Photoactivated Adenylyl Cyclases: Fundamental Properties and Applications.

Photoactivated adenylyl cyclase (PAC) was first discovered to be a sensor for photoavoidance in the flagellate Euglena gracilis. PAC is a flavoprotein that catalyzes the production of cAMP upon illumination with blue light, which enables us to optogenetically manipulate intracellular cAMP levels in various biological systems. Recent progress in genome sequencing has revealed several related proteins in bacteria and ameboflagellates. Among them, the PACs from sulfur bacterium Beggiatoa sp. and cyanobacterium Oscillatoria acuminata have been well characterized, including their crystalline structure. Although there have not been many reported optogenetic applications of PACs so far, they have the potential to be used in various fields within bioscience.

Hydrogen Bonding Environments in the Photocycle Process around the Flavin Chromophore of the AppA-BLUF domain.

Three kinds of photochemical reactions are known in flavins as chromophores of photosensor proteins, reflecting the various catalytic reactions of the flavin in flavoenzymes. Sensor of blue light using the flavin FAD (BLUF) domains exhibit a unique photoreaction compared with other flavin-binding photoreceptors in that the chromophore does not change its chemical structure between unphotolyzed and intermediate states. Rather, the hydrogen bonding environment is altered, whereby the conserved Gln and Tyr residues near FAD play a crucial role. One proposal for this behavior is that the conserved Gln changes its chemical structure from a keto to an enol. We applied light-induced difference Fourier transform infrared (FTIR) spectroscopy to AppA-BLUF. The spectra of AppA-BLUF exhibited a different feature upon 15N-Gln labeling compared with the previously reported spectra from BlrB, a different BLUF domain. The FTIR signals were interpreted from quantum mechanics/molecular mechanics (QM/MM) calculation as the keto-enol tautomerization and rotation of the Gln63 side chain in the AppA-BLUF domain. The former was consistent with the result from BlrB, but the latter was not uniquely determined by the previous study. QM/MM calculation also indicated that the infrared signal shape is influenced depending on whether a Trp side chain forms a hydrogen bond with the Gln side chain. FTIR spectra and QM/MM simulations concluded that Trp104 does not flip out but is maintained in the intermediate state. In contrast, our data revealed that the Trp residue at the corresponding position in BlrB faces outward in both states.

Molecular mechanism of photoactivation of a light-regulated adenylate cyclase.

The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) detects light through a flavin chromophore within the N-terminal BLUF domain. BLUF domains have been found in a number of different light-activated proteins, but with different relative orientations. The two BLUF domains of OaPAC are found in close contact with each other, forming a coiled coil at their interface. Crystallization does not impede the activity switching of the enzyme, but flash cooling the crystals to cryogenic temperatures prevents the signature spectral changes that occur on photoactivation/deactivation. High-resolution crystallographic analysis of OaPAC in the fully activated state has been achieved by cryocooling the crystals immediately after light exposure. Comparison of the isomorphous light- and dark-state structures shows that the active site undergoes minimal changes, yet enzyme activity may increase up to 50-fold, depending on conditions. The OaPAC models will assist the development of simple, direct means to raise the cyclic AMP levels of living cells by light, and other tools for optogenetics.

Photomovement in Euglena.

