| Summary: | 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.
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