Enzymerhodopsins: novel photoregulated catalysts for optogenetics

Shatanik Mukherjee*, Peter Hegemann, & Matthias Broser*

Institute for Biology, Experimental Biophysics, Humboldt-Universität zu Berlin, Germany

*Correspondence should be addressed to: mukherjs@hu-berlin.de or matthias.broser@hu-berlin.de Field Code Changed

Field Code Changed
Abstract
Enzymerhodopsins are a recently discovered class of natural rhodopsin-based
photoreceptors with light-regulated enzyme activity. Currently, three different types of
these fusion proteins with an N-terminal type-1 rhodopsin and a C-terminal enzyme domain
have been identified, but their physiological relevance is mostly unknown. Among these,
histidine kinase rhodopsins (HKR) are photo-regulated two-component-like signaling systems
that trigger a phosphorylation cascade, whereas rhodopsin phosphodiesterase (RhoPDE) or
rhodopsin guanylyl cyclase (RhGC), show either light-activated hydrolysis or production of
cyclic nucleotides. RhGC, the best characterized enzymerhodopsin, is involved in the
phototaxis of fungal zoospores and allows for optically controlled production of cyclic
nucleotides in different cell-types. These photoreceptors have great optogenetic potential
and possess several advantages over the hitherto existing tools to manipulate cyclic-
nucleotide dynamics in living cells.

Introduction
Microbial (type 1) rhodopsins found in archaea, bacteria, and lower eukaryotes are involved
in a myriad of cellular functionalities including phototaxis, photoadaptation, and ATP
synthesis [1,2]. They are typically seven-pass transmembrane photoactive proteins with a
retinal chromophore covalently bound to a conserved lysine residue forming a retinal Schiff
base (RSB). In most cases the RSB is protonated in the dark state and stabilized by conserved
counterion residues containing carboxylate side chains.
Upon absorption of a photon, the canonical primary event is the trans-cis isomerization of
the chromophore around the C13=C14 double bond. This triggers the ‘photocycle’, a cyclic
reaction of the retinal, entailing spectrally distinct intermediates sequentially termed ‘J’, ‘K’,
‘L’, ‘M’, ´N´, and ´O´, before the dark state is recovered. During the photocycle the RSB
undergoes transient deprotonation that shifts retinal absorbance to the near UV (400 nm)
within the M-state. The molecular dynamic of the photocycle is closely linked to the protein
function. For example, in ion-pumping rhodopsin e.g. bacteriorhodopsin (BR), one ion is
actively transported per photocycle, the fast kinetics determines the pump efficacy at high
light intensities. In contrast, non-energizing photosensory rhodopsins often show slow
photocycle to allow for signal-propagation from the signaling state of sensor to the effector.
De- and re-protonation of the RSB is accompanied by conformational changes of the protein
that lead to functionally relevant events e.g. changes of the surface accessibility, opening/
closing of a pore, and activation of transducers [1].
While the involvement of rhodopsins in microbial phototaxis or photo-accumulation in
prokaryotes was known for decades [3–6], the advent of genomic databases propelled the
discovery of many sequestered rhodopsins with a diverse range of functionalities. Especially
the discovery of channelrhodopsin and its impact as an optogenetic tool to precisely

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modulate the membrane potential of cells with light encouraged the search for rhodopsins
with novel functions [7,8]. In 2004, the first sequence encoding a putative fusion protein of a
rhodopsin coupled with a histidine kinase was found and named enzymerhodopsin
(alternatively named rhodopsin-coupled enzymes) [9]. Since then new members have been
discovered with light-regulated phosphodiesterase (RhoPDE), and nucleotide cyclase activity
(RhGC) [10–13](Figure 1). All of the enzymerhodopsins share a modular, and in most cases
homodimeric architecture and an allosteric photoregulation of catalysis. These
enzymerhodopsins are promising tools for precise and non-invasive spatiotemporal control
of cyclic-nucleotide signaling pathways. Here, we summarize the biophysical and
biochemical properties of these novel enzymerhodopsins vis-a-vis other photoactivated
enzymes evaluating their use as next generation optogenetic tools.

Histidine Kinase Rhodopsin (HKR)