Motile microorganisms such as the green Euglena gracilis use a number of external stimuli to orient in their environment. They respond to light with photophobic responses, photokinesis and phototaxis, all of which can result in accumulations of the organisms in suitable habitats. The light responses operate synergistically with gravitaxis, aerotaxis and other responses. Originally the microscopically obvious stigma was thought to be the photoreceptor, but later the paraxonemal body (PAB, paraflagellar body) has been identified as the light responsive organelle, located in the trailing flagellum inside the reservoir. The stigma can aid in light direction perception by shading the PAB periodically when the cell rotates helically in lateral light, but stigmaless mutants can also orient with respect to the light direction, and negative phototaxis does not need the presence of the stigma. The PAB is composed of dichroically oriented chromoproteins which is reflected in a pronounced polarotaxis in polarized light. There was a long debate about the potential photoreceptor molecule in Euglena, including carotenoids, flavins and rhodopsins. This discussion was terminated by the unambiguous proof that the photoreceptor is a 400 kDa photoactivated adenylyl cyclase (PAC) which consists of two α- and two ß-subunits each. Each subunit possesses two BLUF (Blue Light receptor Using FAD) domains binding FAD, which harvest the light energy, and two adenylyl cyclases, which produce cAMP from ATP. The cAMP has been found to activate one of the five protein kinase s found in Euglena (PK.4). This enzyme in turn is thought to phosphorylate proteins inside the flagellum which result in a change in the flagellar beating pattern and thus a course correction of the cell. The involvements of PAC and protein kinase have been confirmed by RNA interference (RNAi). PAC is responsible for step-up photophobic responses as well as positive and negative phototaxis, but not for the step-down photophobic response, even though the action spectrum of this resembles those for the other two responses. Analysis of several colorless Euglena mutants and the closely related Euglena longa (formerly Astasia longa) confirms the results. Photokinesis shows a completely different action spectrum. Some other Euglena species, such as E. sanguinea and the gliding E. mutabilis, have been investigated, again showing totally different action spectra for phototaxis and photokinesis as well as step-up and step-down photophobic responses.

Structural insight into photoactivation of an adenylate cyclase from a photosynthetic cyanobacterium.

Cyclic-AMP is one of the most important second messengers, regulating many crucial cellular events in both prokaryotes and eukaryotes, and precise spatial and temporal control of cAMP levels by light shows great promise as a simple means of manipulating and studying numerous cell pathways and processes. The photoactivated adenylate cyclase (PAC) from the photosynthetic cyanobacterium Oscillatoria acuminata (OaPAC) is a small homodimer eminently suitable for this task, requiring only a simple flavin chromophore within a blue light using flavin (BLUF) domain. These domains, one of the most studied types of biological photoreceptor, respond to blue light and either regulate the activity of an attached enzyme domain or change its affinity for a repressor protein. BLUF domains were discovered through studies of photo-induced movements of Euglena gracilis, a unicellular flagellate, and gene expression in the purple bacterium Rhodobacter sphaeroides, but the precise details of light activation remain unknown. Here, we describe crystal structures and the light regulation mechanism of the previously undescribed OaPAC, showing a central coiled coil transmits changes from the light-sensing domains to the active sites with minimal structural rearrangement. Site-directed mutants show residues essential for signal transduction over 45 Å across the protein. The use of the protein in living human cells is demonstrated with cAMP-dependent luciferase, showing a rapid and stable response to light over many hours and activation cycles. The structures determined in this study will assist future efforts to create artificial light-regulated control modules as part of a general optogenetic toolkit.

Photoactivated adenylyl cyclase (PAC) reveals novel mechanisms underlying cAMP-dependent axonal morphogenesis.

Spatiotemporal regulation of axonal branching and elongation is essential in the development of refined neural circuits. cAMP is a key regulator of axonal growth; however, whether and how intracellular cAMP regulates axonal branching and elongation remain unclear, mainly because tools to spatiotemporally manipulate intracellular cAMP levels have been lacking. To overcome this issue, we utilized photoactivated adenylyl cyclase (PAC), which produces cAMP in response to blue-light exposure. In primary cultures of dentate granule cells transfected with PAC, short-term elevation of intracellular cAMP levels induced axonal branching but not elongation, whereas long-term cAMP elevation induced both axonal branching and elongation. The temporal dynamics of intracellular cAMP levels regulated axonal branching and elongation through the activation of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac), respectively. Thus, using PAC, our study for the first time reveals that temporal cAMP dynamics could regulate axonal branching and elongation via different signaling pathways.

Possible involvement of a tetrahydrobiopterin in photoreception for UV-B-induced anthocyanin synthesis in carrot.

