Phytochromobilin Binding and Specific Amino Acid Residues Near The Chromophore Contribute To Orange Light Perception By The Dualchrome Phytochrome Region.

A novel photoreceptor dualchrome 1 (DUC1), containing a fused structure of cryptochrome and phytochrome, was discovered in the marine green alga Pycnococcus provasolli. The DUC1 phytochrome region (PpDUC1-N) binds to the bilin (linear tetrapyrrole) chromophores, phytochromobilin (PΦB) or phycocyanobilin (PCB), and reversibly photoconverts between the orange-absorbing dark-adapted state and the far-red-absorbing photoproduct state. This contrasts with typical phytochromes, which photoconvert between the red-absorbing dark-adapted and far-red-absorbing photoproduct states. In this study, we examined the molecular mechanism of PpDUC1-N to sense orange light by identifying the chromophore species synthesized by P. provasolli and the amino acid residues within the PpDUC1-N responsible for sensing orange light in the dark-adapted state. We focused on the PcyA homolog of P. provasolli (PpPcyA). Coexpression with the photoreceptors followed by an enzymatic assay revealed that PpPcyA synthesized PCB. Next, we focused on the PpDUC1-N GAF domain responsible for chromophore binding and light sensing. Ten amino acid residues were selected as the mutagenesis target near the chromophore. Replacement of these residues with those conserved in typical phytochromes revealed that three mutations (F290Y/M304S/L353M) resulted in a 23-nm red-shift in the dark-adapted state. Finally, we combined these constructs to obtain the PΦB-binding F290Y/M304S/L353M mutant and a 38-nm red-shift was observed compared with the PCB-binding wild-type PpDUC1. The binding chromophore species and the key residues near the chromophore contribute to blue-shifted orange light sensing in the dark-adapted state of the PpDUC1-N.

Conformational change in an engineered biliverdin-binding cyanobacteriochrome during the photoconversion process.

Cyanobacteriochromes (CBCRs) derived from cyanobacteria are linear-tetrapyrrole-binding photoreceptors related to the canonical red/far-red reversible phytochrome photoreceptors. CBCRs contain chromophore-binding cGMP-specific phosphodiesterase/adenylate cyclase/FhlA (GAF) domains that are highly diverse in their primary sequences and are categorized into many subfamilies. Among this repertoire, the biliverdin (BV)-binding CBCR GAF domains receive considerable attention for their in vivo optogenetic and bioimaging applications because BV is a mammalian intrinsic chromophore and can absorb far-red light that penetrates deep into the mammalian body. The typical BV-binding CBCR GAF domain exhibits reversible photoconversion between far-red-absorbing dark-adapted and orange-absorbing photoproduct states. Herein, we applied various biochemical and spectral studies to identify the details of the conformational change during this photoconversion process. No oligomeric state change was observed, whereas the surface charge would change with a modification of the α-helix structures during the photoconversion process. Combinatorial analysis using partial protease digestion and mass spectrometry identified the region where the conformational change occurred. These results provide clues for the future development of optogenetic tools.

Novel cyanobacteriochrome photoreceptor with the second Cys residue showing atypical orange/blue reversible photoconversion.

Cyanobacteriochromes (CBCRs) are cyanobacterial linear tetrapyrrole-binding photoreceptors distantly related to phytochromes. Only the GAF domain is needed for chromophore incorporation and proper photoconversion of the CBCRs. Most CBCR GAF domains possess the canonical Cys residue stably ligating to the chromophore. DXCF-type CBCR GAF domains also possess a second Cys residue within the DXCF motif. This second Cys residue reversibly ligates to the C10 of the chromophore. The Cys adduct formation is mostly observed for the dark-adapted state but not for the photoproduct state. In this study, we discovered novel CBCR GAF domains with a DXCI motif instead of the DXCF motif. Since these CBCR GAF domains are categorized into two subfamilies (DXCI-1 and DXCI-2), the GAF domains from each subfamily were analyzed. Although the CBCR GAF domain belonging to the DXCI-2 subfamily showed orange/green reversible photoconversion without transient Cys ligation, the CBCR GAF domain belonging to the DXCI-1 subfamily showed reversible photoconversion between an orange-absorbing dark-adapted state and a blue-absorbing photoproduct state. This indicates that the second Cys residue is covalently bound to the C10 of the chromophore in the photoproduct state but not in the dark-adapted state. Since the covalent bond formation in the photoproduct state is atypical, site-directed mutagenesis was conducted to understand the molecular mechanism of this GAF domain. The Ile residue within the DXCI motif may be key for covalent bond formation in the photoproduct state.