The first sequence encoding a putative enzymerhodopsin was identified by searching the
fresh-water algae C. reinhardtii genome against sensory rhodopsin sequences [9]. The
protein sequence (Chlamydomonas opsin-related protein5; COP5, or histidine kinase
rhodopsin1 i.e. HKR1) revealed a modular domain arrangement with the N-terminal
rhodopsin domain followed by a histidine-kinase (HK), a response regulator (RR) and a
guanylyl cyclase (Figure 1). Putative HKR sequences that share this architecture are found in
various organisms, but their native function is unknown. In some algae, HKRs have been
linked to cell differentiation and circadian rhythmicity [14,15]. Of the eight HKRs presumably
present in C. reinhardtii, only the transcription of HKR1, HKR2 and HKR4 have been verified
[16–18]. HKR1 was located within the eyespot [16] whereas HKR4 travels between eyespot
and flagella depending on the light regime [17]. The modular architecture of HKRs resembles
the widely distributed two-component regulatory systems (TCS) that respond to a broad
variety of stimuli [19]. In canonical TCS, activation of the homodimeric membrane-bound
protein leads to trans/cis auto-phosphorylation of a specific histidine residue and
subsequent transfer of the phosphate to a second soluble component, the response-
regulator for cellular response [20]. In HKRs, these components reside putatively within one
polypeptide-chain similar to hybrid histidine kinase of phosphorelay systems [20].
Unlike typical rhodopsins, the photosensor domain of HKRs comprises 8 transmembrane
helices (Figure 1) with both termini located within the cytoplasm [18]. Notably, this topology
is shared by all three types of enzymerhodopsins (see below and Figure 1).
The rhodopsin is connected to the cytoplasmic histidine-kinase by a short 20 amino-acid
long putatively helical linker with no homology to known domains utilized in other signal-
transduction systems (e.g. HAMP domain) [21]. In contrast, the histidine-kinase domain
resembles the sequences of canonical histidine-kinases including a dimerization/histidine
phosphoryl-transfer (DHp) and a catalytic/ATP-binding (CA) domain (Figure 1). The adjacent
receiver-domain is homologous to TCS response-regulator and contains the conserved
phosphate-accepting aspartate. In some HKRs, further potential effector domains, such as
cNMP cyclases are present, albeit some sequences that lack catalysis-relevant residues are
unlikely to produce an active enzyme. Analogous to TCS, light-triggered conformational
changes are postulated to activate a phosphorylation cascade in HKRs that modulates the
enzymatic activity of the cyclase [22] or activates/recruits further proteins for cellular
response.
Recently, HKR2 (termed 2c-Cyclop: two-component cyclase opsins in [18]) from C. reinhardtii
and its orthologue from V. carteri were heterologously expressed in X. laevis oocytes and the
enzymatic function has been demonstrated. Both are light-inhibited guanylyl-cyclases with

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an activity that depends on the presence of ATP. According to mutational studies, the dark
active histidine kinase leads to autophosphorylation and phosphoryl transfer to the acceptor
aspartate that subsequently activates the cyclase. Illumination with green light reduces
cGMP production presumably by inhibiting the kinase function needed to preserve the
phosphorylation state of the protein. Dark-adaptation of illuminated sample allows to
recover the initial dark-activity within 30 s in HKR2, setting an upper limit for the rhodopsin
photocycle. However, the photochemistry and dynamics of the HKR2-rhodopsin is unknown.
In contrast, the photochemistry of the isolated rhodopsin domain of HKR1 was studied in
detail. The photosensor exhibits two thermally stable states absorbing blue (Rh-Bl, max 490
nm) or UV (Rh-UV, max 380 nm) light [16,23,24]. Both states can be interconverted by
switching the wavelength of light between blue and UV [16]. The large color change
associated with photoswitching of HKR1 is related to RSB de- or re-protonation.
Consequently, the photoconversion from Rh-Bl to deprotonated Rh-UV relates to the dark-
state to M-transition of other microbial rhodopsins [25]. Notably HKR1 expressed in X. laevis
oocytes does not produce cNMP, likely due to its non-functional cyclase domain that may
allow catalysis only by dimerization with a so far unknown catalytic counterpart [18].

Rhodopsin Phosphodiesterase (RhoPDE)

Recently, the genome of the choanoflagellate S. rosetta revealed one gene that encodes a
rhodopsin phosphodiesterase fusion protein (RhoPDE) [12]. Similar to HKRs and rhodopsin-
cyclases, the RhoPDE comprises 8 transmembrane helices (Figure 1). Upon heterologous
expression in HEK293 cells and X. laevis oocytes, the protein exhibited a nominal light-
dependent cGMP- and lower amount of cAMP-phosphodiesterase activity [26]. Illumination
of RhoPDE results in a decrease of the Michaelis-Menten constant (Km) while the maximal
velocity (Vmax) increases only slightly. Due to this factor and the high constitutive activity,
light-activation of RhoPDE cannot be observed under saturating substrate conditions. Light-
induced increase of cNMP hydrolyses activity up to 4-fold and 5-fold of the dark-value are
reported for cGMP or cAMP respectively [26,27].
Dark-adapted RhoPDE shows a typical broad rhodopsin spectrum that peaks at 492 nm and
Illumination leads to a very long-living M-state that decays within 7 s to recover the dark
state. This decay coincides with the observed light-induced enzyme activation; therefore
structural rearrangements during the decay of the M intermediate may cause
photoactivation of the enzyme [12]. However, a weak structural coupling between the
rhodopsin and enzyme domain has been suggested based on the finding that the enzymatic
activity upon light stimulation declines one order of magnitude slower than the proposed
duration of the photocycle [28]. Prolonged M-state is found in various microbial rhodopsins,
including sensory rhodopsin II (SRII) and proton-donor mutants (e.g. D96N) of BR. In RhoPDE,
the hydrophobic W175 at D96 homologue position may hinder re-protonation of the RSB as
discussed for SRII (F86, see Figure 2a) [29].

The PDE domain shows high sequence similarity to other PDEs and has been expressed as
soluble protein in E. coli to obtain its crystal structure [27]. The protein resembles the typical
structure comprised of 16 -helices known from other PDEs and arranged as antiparallel
homodimer [27,30]. The two canonical metal centers are occupied by Zn2+ and Mg2+ while a
bound ligand is missing (Figure 2b). Nevertheless, the structure represents the open form of
the enzyme with the so-called H-loop (Figure 2b) tilted outwards to allow the substrate to
access the catalytic center. Accordingly, the isolated enzymatic domain is catalytically active,
albeit with lower turnover rate compared to the full-length protein [27].