Our previous studies of action spectra for UV-B-induced anthocyanin accumulation in cultured carrot cells indicated that a reduced form of pterin, possibly tetrahydrobiopterin, contributes to UV-B photoreception. In this report, we provide additional evidence for the involvement of pterin in UV-B light sensing. UV-B-induced phenylalanine ammonia-lyase (PAL) activity was considerably suppressed by N-acetylserotonin (an inhibitor of tetrahydrobiopterin biosynthesis), and this suppression was partially recovered by adding biopterin or tetrahydrobiobiopterin. In addition, protein(s) specifically bound to biopterin were detected by radiolabeling experiments in N-acetylserotonin-treated cells. Furthermore, diphenyleneiodonium, a potent inhibitor of electron transfer, completely suppressed UV-B-induced PAL activity. These results suggest the occurrence of an unidentified UV-B photoreceptor (other than UVR8, the tryptophan-based UV-B sensor originally identified in Arabidopsis) with reduced pterin in carrot cells. After reexamining published action spectra, we suggest that anthocyanin synthesis is coordinately regulated by these two UV-B sensors.

Identification of photoactivated adenylyl cyclases in Naegleria australiensis and BLUF-containing protein in Naegleria fowleri.

Complete genome sequencing of Naegleria gruberi has revealed that the organism encodes polypeptides similar to photoactivated adenylyl cyclases (PACs). Screening in the N. australiensis genome showed that the organism also encodes polypeptides similar to PACs. Each of the Naegleria proteins consists of a "sensors of blue-light using FAD" domain (BLUF domain) and an adenylyl cyclase domain (AC domain). PAC activity of the Naegleria proteins was assayed by comparing sensitivities of Escherichia coli cells heterologously expressing the proteins to antibiotics in a dark condition and a blue light-irradiated condition. Antibiotics used in the assays were fosfomycin and fosmidomycin. E. coli cells expressing the Naegleria proteins showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light, indicating that the proteins functioned as PACs in the bacterial cells. Analysis of the N. fowleri genome revealed that the organism encodes a protein bearing an amino acid sequence similar to that of BLUF. A plasmid expressing a chimeric protein consisting of the BLUF-like sequence found in N. fowleri and the adenylyl cyclase domain of N. gruberi PAC was constructed to determine whether the BLUF-like sequence functioned as a sensor of blue light. E. coli cells expressing a chimeric protein showed increased fosfomycin sensitivity and fosmidomycin sensitivity when incubated under blue light. These experimental results indicated that the sequence similar to the BLUF domain found in N. fowleri functioned as a sensor of blue light.

Photomanipulation of antibiotic susceptibility and biofilm formation of Escherichia coli heterologously expressing photoactivated adenylyl cyclase.

A cyaA-deficient Escherichia coli strain was transformed by a plasmid carrying the gene for BsPAC, a photoactivated adenylyl cyclase identified from a Beggiatoa sp., and was subjected to an antibiotic susceptibility assay and biofilm formation assay under a light or dark condition. Cells expressing BsPAC that were incubated under blue light (470 nm) were more susceptible to fosfomycin, nalidixic acid and streptomycin than were cells incubated in the dark. Cells expressing BsPAC formed more biofilms when incubated under the light than did cells cultured in the dark. We concluded from these observations that it is possible to determine the importance of cAMP in antibiotic susceptibility and biofilm formation of E. coli by photomanipulating the cellular cAMP level by the use of BsPAC. A site-directed mutant of BsPAC in which Tyr7 was replaced by Phe functioned even in the dark, indicating that Tyr7 plays an important role in photoactivation of BsPAC. Results of mutational analysis of BsPAC should contribute to an understanding of the molecular basis for photoactivation of the protein.

Structural change of a cofactor binding site of flavoprotein detected by single-protein fluorescence spectroscopy at 1.5 k.

The visible fluorescence spectrum of single flavoprotein at a temperature of 1.5 K has been measured by one-photon excitation. The flavoprotein studied was a photoswitchable enzyme, photoactivated adenylyl cyclase. The time course of the spectrum revealed a structural change occurring at a rate of 10(-3) s(-1) around hydrogen bonds at the flavin cofactor binding site.

Blue and red light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus.