Evidence for an early green/red photocycle that precedes the diversification of GAF domain photoreceptor cyanobacteriochromes.

Phytochromes are linear tetrapyrrole-binding photoreceptors in eukaryotes and bacteria, primarily responding to red and far-red light signals reversibly. Among the GAF domain-based phytochrome superfamily, cyanobacteria-specific cyanobacteriochromes show various optical properties covering the entire visible region. It is unknown what physiological demands drove the evolution of cyanobacteriochromes in cyanobacteria. Here, we utilize ancestral sequence reconstruction and biochemical verification to show that the resurrected ancestral cyanobacteriochrome proteins reversibly respond to green and red light signals. pH titration analyses indicate that the deprotonation of the bound phycocyanobilin chromophore is crucial to perceive green light. The ancestral cyanobacteriochromes show only modest thermal reversion to the green light-absorbing form, suggesting that they evolved to sense the incident green/red light ratio. Many cyanobacteria can utilize green light for photosynthesis using phycobilisome light-harvesting complexes. The green/red sensing cyanobacteriochromes may have allowed better acclimation to changing light environments by rearranging the absorption capacity of the phycobilisome through chromatic acclimation.

Role of hypoxanthine-guanine phosphoribosyltransferase in the metabolism of fairy chemicals in rice.

Fairy chemicals (FCs), 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are molecules with many diverse functions in plants. The defined biosynthetic pathway for FCs is a novel purine metabolism in which they are biosynthesized from 5-aminoimidazole-4-carboxamide. Here, we show that one of the purine salvage enzymes, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), recognizes AHX and AOH as substrates. Two novel compounds, AOH ribonucleotide and its ribonucleoside which are the derivatives of AOH, were enzymatically synthesized. The structures were determined by mass spectrometry, 1D and 2D NMR spectroscopy, and X-ray single-crystal diffraction analysis. This report demonstrates the function of HGPRT and the existence of novel purine metabolism associated with the biosynthesis of FCs in rice.

Evolution-inspired design of multicolored photoswitches from a single cyanobacteriochrome scaffold.

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.

Identification of significant residues for intermediate accumulation in phycocyanobilin synthesis.

Phycocyanobilin, the primary pigment of both light perception and light-harvesting in cyanobacteria, is synthesized from biliverdin IXα (BV) through intermediate 181, 182-dihydrobiliverdin (181, 182-DHBV) by a phycocyanobilin:ferredoxin oxidoreductase (PcyA). In our previous study, we discovered two PcyA homologs (AmPcyAc and AmPcyAp) derived from Acaryochloris marina MBIC 11017 (A. marina) that exceptionally uses chlorophyll d as the primary photosynthetic pigment, absorbing longer wavelength far-red light than chlorophyll a, the photosynthetic pigment found in most cyanobacteria. Biochemical characterization of the two PcyA homologs identified functional diversification of these two enzymes: AmPcyAc provides 181, 182-DHBV, and PCB to the cyanobacteriochrome (CBCR) photoreceptors, whereas, AmPcyAp specifically provides PCB to the light-harvesting phycobilisome subunit. In this study, we focused on the residues necessary for 181, 182-DHBV supply to the CBCR photoreceptors by AmPcyAc. Based on the SyPcyA structure, we concentrated on the 30 residues that constitute the substrate-binding pocket. Among them, we discovered that Leu151 and Val225 in AmPcyAc were both substituted with isoleucine. During the enzymatic reaction, the SyPcyA variant molecule, possessing V225I and L151I replacements, accumulates the 181, 182-DHBV and supplies it to a CBCR molecule derived from A. marina. It is worth noting that the substitution of Val225 with isoleucine was specifically conserved among the Acaryochloris genus. Collectively, we propose that the specific evolution of PcyA among the Acaryochloris genus may correlate with the acquisition of Chl. d synthetic ability and growth in long-wavelength far-red light environments.

