Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates
Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still i...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2022-01-01
|
| Series: | Biomolecules |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2218-273X/12/1/88 |
| _version_ | 1827666362833567744 |
|---|---|
| author | Yuehui Tian Shang Yang Georg Nagel Shiqiang Gao |
| author_facet | Yuehui Tian Shang Yang Georg Nagel Shiqiang Gao |
| author_sort | Yuehui Tian |
| collection | DOAJ |
| description | Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from <i>Salpingoeca rosetta</i>, <i>Choanoeca flexa</i> and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in <i>Xenopus</i> oocytes, except the <i>As</i>RhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::<i>As</i>RhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, <i>Sr</i>RhoPDE, <i>Cf</i>RhoPDE1, 4 and <i>Mr</i>RhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering <i>Sr</i>RhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future. |
| first_indexed | 2024-03-10T01:49:41Z |
| format | Article |
| id | doaj.art-062b5a3b2ba641e391f0f23df3860774 |
| institution | Directory Open Access Journal |
| issn | 2218-273X |
| language | English |
| last_indexed | 2024-03-10T01:49:41Z |
| publishDate | 2022-01-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biomolecules |
| spelling | doaj.art-062b5a3b2ba641e391f0f23df38607742023-11-23T13:06:52ZengMDPI AGBiomolecules2218-273X2022-01-011218810.3390/biom12010088Characterization and Modification of Light-Sensitive Phosphodiesterases from ChoanoflagellatesYuehui Tian0Shang Yang1Georg Nagel2Shiqiang Gao3Environmental Microbiomics Research Center, School of Environmental Science and Engineering, Southern Marine Science and Engineering Guangdong Laboratory (Zhuhai), Sun Yat-sen University, Guangzhou 510006, ChinaDepartment of Neurophysiology, Institute of Physiology, Biocenter, University of Wuerzburg, 97070 Wuerzburg, GermanyDepartment of Neurophysiology, Institute of Physiology, Biocenter, University of Wuerzburg, 97070 Wuerzburg, GermanyDepartment of Neurophysiology, Institute of Physiology, Biocenter, University of Wuerzburg, 97070 Wuerzburg, GermanyEnzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from <i>Salpingoeca rosetta</i>, <i>Choanoeca flexa</i> and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in <i>Xenopus</i> oocytes, except the <i>As</i>RhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::<i>As</i>RhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, <i>Sr</i>RhoPDE, <i>Cf</i>RhoPDE1, 4 and <i>Mr</i>RhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering <i>Sr</i>RhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.https://www.mdpi.com/2218-273X/12/1/88choanoflagellatesoptogeneticsrhodopsin phosphodiesterase (RhoPDE)cGMP |
| spellingShingle | Yuehui Tian Shang Yang Georg Nagel Shiqiang Gao Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates Biomolecules choanoflagellates optogenetics rhodopsin phosphodiesterase (RhoPDE) cGMP |
| title | Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates |
| title_full | Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates |
| title_fullStr | Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates |
| title_full_unstemmed | Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates |
| title_short | Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates |
| title_sort | characterization and modification of light sensitive phosphodiesterases from choanoflagellates |
| topic | choanoflagellates optogenetics rhodopsin phosphodiesterase (RhoPDE) cGMP |
| url | https://www.mdpi.com/2218-273X/12/1/88 |
| work_keys_str_mv | AT yuehuitian characterizationandmodificationoflightsensitivephosphodiesterasesfromchoanoflagellates AT shangyang characterizationandmodificationoflightsensitivephosphodiesterasesfromchoanoflagellates AT georgnagel characterizationandmodificationoflightsensitivephosphodiesterasesfromchoanoflagellates AT shiqianggao characterizationandmodificationoflightsensitivephosphodiesterasesfromchoanoflagellates |