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Proceedings of the National Academy of... Jan 2020A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and...
A major barrier to defining the structural intermediates that arise during the reversible photointerconversion of phytochromes between their biologically inactive and active states has been the lack of crystals that faithfully undergo this transition within the crystal lattice. Here, we describe a crystalline form of the cyclic GMP phosphodiesterases/adenylyl cyclase/FhlA (GAF) domain from the cyanobacteriochrome PixJ in assembled with phycocyanobilin that permits reversible photoconversion between the blue light-absorbing Pb and green light-absorbing Pg states, as well as thermal reversion of Pg back to Pb. The X-ray crystallographic structure of Pb matches previous models, including autocatalytic conversion of phycocyanobilin to phycoviolobilin upon binding and its tandem thioether linkage to the GAF domain. Cryocrystallography at 150 K, which compared diffraction data from a single crystal as Pb or after irradiation with blue light, detected photoconversion product(s) based on F - F difference maps that were consistent with rotation of the bonds connecting pyrrole rings C and D. Further spectroscopic analyses showed that phycoviolobilin is susceptible to X-ray radiation damage, especially as Pg, during single-crystal X-ray diffraction analyses, which could complicate fine mapping of the various intermediate states. Fortunately, we found that PixJ crystals are amenable to serial femtosecond crystallography (SFX) analyses using X-ray free-electron lasers (XFELs). As proof of principle, we solved by room temperature SFX the GAF domain structure of Pb to 1.55-Å resolution, which was strongly congruent with synchrotron-based models. Analysis of these crystals by SFX should now enable structural characterization of the early events that drive phytochrome photoconversion.
Topics: Adenylyl Cyclases; Crystallography; Crystallography, X-Ray; Cyanobacteria; Cyclic GMP; Light; Models, Molecular; Phosphoric Diester Hydrolases; Photoreceptor Cells; Phycobilins; Phycocyanin; Phytochrome; Protein Conformation; Protein Domains; Thermosynechococcus; Trans-Activators
PubMed: 31852825
DOI: 10.1073/pnas.1912041116 -
Efficient synthesis of phycocyanobilin in mammalian cells for optogenetic control of cell signaling.Proceedings of the National Academy of... Nov 2017Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced...
Optogenetics is a powerful tool to precisely manipulate cell signaling in space and time. For example, protein activity can be regulated by several light-induced dimerization (LID) systems. Among them, the phytochrome B (PhyB)-phytochrome-interacting factor (PIF) system is the only available LID system controlled by red and far-red lights. However, the PhyB-PIF system requires phycocyanobilin (PCB) or phytochromobilin as a chromophore, which must be artificially added to mammalian cells. Here, we report an expression vector that coexpresses HO1 and PcyA with Ferredoxin and Ferredoxin-NADP+ reductase for the efficient synthesis of PCB in the mitochondria of mammalian cells. An even higher intracellular PCB concentration was achieved by the depletion of biliverdin reductase A, which degrades PCB. The PCB synthesis and PhyB-PIF systems allowed us to optogenetically regulate intracellular signaling without any external supply of chromophores. Thus, we have provided a practical method for developing a fully genetically encoded PhyB-PIF system, which paves the way for its application to a living animal.
Topics: Cell Line, Tumor; Ferredoxin-NADP Reductase; Ferredoxins; Genetic Vectors; HeLa Cells; Heme Oxygenase (Decyclizing); Humans; Light; Optogenetics; Oxidoreductases; Oxidoreductases Acting on CH-CH Group Donors; Phycobilins; Phycocyanin; Signal Transduction
PubMed: 29078307
DOI: 10.1073/pnas.1707190114 -
Biomolecules Mar 2024Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB's antioxidation,...
Phycocyanobilin (PCB) is a natural blue tetrapyrrole chromophore that is found in phycocyanin and plays an essential role in photosynthesis. Due to PCB's antioxidation, anti-inflammatory and anti-cancer properties, it has been utilized in the food, pharmaceutical and cosmetic industries. Currently, the extraction of PCB from involves complex processes, which has led to increasing interest in the biosynthesis of PCB in . However, the PCB titer remains low because of the poor activity of key enzymes and the insufficient precursor supply. Here, the synthesis of PCB was firstly improved by screening the optimal heme oxygenase (HO) from BP-1(HO) and PCB: ferredoxin oxidoreductase from sp. PCC6803 (PcyA). In addition, based on a rational design and the infrared fluorescence method for high-throughput screening, the mutants of HO(F29W/K166D) and PcyA(D220G/H74M) with significantly higher activities were obtained. Furthermore, a DNA scaffold was applied in the assembly of HO and PcyA mutants to reduce the spatial barriers, and the heme supply was enhanced via the moderate overexpression of and , resulting in the highest PCB titer (184.20 mg/L) obtained in a 5 L fermenter. The strategies applied in this study lay the foundation for the industrial production of PCB and its heme derivatives.