Unusual ring D fixation by three crucial residues promotes phycoviolobilin formation in the DXCF-type cyanobacteriochrome without the second Cys.

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. Among them, dual Cys-type cyanobacteriochrome GAF domains possessing not only a highly conserved 'first Cys' but also a 'second Cys' are found from multiple lineages. The first Cys stably attaches to C31 of the A-ring, while the second Cys mostly shows reversible ligation to the C10 of the chromophore. Notably, the position of the second Cys in the primary sequence is diversified, and the most abundant dual Cys-type GAF domains have a 'second Cys' within the DXCF motif, which are called DXCF GAF domains. It has been long known that the second Cys in the DXCF GAF domains not only shows the reversible ligation but also is involved in isomerization activity (reduction in C4=C5 double bond) from the initially incorporated phycocyanobilin to phycoviolobilin. However, comprehensive site-directed mutagenesis on the DXCF GAF domains, AM1_6305g1 and AM1_1499g1, revealed that the second Cys is dispensable for isomerization activity, in which three residues participate by fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is necessary, even though one side of the fixation site is far from this bridge, with the other side at C31 fixed by the first Cys.

Rational conversion of chromophore selectivity of cyanobacteriochromes to accept mammalian intrinsic biliverdin.

Because cyanobacteriochrome photoreceptors need only a single compact domain for chromophore incorporation and for absorption of visible spectra including the long-wavelength far-red region, these molecules have been paid much attention for application to bioimaging and optogenetics. Most cyanobacteriochromes, however, have a drawback to incorporate phycocyanobilin that is not available in the mammalian cells. In this study, we focused on biliverdin (BV) that is a mammalian intrinsic chromophore and absorbs the far-red region and revealed that replacement of only four residues was enough for conversion from BV-rejective cyanobacteriochromes into BV-acceptable molecules. We succeeded in determining the crystal structure of one of such engineered molecules, AnPixJg2_BV4, at 1.6 Å resolution. This structure identified unusual covalent bond linkage, which resulted in deep BV insertion into the protein pocket. The four mutated residues contributed to reducing steric hindrances derived from the deeper insertion. We introduced these residues into other domains, and one of them, NpF2164g5_BV4, produced bright near-infrared fluorescence from mammalian liver in vivo. Collectively, this study provides not only molecular basis to incorporate BV by the cyanobacteriochromes but also rational strategy to open the door for application of cyanobacteriochromes to visualization and regulation of deep mammalian tissues.

Comparison of the Forward and Reverse Photocycle Dynamics of Two Highly Similar Canonical Red/Green Cyanobacteriochromes Reveals Unexpected Differences.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward (15ZPr → 15EPg) and reverse (15EPg → 15ZPr) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-Gf intermediate red-shifted from the 15ZPr photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φp within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.

Phytochromes and Cyanobacteriochromes: Photoreceptor Molecules Incorporating a Linear Tetrapyrrole Chromophore.

In this chapter, we summarize the molecular mechanisms of the linear tetrapyrrole-binding photoreceptors, phytochromes, and cyanobacteriochromes. We especially focus on the color-tuning mechanisms and conformational changes during the photoconversion process. Furthermore, we introduce current status of development of the optogenetic tools based on these molecules. Huge repertoire of these photoreceptors with diverse spectral properties would contribute to development of multiplex optogenetic regulation. Among them, the photoreceptors incorporating the biliverdin IXα chromophore is advantageous for in vivo optogenetics because this is intrinsic in the mammalian cells, and absorbs far-red light penetrating into deep mammalian tissues.