Photophysiological and pharmacological approaches were used to examine light-induced germination of resting spores in the red-tide diatom Leptocylindrus danicus. The equal-quantum action spectrum for photogermination had peaks at about 440 nm (blue light) and 680 nm (red light), which matched the absorption spectrum of the resting spore chloroplast, as well as photosynthetic action spectra reported for other diatoms. DCMU, an inhibitor of photosynthetic electron flow near photosystem II, completely blocked photogermination. These results suggest that the photosynthetic system is involved in the photoreception process of light-induced germination. Results of pharmacological studies of the downstream signal transduction pathway suggested that Ca(2+) influx is the closest downstream neighbor, followed by steps involving calmodulin, nitric oxide synthase, guanylyl cyclase, protein-tyrosine-phosphatase, protein kinase C and actin polymerization and translation.

Differentiation of photocycle characteristics of flavin-binding BLUF domains of α- and ß-subunits of photoactivated adenylyl cyclase of Euglena gracilis.

Photoactivated adenylyl cyclase (PAC), an FAD-containing photoreceptor of Euglena gracilis, appears to be a heterotetrameric structure composed of 2 homologous subunits (PACα and PACß), each with a pair of BLUF domains (F1 and F2). PAC promotes blue light-induced activation of adenylyl cyclase. In our previous report, we demonstrated that a recombinant version of the PACαF2 domain displays blue light-induced photocycle similar to those of prokaryotic BLUFs (Ito et al., Photochem. Photobiol. Sci., 2005, 4, 762-769). Here, we further examine the recombinant PACßF2 domain, which like PACαF2 exhibits a blue light-induced photocycle. The estimated quantum efficiency for the phototransformation of PACßF2 was 0.06-0.08, and the half-life for dark relaxation was 3-6 s while the corresponding values for the PACαF2 were 0.28-0.32 and 34-44 s. The remarkable differences between PACαF2 and PACßF2 may be related to the sensitivity of the photoactivation. In PACαF2, amino acid position 556, which is equivalent to Trp104 in the BLUF domain of the purple bacterial AppA protein, is occupied by a Leu residue, while in PACßF2 the equivalent BLUF domain site is conserved as Trp560. Amino acid substitution at this site in PACßF2-Trp560Leu markedly increased the estimated quantum efficiency (0.23) and accelerated the half-life of the dark-relaxation (2 s). These results indicate that Trp560 in PACßF2 plays a main role in suppressing the quantum efficiency.

Flagellar motions in phototactic steering in a brown algal swarmer.

Using infrared high-speed video microscopy, we observed light-triggered transitory flagellar motions in flagellate reproductive cells (swarmers) of a brown alga, Scytosiphon lomentaria, under primary helical swimming conditions before and during negative phototactic orientation to unilateral actinic light. The posterior flagellum, which is autofluorescent and thought to be light-sensing, was passively dragged in the dark and exhibited one to several rapid lateral beats during orientation changes for phototactic steering. Notably, a brief cessation of anterior flagellar beating was occasionally observed concomitantly with rapid beats of the posterior flagellum. This behavior caused a pause in helical body rotation, which may contribute to the accuracy of phototactic steering. Thus, coordinated regulation of the movement of the two flagella plays a crucial role in phototactic steering.

Origins of a cyanobacterial 6-phosphogluconate dehydrogenase in plastid-lacking eukaryotes.

