@article{TianYangNageletal.2022,
  author    = {Tian, Yuehui and Yang, Shang and Nagel, Georg and Gao, Shiqiang},
  title     = {Characterization and modification of light-sensitive phosphodiesterases from choanoflagellates},
  series = {Biomolecules},
  volume    = {12},
  journal   = {Biomolecules},
  number    = {1},
  issn      = {2218-273X},
  doi       = {10.3390/biom12010088},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254769},
  year      = {2022},
  abstract  = {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 Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, 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.},
  language  = {en}
}