Topics: Phycocyanin; Escherichia coli; Phycobilins; Heme Oxygenase (Decyclizing); Heme
PubMed: 38540721
DOI: 10.3390/biom14030301 -
International Journal of Molecular... Dec 2019Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine,...
Biomarkers engineered on the basis of bacterial phytochromes with biliverdin IXα (BV) cofactor as a chromophore are increasingly used in cell biology and biomedicine, since their absorption and fluorescence spectra lie within the so-called optical "transparency window" of biological tissues. However, the quantum yield of BV fluorescence in these biomarkers does not exceed 0.145. The task of generating biomarkers with a higher fluorescence quantum yield remains relevant. To address the problem, we proposed the use of phycocyanobilin (PCB) as a chromophore of biomarkers derived from bacterial phytochromes. In this work, we characterized the complexes of iRFP713 evolved from BphP2 and its mutant variants with different location of cysteine residues capable of covalent tetrapyrrole attachment with the PCB cofactor. All analyzed proteins assembled with PCB were shown to have a higher fluorescence quantum yield than the proteins assembled with BV. The iRFP713/V256C and iRFP713/C15S/V256C assembled with PCB have a particularly high quantum yield of 0.5 and 0.45, which exceeds the quantum yield of all currently available near-infrared biomarkers. Moreover, PCB has 4 times greater affinity for iRFP713/V256C and iRFP713/C15S/V256C proteins compared to BV. These data establish iRFP713/V256C and iRFP713/C15S/V256C assembled with the PCB chromophore as promising biomarkers for application in vivo. The analysis of the spectral properties of the tested biomarkers allowed for suggesting that the high-fluorescence quantum yield of the PCB chromophore can be attributed to the lower mobility of the D-ring of PCB compared to BV.
Topics: Bacteria; Bacterial Proteins; Biliverdine; Biomarkers; Cysteine; Fluorescence; Luminescent Proteins; Phycobilins; Phycocyanin; Phytochrome; Protein Binding; Tetrapyrroles
PubMed: 31810174
DOI: 10.3390/ijms20236067 -
Journal of the American Chemical Society May 2023The phycobilisome is the primary light-harvesting antenna in cyanobacterial and red algal oxygenic photosynthesis. It maintains near-unity efficiency of energy transfer...
The phycobilisome is the primary light-harvesting antenna in cyanobacterial and red algal oxygenic photosynthesis. It maintains near-unity efficiency of energy transfer to reaction centers despite relying on slow exciton hopping along a relatively sparse network of highly fluorescent phycobilin chromophores. How the complex maintains this high efficiency remains unexplained. Using a two-dimensional electronic spectroscopy polarization scheme that enhances energy transfer features, we directly watch energy flow in the phycobilisome complex of sp. PCC 6803 from the outer phycocyanin rods to the allophycocyanin core. The observed downhill flow of energy, previously hidden within congested spectra, is faster than timescales predicted by Förster hopping along single rod chromophores. We attribute the fast, 8 ps energy transfer to interactions between rod-core linker proteins and terminal rod chromophores, which facilitate unidirectionally downhill energy flow to the core. This mechanism drives the high energy transfer efficiency in the phycobilisome and suggests that linker protein-chromophore interactions have likely evolved to shape its energetic landscape.
Topics: Phycobilisomes; Photosynthesis; Energy Transfer; Synechocystis
PubMed: 37200045
DOI: 10.1021/jacs.3c01799 -
The New Phytologist May 2017Land plant phytochromes perceive red and far-red light to control growth and development, using the linear tetrapyrrole (bilin) chromophore phytochromobilin (PΦB)....