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An activation mechanism has been proposed for human PDE2A in which a GAFA-GAFB
(cGMP-specific phosphodiesterase, adenylyl cyclase, and FhlA) tandem precedes the enzyme
domain. According to this the conformational change induced by the binding of cGMP to the
allosteric center in GAFB is propagated via a coiled-coil linker and alters the position of the
H-loop which releases the PDE monomers from its auto-inhibited state [31]. In RhoPDE, a
similar mechanism is likely to occur in which light-induced movement of transmembrane
helix 7 (Figure 2a) triggers this process [12].

Rhodopsin Gyanylyl-Cyclase (RhGC)

The first enzymerhodopsin with proven enzyme activity was found in the fungus B. emersonii
and represents a rhodopsin coupled to a guanylyl cyclase domain (RhGC, see Figure 1). As
proposed earlier [32], Suely Gomez and colleagues showed that the RhGC is essential for
phototactic behavior of fungal zoospores through a cGMP driven signaling pathway [13,33].
Among several orthologues found in related fungi, two RhGCs, from B. emersonii (BeRhGC)
and C. anguillulae (CaRhGC), sharing 80% sequence similarity have been characterized in
detail [10,11,34,35]. They can be expressed in various heterologous cells and generate cGMP
when activated with green light (530 nm), while other fungal RhGCs have little or no light-
induced enzymatic activity [10]. Importantly, both RhGCs are highly substrate specific
towards GTP and show virtually no dark activity in vivo [10,11,34]. BeRhGC, the first
enzymerhodopsin that has been used as an optogenetic tool, exhibits significant
photobleaching upon strong illumination [11,36] whereas CaRhGC shows higher
photostability and higher GTP turnover when expressed in hippocampal neurons compared
to BeRhGC [34].
The rhodopsin domain shares the hydrophobic retinal-binding pocket with conserved
photochemically important residues of other microbial rhodopsins (Figure 2a). It absorbs
maximally at 530 nm and has a photocycle with an early red shifted K-intermediate after
photoexcitation. In CaRhGC, two further blue-shifted photocycle products L1 and L2, not
observed for BeRhGC, evolve with µs kinetics before the RSB deprotonates within 31 ms to
form the M-state. The M-state decay recovers the initial dark state within 571 ms. In
BeRhGC, the evolution and decay (8 ms and 93 ms respectively) of the M state is significantly
faster. The electrogenic response upon illumination of CaRhGC in neurons (employing cGMP
gated reporter-channels) sets an upper time limit for enzyme activation of 25 ms, rendering
the M-state to be the active state.
RhGCs contain two extra non-canonical alpha helices (helix 0, helix -1, Figure 1) at the N-
terminus preceding the seven transmembrane helical domains of microbial rhodopsins and
Helix 0 has been proven to reside in the cytosol. Truncation of the N-Terminus alters photo-
regulation, thus indicating its functional relevance. Approximately 50 amino acids with
pronounced heptad repeats typical for coiled-coil structures connect the rhodopsin with the
cyclase and mutations interrupting this structure lose photoregulation (Scheib, U. personal
communication). The linker further contains a cyclase transducing element (CTE) [37], a
conserved stretch of 19 amino acids widely present in class III nucleotide cyclases.
The cyclase domain of the RhGCs belongs to class III cNMP cyclases found in prokaryotes to
mammalians (Figure 3a). Similar to other cyclases of this type, RhGCs are much more active
in the presence of Mn2+ over Mg2+ and the isolated cyclase domain expressed in E. coli is
constitutively active [38]. Therefore, the rhodopsin domain suppresses strongly the activity
in darkness to allow the photoregulation of the full-length protein. Interestingly, full-length
RhGCs are 3-5-fold more active in light than the isolated cyclases. The activated rhodopsin

4
domain presumably stabilizes an enzyme conformation different to the rhodopsin-free form
that further promotes catalysis. Full-length CaRhGC has a KM value of 6.1 mM, Vmax of 821
μmol cGMP min−1 μmol−1 protein when illuminated under non-saturating light-conditions at
the pH optimum of 7.5. Lower enzyme activity was found for BeRhGC which may correlate
with the slower cGMP rise seen in neurons.
Despite the high specificity of RhGCs for GTP, they can be converted to an adenylyl cyclase
via a double point mutation of active site glutamate and cysteine (E497K, C566D, see Figure
3a) as in the case of the phytochrome-based chimeric guanylyl cyclase PagC [39,40]. The
engineered RhACs display highly specific catalysis of ATP but show elevated dark-activity in
contrast to the parental proteins. Nevertheless, in particular CaRhAC has been suitable for
optogenetic cAMP control in hippocampal neurons [34].
The loss of light-regulation indicates that base discrimination plays a mechanistic role in
modulating enzyme activity due to the photosensor, since the kinetic properties of the
isolated cyclase domain remains unaffected. Indeed, base discriminating interactions are
believed to occur late during catalysis and proper nucleotide placement may be crucial for
turnover [41].
Crystal structures for the isolated cyclase domain either as wildtype (BeGC/CaGC) or
substrate-specificity determining mutants (E497K, C566D; BeAC/CaAC) have been obtained
[34,42]. All structures exhibit the classical cNMP cyclase class III fold with a central 7
stranded β-sheet shielded by 3 helices, but so far only CaAC co-crystallized with the inhibitor
ATPαS forms the functional homodimeric head-to-tail arrangement (Figure 3b). The CaAC
structure reveals two symmetric active sites at the dimer-interface each occupied by the
inhibitor. Similar to other AC structures, residues anchoring the adenine (in particular
mutated E497K/C566D) and phosphate of ATPαS belong to different monomers (Figure
3a/b). This feature supports a cyclization mechanism that includes movement of the
monomers to facilitate substrate alignment, formation of the transition state, and catalytic
turnover. NTP cyclization proceeds via an intramolecular nucleophilic substitution (SN2)
initiated through the attack of ribose-3′-OH oxygen at Pα [41]. Two metals centers are
involved in catalysis: ion B binds the substrate via the triphosphate while transient ion A
supports deprotonation of the attacking ribose-3´-OH [43]. The ion binding residues are
highly conserved in class III cyclases but only ion B is present in most ligand bound crystal-
structures (Figure 3a).