An Engineered Biliverdin-Compatible Cyanobacteriochrome Enables a Unique Ultrafast Reversible Photoswitching Pathway.

Cyanobacteriochromes (CBCRs) are promising optogenetic tools for their diverse absorption properties with a single compact cofactor-binding domain. We previously uncovered the ultrafast reversible photoswitching dynamics of a red/green photoreceptor AnPixJg2, which binds phycocyanobilin (PCB) that is unavailable in mammalian cells. Biliverdin (BV) is a mammalian cofactor with a similar structure to PCB but exhibits redder absorption. To improve the AnPixJg2 feasibility in mammalian applications, AnPixJg2_BV4 with only four mutations has been engineered to incorporate BV. Herein, we implemented femtosecond transient absorption (fs-TA) and ground state femtosecond stimulated Raman spectroscopy (GS-FSRS) to uncover transient electronic dynamics on molecular time scales and key structural motions responsible for the photoconversion of AnPixJg2_BV4 with PCB (Bpcb) and BV (Bbv) cofactors in comparison with the parent AnPixJg2 (Apcb). Bpcb adopts the same photoconversion scheme as Apcb, while BV4 mutations create a less bulky environment around the cofactor D ring that promotes a faster twist. The engineered Bbv employs a reversible clockwise/counterclockwise photoswitching that requires a two-step twist on ~5 and 35 picosecond (ps) time scales. The primary forward Pfr → Po transition displays equal amplitude weights between the two processes before reaching a conical intersection. In contrast, the primary reverse Po → Pfr transition shows a 2:1 weight ratio of the ~35 ps over 5 ps component, implying notable changes to the D-ring-twisting pathway. Moreover, we performed pre-resonance GS-FSRS and quantum calculations to identify the Bbv vibrational marker bands at ~659,797, and 1225 cm-1. These modes reveal a stronger H-bonding network around the BV cofactor A ring with BV4 mutations, corroborating the D-ring-dominant reversible photoswitching pathway in the excited state. Implementation of BV4 mutations in other PCB-binding GAF domains like AnPixJg4, AM1_1870g3, and NpF2164g5 could promote similar efficient reversible photoswitching for more directional bioimaging and optogenetic applications, and inspire other bioengineering advances.

A photoproduct of DXCF cyanobacteriochromes without reversible Cys ligation is destabilized by rotating ring twist of the chromophore.

Cyanobacteriochrome photoreceptors (CBCRs) ligate linear tetrapyrrole chromophores via their first (canonical) Cys residue and show reversible photoconversion triggered by light-dependent Z/E isomerization of the chromophore. Among the huge repertoire of CBCRs, DXCF CBCRs contain a second Cys residue within the highly conserved Asp-Xaa-Cys-Phe (DXCF) motif. In the typical receptors, the second Cys covalently attaches to the 15Z-chromophore in the dark state and detaches from the 15E-chromophore in the photoproduct state, whereas atypical ones that lack reversible ligation activity show red-shifted absorption in the dark state due to a more extended π-conjugated system. Moreover, some DXCF CBCRs show blue-shifted absorption in the photoproduct state due to the twisted geometry of the rotating ring. During the process of rational color tuning of a certain DXCF CBCR, we unexpectedly found that twisted photoproducts of some variant molecules showed dark reversion to the dark state, which prompted us to hypothesize that the photoproduct is destabilized by the twisted geometry of the rotating ring. In this study, we comprehensively examined the photoproduct stability of the twisted and relaxed molecules derived from the same CBCR scaffolds under dark conditions. In the DXCF CBCRs lacking reversible ligation activity, the twisted photoproducts showed faster dark reversion than the relaxed ones, supporting our hypothesis. By contrast, in the DXCF CBCRs exhibiting reversible ligation activity, the twisted photoproducts showed no detectable photoconversion. Reversible Cys adduct formation thus results in drastic rearrangement of the protein-chromophore interaction in the photoproduct state, which would contribute to the previously unknown photoproduct stability.

The Cruciality of Single Amino Acid Replacement for the Spectral Tuning of Biliverdin-Binding Cyanobacteriochromes.