BACKGROUND: Plastids have inherited their own genomes from a single cyanobacterial ancestor, but the majority of cyanobacterial genes, once retained in the ancestral plastid genome, have been lost or transferred into the eukaryotic host nuclear genome via endosymbiotic gene transfer. Although previous studies showed that cyanobacterial gnd genes, which encode 6-phosphogluconate dehydrogenase, are present in several plastid-lacking protists as well as primary and secondary plastid-containing phototrophic eukaryotes, the evolutionary paths of these genes remain elusive. RESULTS: Here we show an extended phylogenetic analysis including novel gnd gene sequences from Excavata and Glaucophyta. Our analysis demonstrated the patchy distribution of the excavate genes in the gnd gene phylogeny. The Diplonema gene was related to cytosol-type genes in red algae and Opisthokonta, while heterolobosean genes occupied basal phylogenetic positions with plastid-type red algal genes within the monophyletic eukaryotic group that is sister to cyanobacterial genes. Statistical tests based on exhaustive maximum likelihood analyses strongly rejected that heterolobosean gnd genes were derived from a secondary plastid of green lineage. In addition, the cyanobacterial gnd genes from phototrophic and phagotrophic species in Euglenida were robustly monophyletic with Stramenopiles, and this monophyletic clade was moderately separated from those of red algae. These data suggest that these secondary phototrophic groups might have acquired the cyanobacterial genes independently of secondary endosymbioses. CONCLUSION: We propose an evolutionary scenario in which plastid-lacking Excavata acquired cyanobacterial gnd genes via eukaryote-to-eukaryote lateral gene transfer or primary endosymbiotic gene transfer early in eukaryotic evolution, and then lost either their pre-existing or cyanobacterial gene.

Functional transplant of photoactivated adenylyl cyclase (PAC) into Aplysia sensory neurons.

In neural mechanisms of animal learning, intracellular cAMP has been known to play an important role. In the present experiments we attempted functional transplant of a photoactivated adenylyl cyclase (PAC) isolated from Euglena into Aplysia neurons, and explored whether PAC can produce cAMP in the neurons by light stimulation. Serotonergic modulation of mechanoafferent sensory neurons in Aplysia pleural ganglia has been reported to increase intracellular cAMP level and promotes synaptic transmission to motor neurons by increasing spike width of sensory neurons. When cAMP was directly injected into the sensory neurons, spike amplitude temporarily decreased while spike width temporarily increased. This effect was not substituted by injection of 5'AMP, and maintained longer in a bath solution containing IBMX, the phosphodiesterase inhibitor. We, therefore, explored these changes as indicators of appearance of the PAC function. PAC or the PAC expression vector (pNEX-PAC) was injected into cell bodies of sensory neurons. Spike amplitude decreased in both cases and spike width increased in the PAC injection when the neurons were stimulated with light, suggesting that the transplanted PAC works well in Aplysia neurons. These results indicate that we can control cAMP production in specific neurons with light by the functional transplant of PAC.

Homologs of the sexually induced gene 1 (sig1) product constitute the stramenopile mastigonemes.

The tripartite tubular mastigoneme on the anterior flagellum is a morphological feature that characterizes the stramenopiles. Mastigonemes are significant and potentially informative structures not only from the viewpoint of systematics, but also of cell biology. Nevertheless, few biochemical studies have been reported on stramenopile mastigonemes. The flagella of Scytosiphon lomentaria (Phaeophyceae) were successfully isolated and analyzed using SDS-PAGE followed by protein sequencing. The partial amino acid sequence of one flagellar protein (115kDa) showed high similarity with the sexually induced gene 1 (sig1) product of centric diatoms. A polyclonal antibody against the 115-kDa protein reacted not only to the shaft of mastigonemes in Scytosiphon lomentaria, but also another distinctly different stramenopile flagellate, Sulcochrysis biplastida (Dictyochophyceae). Therefore, we propose that the 115-kDa protein (i.e. Sig1 homologs) is a constituent of the tubular shaft of the mastigoneme.

Kinetic analysis of the activation of photoactivated adenylyl cyclase (PAC), a blue-light receptor for photomovements of Euglena.

Photoactivated adenylyl cyclase (PAC) was first purified from a photosensing organelle (the paraflagellar body) of the unicellular flagellate Euglena gracilis, and is regarded as the photoreceptor for the step-up photophobic response. Here, we report the kinetic properties of photoactivation of PAC and a change in intracellular cAMP levels upon blue light irradiation. Activation of PAC was dependent both on photon fluence rate and duration of irradiation, between which reciprocity held well in the range of 2--50 micromol m(-2) s(-1)(total fluence of 1200 micromol m(-2)). Intermittent irradiation also caused activation of PAC in a photon fluence-dependent manner irrespective of cycle periods. Wavelength dependency of PAC activation showed prominent peaks in the UV-B/C, UV-A and blue regions of the spectrum. The time course of the changes in intracellular cAMP levels corresponded well with that of the step-up photophobic response. From this and the kinetic properties of PAC photoactivation, we concluded that an increase in intracellular cAMP levels evoked by photoactivation of PAC is a key event of the step-up photophobic response.