The question regarding the mechanism of the signal transduction for modulation of enzyme
activity after retinal isomerization remains open. Full-length structures for soluble light-
activated adenylyl cyclases from bacteria proposed two competing mechanisms. Either
protein activation is based on changes of the vibrational mode with minimal alteration of
protein conformation or on the allosteric movement inter alia of the β4-β5 loop involved in
base-binding (Figure 3a/b) [44,45]. Notably the β4-β5 loop directly interacts with the
preceding CTE. In absence of CTE as in CaAC and other cyclase structures this loop is flexible
and adopts different conformation depending on its surrounding. This flexibility could
support a potential regulatory role, but a precise analysis of signal propagation of RhGC
depends on the availability of structural data for the full-length protein.

Concluding remarks: why use enzymerhodopsins?

An increased interest in photoreceptor research is motivated by their application as
optogenetic tools. In this regard, enzymerhodopsins possess significant potential especially

5
since several of their properties can be adapted to experimental needs via protein
engineering.
HKR1 with its unique photochemistry as a bimodal photoswitch presents a valuable target to
design new bi-stable optogenetic tools that can be activated/deactivated by applying two
short light pulses of different wavelengths. Different to other approaches utilizing long-lived
photocycle mutants, e.g. step-function rhodopsins, the low spectral overlap enables an
almost complete conversion between the two states [25,46].
Photoactivated cyclases and phosphodiesterases are highly sought after to investigate the
cyclic nucleotide signaling pathways with precise spatial and temporal resolution in living
cells or animals. So far soluble photo-activated adenylyl cyclases from bacteria (e.g. bPAC)
employing BLUF-domains as light sensors (blue-light sensors using FAD) or engineered
chimeric phytochrome-based photoreceptors (e.g. LAPD) provided optical control of cyclic
nucleotide signaling [39,44,47–51]. Other systems rely on the activation of endogenous G
protein by G protein-coupled rhodopsins that mediate either cyclase or PDE activity [52,53].
All these systems possess serious drawbacks that limit their suitability towards optogenetic
approaches: 1) multicomponent systems based on G-proteins are prone to cross-talk with
other signaling events, due to the multiple output of GPCR-signaling and their slow
deactivation resulting in insufficient temporal control, 2) for single component natural PACs,
time resolution is limited by the slow BLUF-photocycle and they exhibit substantial dark-
activity, and 3) engineered light-activated AC/GCs or PDEs suffer from high dark-activity, low
dynamic range, low substrate-specificity, and spectral properties that hinder efficient
deactivation or color tuning [39,48,50,54–57].
RhoPDE shares high dark activity and low dynamic range with LAPD, but its spontaneous
(albeit slow) back conversion clearly is advantageous [12,26,50]. Nevertheless, RhoPDE
variants with improved properties, particularly lower dark-activity, either from protein-
engineering or from natural sources, will be crucial for optogenetic applications.
In contrast, RhGC provide significant benefit over previously reported photo-activated
cyclases (PAC) systems due to virtually non-existing dark-activity and millisecond-scale off-
kinetics [10,11]. Consequently, RhGC possess an excellent dynamic range, albeit the maximal
turnover is lower than for other GCs (e.g. RetGC) and may be modulated via active-site
engineering [58]. The cAMP-producing mutants of RhGC show elevated dark-activity,
nevertheless their dynamic range is only moderately lower than found for bPAC, whose
activity increases 300-fold under illumination [34,45,47]. An attempt to mimic the binding
pocket of mammalian transmembrane adenylyl cyclase tmACs (Figure 3a) by point mutations
on CaRhAC results in lower dark activity but the maximal activity in light decreases as well
[34]. Since the activity of RhGC is unaffected by Ca2+ ions (Scheib, U; Mukherjee, S.; Personal
communication), the enzyme may be suited for unravelling the interplay between
intracellular cAMP, cGMP, and Ca2+ ion dynamics. Additionally, because of the
compartmentalized nature of cyclic-nucleotide signaling in cells, utilizing membrane-located
optical modulators are less likely to perturb intracellular cyclic-nucleotide dynamics.
Multitude of strategies are known that change the photochemical properties of microbial
rhodopsins, thus allowing color-tuning and kinetic adaptation [46,59–63] to modify spectral
properties and apparent photosensitivity. First attempts to alter counterion residues in
RhGCs results in a moderate 5-8 nm blue shift [35], that likely can be expanded to obtain an
extended color palette for multi-color experiments with co-expressed second messenger
sensors in animals e.g. C. elegans [64]. This proves that RhGCs are amenable to protein
engineering strategies based on other rhodopsins. Notably, all enzymerhodopsins rely on the
allosteric regulation within the most likely homodimeric protein complex. Thus, standard