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.

Molecular characterization of DXCF cyanobacteriochromes from the cyanobacterium Acaryochloris marina identifies a blue-light power sensor.

Cyanobacteriochromes (CBCRs) are linear tetrapyrrole-binding photoreceptors that sense a wide range of wavelengths from ultraviolet to far-red. The primary photoreaction in these reactions is a Z/E isomerization of the double bond between rings C and D. After this isomerization, various color-tuning events establish distinct spectral properties of the CBCRs. Among the various CBCRs, the DXCF CBCR lineage is widely distributed among cyanobacteria. Because the DXCF CBCRs from the cyanobacterium Acaryochloris marina vary widely in sequence, we focused on these CBCRs in this study. We identified seven DXCF CBCRs in A. marina and analyzed them after isolation from Escherichia coli that produces phycocyanobilin, a main chromophore for the CBCRs. We found that six of these CBCRs covalently bound a chromophore and exhibited variable properties, including blue/green, blue/teal, green/teal, and blue/orange reversible photoconversions. Notably, one CBCR, AM1_1870g4, displayed unidirectional photoconversion in response to blue-light illumination, with a rapid dark reversion that was temperature-dependent. Furthermore, the photoconversion took place without Z/E isomerization. This observation indicated that AM1_1870g4 likely functions as a blue-light power sensor, whereas typical CBCRs reversibly sense two light qualities. We also found that AM1_1870g4 possesses a GDCF motif in which the Asp residue is swapped with the next Gly residue within the DXCF motif. Site-directed mutagenesis revealed that this swap is essential for the light power-sensing function of AM1_1870g4. This is the first report of a blue-light power sensor from the CBCR superfamily and of photoperception without Z/E isomerization among the bilin-based photoreceptors.

Genetic identification of factors for extracellular cellulose accumulation in the thermophilic cyanobacterium Thermosynechococcus vulcanus: proposal of a novel tripartite secretion system.

Cells of the thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN (NIES-2134) aggregate and produce extracellular cellulose under induced conditions of blue light and low temperature, and both aggregation and cellulose production require the cellulose synthase Tll0007 (XcsA) and photosensory diguanylate cyclases. However, overexpression of both the cellulose synthase and a constitutively active diguanylate cyclase was not sufficient to induce cellulose-mediated cell aggregation under normal growth conditions. Synteny analysis and gene knockout revealed that two putative genes, hlyD-like tlr0903 (xcsB) and endoglucanase-like tlr1902 (xcsC), are linked to tll0007, although they are located apart from tll0007 in the T. vulcanus genome. Gene knockdown revealed that tlr1605 (tolC) was essential for the cellulose-mediated cell aggregation. Low temperature induced marked upregulation of tlr0903, and overexpression of both tlr0903 (but not tlr1902) and diguanylate cyclase resulted in the strong cell aggregation and cellulose accumulation under normal conditions. Based on these and phylogenetic analysis, we propose that the cyanobacterial extracellular cellulose production is due to a novel variant of the bacterial tripartite secretion system.

CyanoBase: a large-scale update on its 20th anniversary.

The first ever cyanobacterial genome sequence was determined two decades ago and CyanoBase (http://genome.microbedb.jp/cyanobase), the first database for cyanobacteria was simultaneously developed to allow this genomic information to be used more efficiently. Since then, CyanoBase has constantly been extended and has received several updates. Here, we describe a new large-scale update of the database, which coincides with its 20th anniversary. We have expanded the number of cyanobacterial genomic sequences from 39 to 376 species, which consists of 86 complete and 290 draft genomes. We have also optimized the user interface for large genomic data to include the use of semantic web technologies and JBrowse and have extended community-based reannotation resources through the re-annotation of Synechocystis sp. PCC 6803 by the cyanobacterial research community. These updates have markedly improved CyanoBase, providing cyanobacterial genome annotations as references for cyanobacterial research.

Protein Engineering of Dual-Cys Cyanobacteriochrome AM1_1186g2 for Biliverdin Incorporation and Far-Red/Blue Reversible Photoconversion.

