Molecular Properties of New Enzyme Rhodopsins with Phosphodiesterase Activity

[Image: see text] The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme (SrRh-PDE), which decreases the concentrations of cyclic nucleotides such as cGMP and cAMP in light, is a useful tool in optogenetics. Recently, eight additional Rh-PDE enzymes were found in choanoflagellate species, four from Choanoeca flexa and the other four from other species. In this paper, we studied the molecular properties of these new Rh-PDEs, which were compared with SrRh-PDE. Upon expression in HEK293 cells, four Rh-PDE proteins, including CfRh-PDE2 and CfRh-PDE3, exhibited no PDE activity when assessed by in-cell measurements and in vitro HPLC analysis. On the other hand, CfRh-PDE1 showed light-dependent PDE activity toward cGMP, which absorbed maximally at 491 nm. Therefore, CfRh-PDE1 is presumably responsible for colony inversion in C. flexa by absorbing blue-green light. The molecular properties of MrRh-PDE were similar to those of SrRh-PDE, although the λ(max) of MrRh-PDE (516 nm) was considerably red-shifted from that of SrRh-PDE (492 nm). One Rh-PDE, AsRh-PDE, did not contain the retinal-binding Lys at TM7 and showed cAMP-specific PDE activity both in the dark and light. These results provide mechanistic insight into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.