Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i>
Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium <i>Deinococcus radiodurans</i> is, like some other homol...
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MDPI AG
2024-01-01
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| author | Kelly Frade Célia M. Silveira Bruno A. Salgueiro Sónia Mendes Lígia O. Martins Carlos Frazão Smilja Todorovic Elin Moe |
| author_facet | Kelly Frade Célia M. Silveira Bruno A. Salgueiro Sónia Mendes Lígia O. Martins Carlos Frazão Smilja Todorovic Elin Moe |
| author_sort | Kelly Frade |
| collection | DOAJ |
| description | Dye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium <i>Deinococcus radiodurans</i> is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H<sub>2</sub>O<sub>2</sub> detected at lower pH. A sequence alignment with other DyPs reveals that <i>Dr</i>DyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting <i>Dr</i>DyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of <i>Dr</i>DyP and <i>Dr</i>DyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of <i>Dr</i>DyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H<sub>2</sub>O<sub>2</sub> substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to <i>Dr</i>DyP. |
| first_indexed | 2024-03-08T09:49:42Z |
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| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-08T09:49:42Z |
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| spelling | doaj.art-68b8a1acb7194b778f4a757f1a0f6e752024-01-29T14:07:50ZengMDPI AGMolecules1420-30492024-01-0129235810.3390/molecules29020358Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i>Kelly Frade0Célia M. Silveira1Bruno A. Salgueiro2Sónia Mendes3Lígia O. Martins4Carlos Frazão5Smilja Todorovic6Elin Moe7Instituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalInstituto de Tecnologia Química e Biológica António Xavier (ITQB-NOVA), Universidade Nova de Lisboa, Av. da Republica (EAN), 2780-157 Oeiras, PortugalDye-decolorizing peroxidases (DyPs) are heme proteins with distinct structural properties and substrate specificities compared to classical peroxidases. Here, we demonstrate that DyP from the extremely radiation-resistant bacterium <i>Deinococcus radiodurans</i> is, like some other homologues, inactive at physiological pH. Resonance Raman (RR) spectroscopy confirms that the heme is in a six-coordinated-low-spin (6cLS) state at pH 7.5 and is thus unable to bind hydrogen peroxide. At pH 4.0, the RR spectra of the enzyme reveal the co-existence of high-spin and low-spin heme states, which corroborates catalytic activity towards H<sub>2</sub>O<sub>2</sub> detected at lower pH. A sequence alignment with other DyPs reveals that <i>Dr</i>DyP possesses a Methionine residue in position five in the highly conserved GXXDG motif. To analyze whether the presence of the Methionine is responsible for the lack of activity at high pH, this residue is substituted with a Glycine. UV-vis and RR spectroscopies reveal that the resulting <i>Dr</i>DyPM190G is also in a 6cLS spin state at pH 7.5, and thus the Methionine does not affect the activity of the protein. The crystal structures of <i>Dr</i>DyP and <i>Dr</i>DyPM190G, determined to 2.20 and 1.53 Å resolution, respectively, nevertheless reveal interesting insights. The high-resolution structure of <i>Dr</i>DyPM190G, obtained at pH 8.5, shows that one hydroxyl group and one water molecule are within hydrogen bonding distance to the heme and the catalytic Asparagine and Arginine. This strong ligand most likely prevents the binding of the H<sub>2</sub>O<sub>2</sub> substrate, reinforcing questions about physiological substrates of this and other DyPs, and about the possible events that can trigger the removal of the hydroxyl group conferring catalytic activity to <i>Dr</i>DyP.https://www.mdpi.com/1420-3049/29/2/358heme proteindye-decolorizing peroxidasesresonance Raman spectroscopyX-ray crystallography |
| spellingShingle | Kelly Frade Célia M. Silveira Bruno A. Salgueiro Sónia Mendes Lígia O. Martins Carlos Frazão Smilja Todorovic Elin Moe Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i> Molecules heme protein dye-decolorizing peroxidases resonance Raman spectroscopy X-ray crystallography |
| title | Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i> |
| title_full | Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i> |
| title_fullStr | Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i> |
| title_full_unstemmed | Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i> |
| title_short | Biochemical, Biophysical, and Structural Analysis of an Unusual DyP from the Extremophile <i>Deinococcus radiodurans</i> |
| title_sort | biochemical biophysical and structural analysis of an unusual dyp from the extremophile i deinococcus radiodurans i |
| topic | heme protein dye-decolorizing peroxidases resonance Raman spectroscopy X-ray crystallography |
| url | https://www.mdpi.com/1420-3049/29/2/358 |
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