6
engineering approaches e.g. linker-variant library, chimera generation, domain swapping,
and directed evolution are likely to succeed in generating novel optogenetic tools. The
elusive structure of a full length enzymerhodopsin will not only unravel the signal
transduction mechanism of such a complex protein but also boost rational protein
engineering approaches.

Acknowledgements:

S.M. is supported by the EU (ERC grant MERA, P.H.), M.B. is supported by the DFG (SFB1078,
B1, P.H.). P.H. is Hertie Senior Professor for Neuroscience and supported by the Hertie
Foundation.
We thank Jonas Wietek and Christiane Grimm for comments and suggestions on the paper.

References

• special interest
• • outstanding interest

1. Govorunova EG, Sineshchekov OA, Li H, Spudich JL: Microbial Rhodopsins: Diversity,

Mechanisms, and Optogenetic Applications. Annu Rev Biochem 2017, 86:845–872.
2. Boeuf D, Audic S, Brillet-Guéguen L, Caron C, Jeanthon C: MicRhoDE: A curated
database for the analysis of microbial rhodopsin diversity and evolution. Database
2015, 2015:1–8.
3. Foster KW, Saranak J, Patel N, Zarilli G, Okabe M, Kline T, Nakanishi K: A rhodopsin is
the functional photoreceptor for phototaxis in the unicellular eukaryote
Chlamydomonas. Nature 1984, 311:756–759.
4. Sineshchekov OA, Govorunova EG, Jung KH, Zauner S, Maier UG, Spudich JL:
Rhodopsin-mediated photoreception in cryptophyte flagellates. Biophys J 2005,
89:4310–4319.
5. Hegemann P: Vision in microalgae. Planta 1997, doi:10.1007/s004250050191.
6. Kröger P, Hegemann P: Photophobic responses and phototaxis in Chlamydomonas
are triggered by a single rhodopsin photoreceptor. FEBS Lett 1994, 341:5–9.
7. Yin T, Wu YI: Guiding lights: Recent developments in optogenetic control of
biochemical signals. Pflugers Arch Eur J Physiol 2013, doi:10.1007/s00424-013-1244-x.
8. Govorunova EG, Koppel LA: The road to optogenetics: Microbial rhodopsins. Biochem
2016, 81:928–940.

7
9. Kateriya S: “Vision” in Single-Celled Algae. News Physiol Sci 2004, 19:133–137.
10. Gao S, Nagpal J, Schneider MW, Kozjak-Pavlovic V, Nagel G, Gottschalk A: Optogenetic
manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-
cyclase opsin CyclOp. Nat Commun 2015, 6:1–12.
11. Scheib U, Stehfest K, Gee CE, Körschen HG, Fudim R, Oertner TG, Hegemann P: The
rhodopsin-guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables
fast optical control of cGMP signaling. Sci Signal 2015, 8:1–9.
12. Yoshida K, Tsunoda SP, Brown LS, Kandori H: A unique choanoflagellate enzyme
rhodopsin exhibits lightdependent cyclic nucleotide phosphodiesterase activity. J
Biol Chem 2017, 292:7531–7541.
13. Avelar GM, Schumacher RI, Zaini PA, Leonard G, Richards TA, Gomes SL: A Rhodopsin-
Guanylyl cyclase gene fusion functions in visual perception in a fungus. Curr Biol
2014, 24:1234–1240.
14. Kianianmomeni A, Hallmann A: Transcriptional analysis of Volvox photoreceptors
suggests the existence of different cell-type specific light-signaling pathways. Curr
Genet 2014, 61:3–18.
15. Pfeuty B, Thommen Q, Corellou F, Djouani-Tahri EB, Bouget FY, Lefranc M: Circadian
clocks in changing weather and seasons: Lessons from the picoalga ostreococcus
tauri. BioEssays 2012, 34:781–790.
16. Luck M, Mathes T, Bruun S, Fudim R, Hagedorn R, Nguyen TMT, Kateriya S, Kennis
JTM, Hildebrandt P, Hegemann P: A photochromic histidine kinase rhodopsin (HKR1)
that is bimodally switched by ultraviolet and blue light. J Biol Chem 2012,
287:40083–40090.
17. Awasthi M, Ranjan P, Sharma K, Veetil SK, Kateriya S: The trafficking of bacterial type
rhodopsins into the Chlamydomonas eyespot and flagella is IFT mediated. Sci Rep
2016, 6.
18. Tian Y, Gao S, von der Heyde EL, Hallmann A, Nagel G: Two-component cyclase opsins
of green algae are ATP-dependent and light-inhibited guanylyl cyclases. BMC Biol
2018, 16:144.
19. Berntsson O, Diensthuber RP, Panman MR, Björling A, Gustavsson E, Hoernke M,
Hughes AJ, Henry L, Niebling S, Takala H, et al.: Sequential conformational transitions
and α-helical supercoiling regulate a sensor histidine kinase. Nat Commun 2017,
8:284.
20. Gao R, Stock AM: Biological Insights from Structures of Two-Component Proteins.
Annu Rev Microbiol 2009, 63:133–154.
21. Dunin-Horkawicz S, Lupas AN: Comprehensive analysis of HAMP domains:
Implications for transmembrane signal transduction. J Mol Biol 2010, 397:1156–
1174.
22. Kianianmomeni A, Hallmann A: Algal photoreceptors: In vivo functions and potential
applications. Planta 2014, 239:1–26.
23. Luck M, Bruun S, Keidel A, Hegemann P, Hildebrandt P: Photochemical chromophore
isomerization in histidine kinase rhodopsin HKR1. FEBS Lett 2015, 589:1067–1071.
24. Penzkofer A, Luck M, Mathes T, Hegemann P: Bistable retinal Schiff base
photodynamics of histidine kinase rhodopsin HKR1 from chlamydomonas
reinhardtii. Photochem Photobiol 2014, 90:773–785.
25. Luck M, Hegemann P: The two parallel photocycles of the Chlamydomonas sensory
photoreceptor histidine kinase rhodopsin 1. J Plant Physiol 2017, 217:77-84.
26. Tian Y, Gao S, Yang S, Nagel G: A novel rhodopsin phosphodiesterase from

