Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity
Glutathione peroxidases (GPXs) are antioxidant selenoenzymes, which catalyze the reduction of hydroperoxides via glutathione (GSH), providing protection to cells against oxidative stress metabolites. The present study aims to create an efficient semisynthetic GPX based on the scaffold of tau class g...
| Main Authors: | , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2023-12-01
|
| Series: | Antioxidants |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2076-3921/13/1/41 |
| _version_ | 1797344694733111296 |
|---|---|
| author | Irene Axarli Farid Ataya Nikolaos E. Labrou |
| author_facet | Irene Axarli Farid Ataya Nikolaos E. Labrou |
| author_sort | Irene Axarli |
| collection | DOAJ |
| description | Glutathione peroxidases (GPXs) are antioxidant selenoenzymes, which catalyze the reduction of hydroperoxides via glutathione (GSH), providing protection to cells against oxidative stress metabolites. The present study aims to create an efficient semisynthetic GPX based on the scaffold of tau class glutathione transferase (GSTU). A library of GSTs was constructed via DNA shuffling, using three homologue GSTUs from <i>Glycine max</i> as parent sequences. The DNA library of the shuffled genes was expressed in <i>E. coli</i> and the catalytic activity of the shuffled enzymes was screened using cumene hydroperoxide (CuOOH) as substrate. A chimeric enzyme variant (named Sh14) with 4-fold enhanced GPX activity, compared to the wild-type enzyme, was identified and selected for further study. Selenocysteine (Sec) was substituted for the active-site Ser13 residue of the Sh14 variant via chemical modification. The GPX activity (k<sub>cat</sub>) and the specificity constant (k<sub>cat</sub>/Κ<sub>m</sub>) of the evolved seleno-Sh14 enzyme (SeSh14) was increased 177- and 2746-fold, respectively, compared to that of the wild-type enzyme for CuOOH. Furthermore, SeSh14 effectively catalyzed the reduction of hydrogen peroxide, an activity that is completely undetectable in all GSTs. Such an engineered GPX-like biocatalyst based on the GSTU scaffold might serve as a catalytic bioscavenger for the detoxification of hazardous hydroperoxides. Furthermore, our results shed light on the evolution of GPXs and their structural and functional link with GSTs. |
| first_indexed | 2024-03-08T11:07:22Z |
| format | Article |
| id | doaj.art-83015a6ac6604e96a1606d0e7bb110ac |
| institution | Directory Open Access Journal |
| issn | 2076-3921 |
| language | English |
| last_indexed | 2024-03-08T11:07:22Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Antioxidants |
| spelling | doaj.art-83015a6ac6604e96a1606d0e7bb110ac2024-01-26T14:40:34ZengMDPI AGAntioxidants2076-39212023-12-011314110.3390/antiox13010041Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase ActivityIrene Axarli0Farid Ataya1Nikolaos E. Labrou2Laboratory of Enzyme Technology, School of Applied Biology and Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, GreeceDepartment of Biochemistry, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi ArabiaLaboratory of Enzyme Technology, School of Applied Biology and Biotechnology, Agricultural University of Athens, 75 Iera Odos Street, GR-11855 Athens, GreeceGlutathione peroxidases (GPXs) are antioxidant selenoenzymes, which catalyze the reduction of hydroperoxides via glutathione (GSH), providing protection to cells against oxidative stress metabolites. The present study aims to create an efficient semisynthetic GPX based on the scaffold of tau class glutathione transferase (GSTU). A library of GSTs was constructed via DNA shuffling, using three homologue GSTUs from <i>Glycine max</i> as parent sequences. The DNA library of the shuffled genes was expressed in <i>E. coli</i> and the catalytic activity of the shuffled enzymes was screened using cumene hydroperoxide (CuOOH) as substrate. A chimeric enzyme variant (named Sh14) with 4-fold enhanced GPX activity, compared to the wild-type enzyme, was identified and selected for further study. Selenocysteine (Sec) was substituted for the active-site Ser13 residue of the Sh14 variant via chemical modification. The GPX activity (k<sub>cat</sub>) and the specificity constant (k<sub>cat</sub>/Κ<sub>m</sub>) of the evolved seleno-Sh14 enzyme (SeSh14) was increased 177- and 2746-fold, respectively, compared to that of the wild-type enzyme for CuOOH. Furthermore, SeSh14 effectively catalyzed the reduction of hydrogen peroxide, an activity that is completely undetectable in all GSTs. Such an engineered GPX-like biocatalyst based on the GSTU scaffold might serve as a catalytic bioscavenger for the detoxification of hazardous hydroperoxides. Furthermore, our results shed light on the evolution of GPXs and their structural and functional link with GSTs.https://www.mdpi.com/2076-3921/13/1/41chemical modificationdirected evolutionenzyme engineeringglutathione transferaseglutathione peroxidase |
| spellingShingle | Irene Axarli Farid Ataya Nikolaos E. Labrou Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity Antioxidants chemical modification directed evolution enzyme engineering glutathione transferase glutathione peroxidase |
| title | Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity |
| title_full | Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity |
| title_fullStr | Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity |
| title_full_unstemmed | Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity |
| title_short | Repurposing Glutathione Transferases: Directed Evolution Combined with Chemical Modification for the Creation of a Semisynthetic Enzyme with High Hydroperoxidase Activity |
| title_sort | repurposing glutathione transferases directed evolution combined with chemical modification for the creation of a semisynthetic enzyme with high hydroperoxidase activity |
| topic | chemical modification directed evolution enzyme engineering glutathione transferase glutathione peroxidase |
| url | https://www.mdpi.com/2076-3921/13/1/41 |
| work_keys_str_mv | AT ireneaxarli repurposingglutathionetransferasesdirectedevolutioncombinedwithchemicalmodificationforthecreationofasemisyntheticenzymewithhighhydroperoxidaseactivity AT faridataya repurposingglutathionetransferasesdirectedevolutioncombinedwithchemicalmodificationforthecreationofasemisyntheticenzymewithhighhydroperoxidaseactivity AT nikolaoselabrou repurposingglutathionetransferasesdirectedevolutioncombinedwithchemicalmodificationforthecreationofasemisyntheticenzymewithhighhydroperoxidaseactivity |