/KM (min−1 M−1) |
---|---|---|---|---|
Sec-TrxR | 2-thioHis-1O2 | 228 ± 36 | 1306 ± 93 | 5.73 × 106 |
EGT-1O2 | 91 ± 15 | 335 ± 50 | 3.68 × 106 |
Substrate | Enzyme | Normalized Velocity (mol NADPH/min/mol TrxR) | Percent Decrease in Activity Compared to Sec-TrxR (%) |
---|---|---|---|
400 μM ESSE | 10 nM Sec-TrxR pH 7.0 | 1430 ± 40 | - |
5 nM Sec-TrxR pH 8.0 | 2670 ± 50 | - | |
114 nM TrxR-GCCG pH 7.0 | 23 ± 0.6 | 98.4 | |
68 nM TrxR-GCCG pH 8.0 | 24 ± 0.3 | 99.1 * | |
300 nM TrxR∆3 pH 7.0 | 2 ± 0.3 | 99.9 | |
300 nM TrxR∆3 pH 8.0 | 1 ± 0.3 | 99.9 * | |
80 nM DmTrxR pH 7.0 | 87 ± 9 | 93.9 | |
80 nM DmTrxR pH 8.0 | 72 ± 9 | 97.3 * |
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Jenny, K.A.; Mose, G.; Haupt, D.J.; Hondal, R.J. Oxidized Forms of Ergothioneine Are Substrates for Mammalian Thioredoxin Reductase. Antioxidants 2022, 11, 185. https://doi.org/10.3390/antiox11020185
Jenny KA, Mose G, Haupt DJ, Hondal RJ. Oxidized Forms of Ergothioneine Are Substrates for Mammalian Thioredoxin Reductase. Antioxidants. 2022; 11(2):185. https://doi.org/10.3390/antiox11020185
Chicago/Turabian StyleJenny, Kaelyn A., Gracyn Mose, Daniel J. Haupt, and Robert J. Hondal. 2022. "Oxidized Forms of Ergothioneine Are Substrates for Mammalian Thioredoxin Reductase" Antioxidants 11, no. 2: 185. https://doi.org/10.3390/antiox11020185