8
 Salpingoeca rosetta shows light-enhanced substrate affinity. Biochem J 2018,
475:1121–1128.
27. Lamarche LB, Kumar RP, Trieu MM, Devine EL, Cohen-Abeles LE, Theobald DL, Oprian
DD: Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase
Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate
Salpingoeca rosetta. Biochemistry 2017, 56:5812–5822.
28. Watari M, Ikuta T, Yamada D, Shihoya W, Yoshida K, Tsunoda SP, Nureki O, Kandori H:
Spectroscopic study of the transmembrane domain of a rhodopsin–
phosphodiesterase fusion protein from a unicellular eukaryote. J Biol Chem 2019,
doi:10.1074/jbc.RA118.006277.
29. Luecke H, Schobert B, Lanyi JK, Spudich EN, Spudich JL: Crystal structure of sensory
rhodopsin II at 2.4 angstroms: Insights into color tuning and transducer interaction.
Science (80- ) 2001, 293:1499–1503.
30. Huai Q, Wang H, Zhang W, Colman RW, Robinson H, Ke H: Crystal structure of
phosphodiesterase 9 shows orientation variation of inhibitor 3-isobutyl-1-
methylxanthine binding. Proc Natl Acad Sci 2004, 101:9624–9629.
31. Pandit J, Forman MD, Fennell KF, Dillman KS, Menniti FS: Mechanism for the allosteric
regulation of phosphodiesterase 2A deduced from the X-ray structure of a near full-
length construct. Proc Natl Acad Sci 2009, 106:18225–18230.
32. Saranak J, Foster KW: Rhodopsin guides fungal phototaxis [7]. Nature 1997, 387:465–
466.
33. Avelar GM, Glaser T, Leonard G, Richards TA, Ulrich H, Gomes SL: A cyclic GMP-
dependent K+channel in the blastocladiomycete fungus Blastocladiella emersonii.
Eukaryot Cell 2015, 14:958–963.
34. Scheib U, Broser M, Constantin OM, Yang S, Gao S, Mukherjee S, Stehfest K, Nagel G,
Gee CE, Hegemann P: Rhodopsin-cyclases for photocontrol of cGMP/cAMP and 2.3 Å
structure of the adenylyl cyclase domain. Nat Commun 2018, 9:2046.
35. Trieu MM, Devine EL, Lamarche LB, Ammerman AE, Greco JA, Birge RR, Theobald DL,
Oprian DD: Expression, purification, and spectral tuning of RhoGC, a
retinylidene/guanylyl cyclase fusion protein and optogenetics tool from the aquatic
fungus Blastocladiella emersonii. J Biol Chem 2017, 292:10379–10389.
36. Penzkofer A, Scheib U, Stehfest K, Hegemann P: Absorption and emission
spectroscopic investigation of thermal dynamics and photo-dynamics of the
rhodopsin domain of the rhodopsin-guanylyl cyclase from the nematophagous
fungus Catenaria anguillulae. Int J Mol Sci 2017, 18.
37. Ziegler M, Bassler J, Beltz S, Schultz A, Lupas AN, Schultz JE: Characterization of a
novel signal transducer element intrinsic to class IIIa/b adenylate cyclases and
guanylate cyclases. FEBS J 2017, 284:1204–1217.
38. Rauch A, Leipelt M, Russwurm M, Steegborn C: Crystal structure of the guanylyl
cyclase Cya2. Proc Natl Acad Sci 2008, 105:15720–15725.
39. Etzl S, Lindner R, Nelson MD, Winkler A: Structure-guided design and functional
characterization of an artificial red light–regulated guanylate/adenylate cyclase for
optogenetic applications. J Biol Chem 2018, 293:9078–9089.
40. Sunahara RK, Beuve A, Tesmer JJG, Sprang SR, Garbers DL, Gilman AG: Exchange of
substrate and inhibitor specificities between adenylyl and guanylyl cyclases. J Biol
Chem 1998, 273:16332–8.
41. Steegborn C: Structure, mechanism, and regulation of soluble adenylyl cyclases -
similarities and differences to transmembrane adenylyl cyclases. Biochim Biophys