Photocycle features of heterologously expressed and assembled eukaryotic flavin-binding BLUF domains of photoactivated adenylyl cyclase (PAC), a blue-light receptor in Euglena gracilis.

Photoactivated adenylyl cyclase (PAC) is a recently discovered blue-light photoreceptor that mediates photomovement in Euglena gracilis(Iseki et al., Nature, 2002, 415, 1047--1051). PAC appears to be a heterotetramer composed of two FAD-binding subunits (PACalpha and PACbeta). Both subunits have a pair of homologous regions (F1 and F2) which show homology with prokaryotic "sensors of blue-light using FAD"(BLUF) domains. The F1 and F2 domains of PAC are the only eukaryotic BLUF domains found thus far. We obtained soluble recombinant F1 and F2 proteins in PACalpha by heterologous expression with fused glutathione-S-transferase (GST) in E. coli. The expressed F1 samples did not bind flavins, but the F2 samples contained both FAD and FMN with trace amounts of riboflavin. We also assembled the histidine-tagged recombinant F2 (6His-F2) from inclusion bodies in E. coli with exogenous FAD or FMN. Blue-light-induced changes in absorption spectra of these assembled samples were highly similar to those reported for prokaryotic BLUF domains. The FAD- or FMN-assembled 6His-F2 photocycled with nearly the same rate constants of light-reaction and dark-relaxation, which were slightly lower than those of GST-cleaved F2. The estimated quantum efficiency for the phototransformation was 0.28--0.32, and the half-life was 34--44 s at 25 degrees C for the recombinant PACalpha F2, whereas that reported for prokaryotic BLUF domains varied from ca. 3.5 s (Tll0078) to ca. 900 s (AppA). The mutated recombinant Y472F and Q514G of PACalpha F2 and the F2 domain of the PACalpha homologue from Eutreptiella gymnastica, which lacks the Gln residue conserved in other BLUF domains, showed no photoinduced transformation.

The origin of photoactivated adenylyl cyclase (PAC), the Euglena blue-light receptor: phylogenetic analysis of orthologues of PAC subunits from several euglenoids and trypanosome-type adenylyl cyclases from Euglena gracilis.

Photoactivated adenylyl cyclase (PAC) is the blue-light receptor flavoprotein recently identified as a photoreceptor for photoavoidance of the unicellular flagellate, Euglena gracilis. To gain an insight into the evolution of this unique protein, similar sequences were searched for in several euglenoids by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers. Two similar transcripts were detected in each of the four phototrophic euglenoids, Euglena stellata, Colacium sideropus, Eutreptia viridis, Eutreptiella gymnastica, and in an osmotrophic (i.e., obtaining nutrients by absorption) one, Khawkinea quartana, but not in a phagotrophic euglenoid, Petalomonas cantuscygni. Each of them seemed to be orthologous to PACalpha and PACbeta, respectively, and had the same domain structure as PAC subunits each of which is composed of two flavin binding domains, F1 and F2, each followed by an adenylyl cyclase catalytic domain, C1 and C2, respectively. This fact implies that they constitute a functional photoactivated adenylyl cyclase like PAC. Phylogenetic analysis of the adenylyl cyclase catalytic domains revealed that they belong to a bacterial cluster, not to a trypanosomal one. In addition, two trypanosome-type adenylyl cyclases were discovered in E. gracilis. In contrast to PAC, deduced amino acid sequences of the trypanosome-type adenylyl cyclases indicated that they are integral membrane proteins with a membrane spanning region at the midpoint of them, followed by an adenylyl cyclase catalytic domain which seems cytoplasmic. Overall, we propose that PAC might have been transferred to euglenoids on the occasion of secondary endosymbiosis.