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 light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, lightinduced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the ␣-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase (Rh-GC) and photosensitive adenylyl cyclase (PAC). Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The 2.6 Å resolution crystal structure of the transmembrane domain revealed a new topology of rhodopsin, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the transmembrane and PDE domain (2.1 Å) with their connecting linkers. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins. -3 - Main text IntroductionMicrobial rhodopsins are photoreceptive membrane proteins with an all-trans retinylidene chromophore 1,2 (all-trans retinal). All microbial rhodopsins share a seven transmembrane topology, in which all-trans retinal chromophore is bound to a conserved lysine residue via Schiff base in the transmembrane helix (TM) 7. Most microbial rhodopsins function as light-driven ion pumps that actively transport various ions 1 (H + , Na + , Cl -, etc.). Some microbial rhodopsins function as light-gated ion channels (channelrhodopsins 3,4 ), which are used as optogenetic tools in neuroscience 5,6 . Enzyme rhodopsins are a group of newly discovered microbial rhodopsins 7 , found in eukaryotes such as fungi and green algae, and choanoflagellates. Enzyme rhodopsins comprise the N-terminal rhodopsin domain and C-terminal enzyme domain, connected by a linker, and function as light-activated enzymes. Two types of enzyme rhodopsins were initially discovered, the histidine kinase rhodopsins 8 (HKRs) and rhodopsin guanylyl cyclase 9 (Rh-GC). While HKRs function as ATP-dependent light-inhibited guanylyl cyclase 10 , Rh-GC from Blastocladiella emersonii (BeGC1) induces a rapid light-triggered cGMP increase in heterologous cells (~5,000-fold) and could be utilized as an optogenetic tool to rapidly manipulate cGMP levels in cells and animals 11,12 .In 2017, rhodopsin phosphodiesterase 13 (Rh-PDE), a novel type of enzyme rhodopsin, was discovered. Rh-PDE derived from Salpingoeca rosetta (SrRh-PDE) was first identified and exhibits light-dependent hydrolytic activity for both of cAMP and cGMP, whereas it lacks pump or channel activity. Moreover, four Rh-PDE homologs were recently discovered in Choanoeca flexa 14 . Thes...