9
 Acta - Mol Basis Dis 2014, 1842:2535-2547.
42. Kumar RP, Morehouse BR, Fofana J, Trieu MM, Zhou DH, Lorenz MO, Oprian DD:
Structure and monomer/dimer equilibrium for the guanylyl cyclase domain of the
optogenetics protein RhoGC. J Biol Chem 2017, 292:21578–21589.
43. Tesmer JJ, Sunahara RK, Johnson RA, Gosselin G, Gilman AG, Sprang SR: Two-metal-
Ion catalysis in adenylyl cyclase. Science 1999, 285:756–60.
44. Ohki M, Sato-Tomita A, Matsunaga S, Iseki M, Tame JRH, Shibayama N, Park S-Y:
Molecular mechanism of photoactivation of a light-regulated adenylate cyclase.
Proc Natl Acad Sci 2017, 114:8562–8567.
45. Lindner R, Hartmann E, Tarnawski M, Winkler A, Frey D, Reinstein J, Meinhart A,
Schlichting I: Photoactivation Mechanism of a Bacterial Light-Regulated Adenylyl
Cyclase. J Mol Biol 2017, 429:1336–1351.
46. Berndt A, Yizhar O, Gunaydin LA, Hegemann P, Deisseroth K: Bi-stable neural state
switches. Nat Neurosci 2009, 12:229–234.
47. Stierl M, Stumpf P, Udwari D, Gueta R, Hagedorn R, Losi A, Gärtner W, Petereit L,
Efetova M, Schwarzel M, et al.: Light modulation of cellular cAMP by a small
bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa. J
Biol Chem 2011, 286:1181–1188.
48. Tanwar M, Sharma K, Moar P, Kateriya S: Biochemical Characterization of the
Engineered Soluble Photoactivated Guanylate Cyclases from Microbes Expands
Optogenetic Tools. Appl Biochem Biotechnol 2018, 185:1014–1028.
49. Tanwar M, Khera L, Haokip N, Kaul R, Naorem A, Kateriya S: Modulation of cyclic
nucleotide-mediated cellular signaling and gene expression using photoactivated
adenylyl cyclase as an optogenetic tool. Sci Rep 2017, 7:1–13.
50. Gasser C, Taiber S, Yeh C-M, Wittig CH, Hegemann P, Ryu S, Wunder F, Moglich A:
Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase.
Proc Natl Acad Sci 2014, 111:8803–8808.
51. Schröder-Lang S, Schwärzel M, Seifert R, Strünker T, Kateriya S, Looser J, Watanabe M,
Kaupp UB, Hegemann P, Nagel G: Fast manipulation of cellular cAMP level by light in
vivo. Nat Methods 2007, 4:39–42.
52. Koyanagi M, Takada E, Nagata T, Tsukamoto H, Terakita A: Homologs of vertebrate
Opn3 potentially serve as a light sensor in nonphotoreceptive tissue. Proc Natl Acad
Sci 2013, 110:4998–5003.
53. Bailes HJ, Zhuang LY, Lucas RJ: Reproducible and sustained regulation of Gαs
signalling using a metazoan opsin as an optogenetic tool. PLoS One 2012, 7.
54. Kim T, Folcher M, Baba MD El, Fussenegger M: A synthetic erectile optogenetic
stimulator enabling blue-light-inducible penile erection. Angew Chemie - Int Ed 2015,
54:5933–5938.
55. Ryu M-H, Kang I-H, Nelson MD, Jensen TM, Lyuksyutova AI, Siltberg-Liberles J, Raizen
DM, Gomelsky M: Engineering adenylate cyclases regulated by near-infrared
window light. Proc Natl Acad Sci 2014, 111:10167–10172.
56. Ryu MH, Moskvin O V., Siltberg-Liberles J, Gomelsky M: Natural and engineered
photoactivated nucleotidyl cyclases for optogenetic applications. J Biol Chem 2010,
285:41501–41508.
57. Blain-Hartung M, Rockwell NC, Moreno M V., Martin SS, Gan F, Bryant DA, Lagarias JC:
Cyanobacteriochrome-based photoswitchable adenylyl cyclases (cPACs) for broad
spectrum light regulation of cAMP levels in cells. J Biol Chem 2018, 293:8473–8483.
58. Peshenko I V., Olshevskaya E V., Savchenko AB, Karan S, Palczewski K, Baehr W,