Land plant phytochromes perceive red and far-red light to control growth and development, using the linear tetrapyrrole (bilin) chromophore phytochromobilin (PΦB). Phytochromes from streptophyte algae, sister species to land plants, instead use phycocyanobilin (PCB). PCB and PΦB are synthesized by different ferredoxin-dependent bilin reductases (FDBRs): PΦB is synthesized by HY2, whereas PCB is synthesized by PcyA. The pathway for PCB biosynthesis in streptophyte algae is unknown. We used phylogenetic analysis and heterologous reconstitution of bilin biosynthesis to investigate bilin biosynthesis in streptophyte algae. Phylogenetic results suggest that PcyA is present in chlorophytes and prasinophytes but absent in streptophytes. A system reconstituting bilin biosynthesis in Escherichia coli was modified to utilize HY2 from the streptophyte alga Klebsormidium flaccidum (KflaHY2). The resulting bilin was incorporated into model cyanobacterial photoreceptors and into phytochrome from the early-diverging streptophyte alga Mesostigma viride (MvirPHY1). All photoreceptors tested incorporate PCB rather than PΦB, indicating that KflaHY2 is sufficient for PCB synthesis without any other algal protein. MvirPHY1 exhibits a red-far-red photocycle similar to those seen in other streptophyte algal phytochromes. These results demonstrate that streptophyte algae use HY2 to synthesize PCB, consistent with the hypothesis that PΦB synthesis arose late in HY2 evolution.
Topics: Algal Proteins; Chlorophyta; Escherichia coli; Ferredoxins; Oxidoreductases; Phycobilins; Phycocyanin; Phylogeny; Phytochrome; Protein Denaturation
PubMed: 28106912
DOI: 10.1111/nph.14422 -
Frontiers in Microbiology 2022Marine efficiently harvest available light for photosynthesis using complex antenna systems, called phycobilisomes, composed of an allophycocyanin core surrounded by...
The phycoerythrobilin isomerization activity of MpeV in sp. WH8020 is prevented by the presence of a histidine at position 141 within its phycoerythrin-I β-subunit substrate.
Marine efficiently harvest available light for photosynthesis using complex antenna systems, called phycobilisomes, composed of an allophycocyanin core surrounded by rods, which in the open ocean are always constituted of phycocyanin and two phycoerythrin (PE) types: PEI and PEII. These cyanobacteria display a wide pigment diversity primarily resulting from differences in the ratio of the two chromophores bound to PEs, the green-light absorbing phycoerythrobilin and the blue-light absorbing phycourobilin. Prior to phycobiliprotein assembly, bilin lyases post-translationally catalyze the ligation of phycoerythrobilin to conserved cysteine residues on α- or β-subunits, whereas the closely related lyase-isomerases isomerize phycoerythrobilin to phycourobilin during the attachment reaction. MpeV was recently shown in sp. RS9916 to be a lyase-isomerase which doubly links phycourobilin to two cysteine residues (C50 and C61; hereafter C50, 61) on the β-subunit of both PEI and PEII. Here we show that sp. WH8020, which belongs to the same pigment type as RS9916, contains MpeV that demonstrates lyase-isomerase activity on the PEII β-subunit but only lyase activity on the PEI β-subunit. We also demonstrate that occurrence of a histidine at position 141 of the PEI β-subunit from WH8020, instead of a leucine in its counterpart from RS9916, prevents the isomerization activity by WH8020 MpeV, showing for the first time that both the substrate and the enzyme play a role in the isomerization reaction. We propose a structural-based mechanism for the role of H141 in blocking isomerization. More generally, the knowledge of the amino acid present at position 141 of the β-subunits may be used to predict which phycobilin is bound at C50, 61 of both PEI and PEII from marine strains.
PubMed: 36458192
DOI: 10.3389/fmicb.2022.1011189 -
Marine Drugs Mar 2020Organisms belonging to sp. genera are observed in all freshwater, brackish, and marine waters of the world. They play a relevant role in these ecosystems, since they...
