A novel rhodopsin phosphodiesterase from Salpingoeca rosetta shows light-enhanced substrate affinity

Abstract

It is since many years textbook knowledge that the concentration of the second messenger cGMP is regulated in animal rod and cone cells by type II rhodopsins via a G-protein signaling cascade. Microbial rhodopsins with enzymatic activity for regulation of cGMP concentration were only recently discovered: in 2014 light-activated guanylyl-cyclase opsins in fungi and in 2017 a novel rhodopsin phosphodiesterase (RhoPDE) in the protist (RhoPDE). The light regulation of RhoPDE, however, seemed very weak or absent. Here, we present strong evidence for light regulation by studyingRhoPDE, expressed in oocytes, at different substrate concentrations. Hydrolysis of cGMP shows an ∼100-fold higher turnover than that of cAMP. Light causes a strong decrease in the value for cGMP from 80 to 13 µM but increases the maximum turnover only by ∼30%. The PDE activity for cAMP is similarly enhanced by light at low substrate concentrations. Illumination does not affect the cGMP degradation of Lys296 mutants that are not able to form a covalent bond of Schiff base type to the chromophore retinal. We demonstrate that RhoPDE shows cytosolic N- and C-termini, most likely via an eight-transmembrane helix structure.RhoPDE is a new optogenetic tool for light-regulated cGMP manipulation which might be further improved by genetic engineering.

Light-regulated collective contractility in a multicellular choanoflagellate

“…Although choanoflagellates are unpigmented and transparent, at least four choanoflagellates (9) encode a choanoflagellate-specific rhodopsin-phosphodiesterase fusion protein ( Fig. S2), RhoPDE, that has been investigated for its potential as an optogenetic tool (20)(21)(22)(23). RhoPDE proteins consist of an N-terminal type I (bacterial) rhodopsin (a class of photosensitive transmembrane proteins broadly involved in light detection (24)) fused to a Cterminal phosphodiesterase (PDE) that catalyzes light-dependent cyclic nucleotide hydrolysis (25), which act on the timescale of transcriptional regulation (at least several minutes (26,27)) and therefore appear unlikely to mediate the light-to-dark transition response.…”

“…3A). Based on in vitro studies(20)(21)(22)(23), RhoPDE from S. rosetta appears capable of converting a photic stimulus into a biochemical signal within seconds, similar to the time scale of the C. flexa response to light-to-dark transitions. To test for the presence of RhoPDE or other candidate photosensitive proteins in C. flexa, we sequenced and assembled the C. flexa transcriptome (Figshare DOI 10.6084/m9.figshare.8216291).…”

Engineered Functional Recovery of Microbial Rhodopsin Without Retinal‐Binding Lysine

“…While animal rhodopsins are mainly photosensory receptors as a specialized subset of G-protein-coupled receptors (1)(2)(3)(4), microbial rhodopsins have various functions, including photosensory receptor, light switch for gene expression, photoactivated enzyme, light-driven ion pump and light-gated ion channel (1,(5)(6)(7)(8)(9)(10). Animal and microbial rhodopsins share a common architecture of seven transmembrane a-helices, though presence of an extra N-terminal transmembrane helix was reported for enzyme rhodopsins (11)(12)(13). Animal and microbial rhodopsins have no significant sequence similarity to warrant a derivation from a common ancestor.…”

Spectroscopic study of the transmembrane domain of a rhodopsin–phosphodiesterase fusion protein from a unicellular eukaryote

“…A putative molecular mechanism to explain Rh-PDE activation was proposed based on a series of studies that examined the full-length protein. Two recent papers revealed that Rh-PDE possesses eight transmembrane helices, not seven, such that both the N and C terminus face the cytoplasmic side (16,17). In addition, the crystal structure of the PDE domain was determined (16), and a better construct than the WT for optogenetic application was reported (17).…”

“…Two recent papers revealed that Rh-PDE possesses eight transmembrane helices, not seven, such that both the N and C terminus face the cytoplasmic side (16,17). In addition, the crystal structure of the PDE domain was determined (16), and a better construct than the WT for optogenetic application was reported (17). The light-induced mechanism of activation of Rh-PDE enzymatic activity is particularly intriguing, and to assess it, in vivo and in vitro studies have been extensively initiated.…”