10
 Dizhoor AM: Enzymatic properties and regulation of the native isozymes of retinal
membrane guanylyl cyclase (RetGC) from mouse photoreceptors. Biochemistry 2011,
50:5590–5600.
59. Karasuyama M, Inoue K, Nakamura R, Kandori H, Takeuchi I: Understanding Colour
Tuning Rules and Predicting Absorption Wavelengths of Microbial Rhodopsins by
Data-Driven Machine-Learning Approach. Sci Rep 2018, 8:15580.
60. Kaneko A, Inoue K, Kojima K, Kandori H, Sudo Y: Conversion of microbial rhodopsins:
insights into functionally essential elements and rational protein engineering.
Biophys Rev 2017, 9:861–876.
61. Wietek J, Prigge M: Optogenetics. 2016, 1408.
62. Shen YC, Sasaki T, Matsuyama T, Yamashita T, Shichida Y, Okitsu T, Yamano Y, Wada A,
Ishizuka T, Yawo H, et al.: Red-Tuning of the Channelrhodopsin Spectrum Using Long
Conjugated Retinal Analogues. Biochemistry 2018, 57:5544–5556.
63. Oda K, Vierock J, Oishi S, Rodriguez-Rozada S, Taniguchi R, Yamashita K, Wiegert JS,
Nishizawa T, Hegemann P, Nureki O: Crystal structure of the red light-activated
channelrhodopsin Chrimson. Nat Commun 2018, 9:3949.
64. Woldemariam S, Nagpal J, Li J, Schneider MS, Shankar R, Futey M, Varshney A,
Andersen K, Barsi-Rhyne B, Tran A, et al.: Robust and sensitive GFP-based cGMP
sensor for real time imaging in intact Caenorhabditis elegans. bioRxiv 2018,
doi:10.1101/433425.
65. Ryu MH, Youn H, Kang IH, Gomelsky M: Identification of bacterial guanylate cyclases.
Proteins Struct Funct Bioinforma 2015, 83:799–804.
66. Winger JA, Derbyshire ER, Lamers MH, Marletta MA, Kuriyan J: The crystal structure
of the catalytic domain of a eukaryotic guanylate cyclase. BMC Struct Biol 2008, 8:42.

Figures:

Figure 1 Representative schematic of the known enzymerhodopsins including the

domain arragment, and their soluble counterparts drawn as plain symbols with
simplified chromophore structure. HKR, histidine kinase rhodopsin, green-light
inhibited hybrid histidine kinase two-component-like system including a guanylyl
cyclase effector domain [18]; RhGC, naturally occurring rhodopsin guanylyl cyclase
activated by green light and its domain architecture [13]; RhoPDE, a blue-light

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activated rhodopsin-phosphodiesterase fusion protein [12]; SHK (sensory histidine
kinase)[19], a blue light-activated chimeric sensor histidine kinase (YF1) based on LOV
(light-oxygen-voltage) domain photoreceptor; PAC/blGC are natural or engineered
adenylyl/guanylyl cyclases containing BLUF (blue-light sensor using FAD) domain
[47,65] ; PaaC/PagC, chimera containing phytochrome as photoreceptor and a
cyanobacterial cyclase domain [39]; LAPD (light-activated phosphodiesterase), an
engineered phytochrome-based red-light activated cAMP/cGMP phosphodiesterase
[50].

Figure 2 Sequence alignment of selected microbial rhodopsins and structure of the

enzyme domain of RhoPDE. (a) Rhodopsin sequences (RhoPDE (S. rosetta), RhGC (C.
anguillulae), Channelrhodopsin 2 (ChR2) and HKR1 (C. reinhardtii), Sensory rhodopsin
II (SRII, N. pharaonis) and Bacteriorhodopsin (BR, H. salinarum)) assigned to the
membrane topology of the 7 canonical transmembrane-helices. Conserved residues
functionally involved in the photocycle are highlighted: black: conserved retinal-
binding Lysine, red: counterion / proton acceptor of RSB, orange: proton-release
complex, green DC-gate. (b) Crystal structure of the isolated homodimeric PDE
domain of RhoPDE (PDB-code: 5VYD)[27]. The protein shown as cartoon with
monomer 1 colored red/yellow and monomer 2 colored green/blue. Metal centers
Mg2+ (magenta) and Zn2+ (green) are drawn as spheres. Superimposed structure of
human PDE9 (PDB-code: 2HD1) is shown in grey [30]. H-loops are marked by arrows.

Figure 3 Nucleotide binding pocket of class III cNMP cyclases and structure of an
RhGC enzyme domain with converted substrate specificity. (a) Schema of the
nucleotide binding as deduced from crystal structures. Secondary structure elements
are represented as arrows (beta-sheet) or cylinders (alpha-helix) with sequences
aligned for RhGC (C. anguillulae, GB AVZ03094.1); GC (algae) (guanylyl cyclase, C.
reinhardtii); RetGC (Retinal guanylyl cyclase, human); tmAC C2 (transmembrane
adenylyl cyclase, rat); AC (Nostoc sp.) (adenylyl cyclase, Nostoc sp. PCC7120) and
from bPAC (bacterial photoactivated adenylyl cyclase, B. alba). Conserved residues
involved in metal- (green), sugar- (cyan) and phosphate-binding (blue) are
highlighted. Base-discriminating residues (red box) with guanine- or adenine-binding
are drawn in red or blue, note that base-binding K/E on β2-β3 belong to the
symmetry related site. (b) Structure of homodimeric CaAC (PDB-code 5OYH) [34] :
Protein is shown as cartoon and colored: monomer 1 red/yellow; monomer 2
blue/green. Structure of eukaryotic GC (C. reinhardtii, PDB-code 3ET6) [66]
superimposed to monomer 2 (grey). Inhibitor ATPαS (red: adenine; cyan: ribose;
blue: phosphate) and Ca2+ (green) are shown as spheres.

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