Molecular Properties of New Enzyme Rhodopsins with Phosphodiesterase Activity

Masahiro Sugiura; Satoshi P Tsunoda; Masahiko Hibi; Hideki Kandori

doi:10.1021/acsomega.0c01113

Molecular Properties of New Enzyme Rhodopsins with Phosphodiesterase Activity

ACS Omega. 2020 Apr 27;5(18):10602-10609. doi: 10.1021/acsomega.0c01113. eCollection 2020 May 12.

Authors

Masahiro Sugiura¹, Satoshi P Tsunoda^{1

2

3}, Masahiko Hibi⁴, Hideki Kandori^{1

2}

Affiliations

¹ Department of Life Science and Applied Chemistry, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
² OptoBioTechnology Research Center, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
³ PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan.
⁴ Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya 464-8602, Japan.

Abstract

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.