Organisms belonging to sp. genera are observed in all freshwater, brackish, and marine waters of the world. They play a relevant role in these ecosystems, since they are one of the main primary producers, especially in open ocean. Eventually, they form mass blooms in coastal areas, which are potentially dangerous for the functioning of marine ecosystems. Allelopathy could be an important factor promoting the proliferation of these organisms. According to the authors' best knowledge, there is no information on the allelopathic activity and allelopathic compounds exhibited by different sp. phenotypes. Therefore, the research conducted here aimed to study the bioactivity of compounds produced by three phenotypes of sp. by studying their influence on the growth, chlorophyll fluorescence, and photosynthetic pigments of eighteen cyanobacteria and microalgae species. We demonstrated that three different sp. phenotypes, including a phycocyanin (PC)-rich strain (Type 1; green strain) and phycoerythrin (PE)-rich strains containing phycoerythrobilin (PEB) and phycocyanobilin (PCB) (Type 2; red strain and Type 3a; brown strain), had a significant allelopathic effect on the selected species of cyanobacteria, diatoms, and green algae. For all green algae, a decrease in cell abundance under the influence of phenotypes of donor cyanobacteria was shown, whereas, among some target cyanobacteria and diatom species, the cell-free filtrate was observed to have a stimulatory effect. Our estimates of the stress on photosystem II () showed a similar pattern, although for some diatoms, there was an effect of stress on photosynthesis, while a stimulatory effect on growth was also displayed. The pigment content was affected by allelopathy in most cases, particularly for chlorophyll , whilst it was a bit less significant for carotenoids. Our results showed that sp. Type 3a had the strongest effect on target species, while sp. Type 1 had the weakest allelopathic effect. Furthermore, GC-MS analysis produced different biochemical profiles for the strains. For every phenotype, the most abundant compound was different, with oxime-, methoxy-phenyl- being the most abundant substance for Type 1, eicosane for Type 2, and silanediol for Type 3a.
Topics: Allelopathy; Cell Proliferation; Harmful Algal Bloom; Pheromones; Photosynthesis; Phycobilins; Phycocyanin; Phycoerythrin; Phytoplankton; Silanes; Synechococcus; Water Microbiology
PubMed: 32230878
DOI: 10.3390/md18040179 -
The Journal of Biological Chemistry 2021Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to...
Synechococcus cyanobacteria are widespread in the marine environment, as the extensive pigment diversity within their light-harvesting phycobilisomes enables them to utilize various wavelengths of light for photosynthesis. The phycobilisomes of Synechococcus sp. RS9916 contain two forms of the protein phycoerythrin (PEI and PEII), each binding two chromophores, green-light absorbing phycoerythrobilin and blue-light absorbing phycourobilin. These chromophores are ligated to specific cysteines via bilin lyases, and some of these enzymes, called lyase isomerases, attach phycoerythrobilin and simultaneously isomerize it to phycourobilin. MpeV is a putative lyase isomerase whose role in PEI and PEII biosynthesis is not clear. We examined MpeV in RS9916 using recombinant protein expression, absorbance spectroscopy, and tandem mass spectrometry. Our results show that MpeV is the lyase isomerase that covalently attaches a doubly linked phycourobilin to two cysteine residues (C50, C61) on the β-subunit of both PEI (CpeB) and PEII (MpeB). MpeV activity requires that CpeB or MpeB is first chromophorylated by the lyase CpeS (which adds phycoerythrobilin to C82). Its activity is further enhanced by CpeZ (a homolog of a chaperone-like protein first characterized in Fremyella diplosiphon). MpeV showed no detectable activity on the α-subunits of PEI or PEII. The mechanism by which MpeV links the A and D rings of phycourobilin to C50 and C61 of CpeB was also explored using site-directed mutants, revealing that linkage at the A ring to C50 is a critical step in chromophore attachment, isomerization, and stability. These data provide novel insights into β-PE biosynthesis and advance our understanding of the mechanisms guiding lyase isomerases.
Topics: Amino Acid Sequence; Bacterial Proteins; Chromatography, Liquid; Isomerases; Marine Biology; Phycobilins; Phycoerythrin; Phylogeny; Recombinant Proteins; Synechococcus; Tandem Mass Spectrometry; Urobilin
PubMed: 33154169
DOI: 10.1074/jbc.RA120.015289 -
Nucleic Acids Research Sep 2017Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic...
Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana. Robust gene expression and high protein levels are achieved by combining genome integrated red light-sensing components with an episomal high-copy reporter construct. The gene of interest as well as the synTALE DNA-binding domain can be easily exchanged, allowing the flexible regulation of any desired gene by targeting endogenous or heterologous promoter regions. To allow low-cost induction of gene expression for industrial fermentation processes, we engineered yeast to endogenously produce the chromophore required for the effective dimerization of PhyB and PIF3. Time course experiments demonstrate high-level induction over a period of at least 48 h.
Topics: Arabidopsis; Arabidopsis Proteins; Basic Helix-Loop-Helix Transcription Factors; DNA-Binding Proteins; Gene Expression; Genetic Engineering; Genetic Vectors; Green Fluorescent Proteins; Homeodomain Proteins; Light; Light Signal Transduction; Phycobilins; Phycocyanin; Phytochrome B; Plasmids; Promoter Regions, Genetic; Protein Multimerization; Saccharomyces cerevisiae
PubMed: 28911120
DOI: 10.1093/nar/gkx610