Cyanobacteria have cyanobacteriochromes (CBCRs), which are photoreceptors that bind to a linear tetrapyrrole chromophore and sense UV-to-visible light. A recent study revealed that the dual-Cys CBCR AM1_1186g2 covalently attaches to phycocyanobilin and exhibits unique photoconversion between a Pr form (red-absorbing dark state, λmax = 641 nm) and Pb form (blue-absorbing photoproduct, λmax = 416 nm). This wavelength separation is larger than those of the other CBCRs, which is advantageous for optical tools. Nowadays, bioimaging and optogenetics technologies are powerful tools for biological research. In particular, the utilization of far-red and near-infrared light sources is required for noninvasive applications to mammals because of their high potential to penetrate into deep tissues. Biliverdin (BV) is an intrinsic chromophore and absorbs the longest wavelength among natural linear tetrapyrrole chromophores. Although the BV-binding photoreceptors are promising platforms for developing optical tools, AM1_1186g2 cannot efficiently attach BV. Herein, by rationally introducing several replacements, we developed a BV-binding AM1_1186g2 variant, KCAP_QV, that exhibited reversible photoconversion between a Pfr form (far-red-absorbing dark state, λmax = 691 nm) and Pb form (λmax = 398 nm). This wavelength separation reached 293 nm, which is the largest among the known phytochrome and CBCR photoreceptors. In conclusion, the KCAP_QV molecule developed in this study can offer an alternative platform for the development of unique optical tools.

Three cyanobacteriochromes work together to form a light color-sensitive input system for c-di-GMP signaling of cell aggregation.

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that have diverse spectral properties and domain compositions. Although large numbers of CBCR genes exist in cyanobacterial genomes, no studies have assessed whether multiple CBCRs work together. We recently showed that the diguanylate cyclase (DGC) activity of the CBCR SesA from Thermosynechococcus elongatus is activated by blue-light irradiation and that, when irradiated, SesA, via its product cyclic dimeric GMP (c-di-GMP), induces aggregation of Thermosynechococcus vulcanus cells at a temperature that is suboptimum for single-cell viability. For this report, we first characterize the photobiochemical properties of two additional CBCRs, SesB and SesC. Blue/teal light-responsive SesB has only c-di-GMP phosphodiesterase (PDE) activity, which is up-regulated by teal light and GTP. Blue/green light-responsive SesC has DGC and PDE activities. Its DGC activity is enhanced by blue light, whereas its PDE activity is enhanced by green light. A ΔsesB mutant cannot suppress cell aggregation under teal-green light. A ΔsesC mutant shows a less sensitive cell-aggregation response to ambient light. ΔsesA/ΔsesB/ΔsesC shows partial cell aggregation, which is accompanied by the loss of color dependency, implying that a nonphotoresponsive DGC(s) producing c-di-GMP can also induce the aggregation. The results suggest that SesB enhances the light color dependency of cell aggregation by degrading c-di-GMP, is particularly effective under teal light, and, therefore, seems to counteract the induction of cell aggregation by SesA. In addition, SesC seems to improve signaling specificity as an auxiliary backup to SesA/SesB activities. The coordinated action of these three CBCRs highlights why so many different CBCRs exist.

Structural heterogeneity in a parent ground-state structure of AnPixJg2 revealed by theory and spectroscopy.

We investigated the red absorbing, dark stable state (Pr state) of the second GAF domain of the cyanobacteriochrome AnPixJ (AnPixJg2) by a molecular dynamics simulation of 1 µs duration. Our results reveal two distinct conformational isoforms of the chromophore, from which only one was known from crystallographic experiments. The interconversion between both isoforms is accompanied by alterations in the hydrogen bond pattern between the chromophore and the protein and the solvation structure of the chromophore binding pocket. The existence of sub-states in the Pr form of AnPixJg2 is supported by the results from experimental 13C MAS NMR spectroscopy. Our finding is consistent with the observation of structural heterogeneity in other cyanobacteriochromes and phytochromes.