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The Plant Journal : For Cell and... Nov 2020The photosynthetic bacterial phycobiliprotein lyases, also called CpcT lyases, catalyze the biogenesis of phycobilisome, a light-harvesting antenna complex, through the...
The photosynthetic bacterial phycobiliprotein lyases, also called CpcT lyases, catalyze the biogenesis of phycobilisome, a light-harvesting antenna complex, through the covalent attachment of chromophores to the antenna proteins. The Arabidopsis CRUMPLED LEAF (CRL) protein is a homolog of the cyanobacterial CpcT lyase. Loss of CRL leads to multiple lesions, including localized foliar cell death, constitutive expression of stress-related nuclear genes, abnormal cell cycle, and impaired plastid division. Notwithstanding the apparent phenotypes, the function of CRL still remains elusive. To gain insight into the function of CRL, we examined whether CRL still retains the capacity to bind with the bacterial chromophore phycocyanobilin (PCB) and its plant analog phytochromobilin (PΦB). The revealed structure of the CpcT domain of CRL is comparable to that of the CpcT lyase, despite the low sequence identity. The subsequent in vitro biochemical assays found, as shown for the CpcT lyase, that PCB/PΦB binds to the CRL dimer. However, some mutant forms of CRL, substantially compromised in their bilin-binding ability, still restore the crl-induced multiple lesions. These results suggest that although CRL retains the bilin-binding pocket, it seems not functionally associated with the crl-induced multiple lesions.
Topics: Arabidopsis; Arabidopsis Proteins; Bile Pigments; Cell Division; Cyanobacteria; Lyases; Mutation; Phenotype; Phycobilins; Phycobiliproteins; Phycobilisomes; Phycocyanin; Plastids; Protein Binding
PubMed: 32860438
DOI: 10.1111/tpj.14974 -
Biochimica Et Biophysica Acta.... Dec 2020Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae....
Bilin lyases are enzymes which ligate linear tetrapyrrole chromophores to specific cysteine residues on light harvesting proteins present in cyanobacteria and red algae. The lyases responsible for chromophorylating the light harvesting protein phycoerythrin (PE) have not been fully characterized. In this study, we explore the role of CpeT, a putative bilin lyase, in the biosynthesis of PE in the cyanobacterium Fremyella diplosiphon. Recombinant protein studies show that CpeT alone can bind phycoerythrobilin (PEB), but CpeZ, a chaperone-like protein, is needed in order to correctly and efficiently attach PEB to the β-subunit of PE. MS analyses of the recombinant β-subunit of PE coexpressed with CpeT and CpeZ show that PEB is attached at Cys-165. Purified phycobilisomes from a cpeT knockout mutant and wild type (WT) samples from F. diplosiphon were analyzed and compared. The cpeT mutant contained much less PE and more phycocyanin than WT cells grown under green light, conditions which should maximize the production of PE. In addition, Northern blot analyses showed that the cpeCDESTR operon mRNAs were upregulated while the cpeBcpeA mRNAs were downregulated in the cpeT mutant strain when compared with WT, suggesting that CpeT may also play a direct or indirect regulatory role in transcription of these operons or their mRNA stability, in addition to its role as a PEB lyase for Cys-165 on β-PE.
Topics: Amino Acid Sequence; Bacterial Proteins; Cyanobacteria; Cysteine; Gene Deletion; Genes, Bacterial; Lyases; Molecular Chaperones; Mutant Proteins; Operon; Peptides; Phenotype; Phycobilins; Phycoerythrin; Recombinant Proteins
PubMed: 32777305
DOI: 10.1016/j.bbabio.2020.148284 -
Photosynthesis Research Jun 2020The crystal structure of phycocyanin (pr-PC) isolated from Phormidium rubidum A09DM (P. rubidum) is described at a resolution of 1.17 Å. Electron density maps derived...
The crystal structure of phycocyanin (pr-PC) isolated from Phormidium rubidum A09DM (P. rubidum) is described at a resolution of 1.17 Å. Electron density maps derived from crystallographic data showed many clear differences in amino acid sequences when compared with the previously obtained gene-derived sequences. The differences were found in 57 positions (30 in α-subunit and 27 in β-subunit of pr-PC), in which all residues except one (β145Arg) are not interacting with the three phycocyanobilin chromophores. Highly purified pr-PC was then sequenced by mass spectrometry (MS) using LC-MS/MS. The MS data were analyzed using two independent proteomic search engines. As a result of this analysis, complete agreement between the polypeptide sequences and the electron density maps was obtained. We attribute the difference to multiple genes in the bacterium encoding the phycocyanin apoproteins and that the gene sequencing sequenced the wrong ones. We are not implying that protein sequencing by mass spectrometry is more accurate than that of gene sequencing. The final 1.17 Å structure of pr-PC allows the chromophore interactions with the protein to be described with high accuracy.
Topics: Amino Acid Sequence; Chromatography, Liquid; Crystallography; Phormidium; Phycobilins; Phycocyanin; Proteomics; Sequence Analysis, Protein; Tandem Mass Spectrometry
PubMed: 32303893
DOI: 10.1007/s11120-020-00746-7 -
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 -
PloS One 2020Upwelling is an important source of inorganic nutrients in marine systems, yet little is known about how gradients in upwelling affect primary producers on coral reefs....
Upwelling is an important source of inorganic nutrients in marine systems, yet little is known about how gradients in upwelling affect primary producers on coral reefs. The Southern Line Islands span a natural gradient of inorganic nutrient concentrations across the equatorial upwelling region in the central Pacific. We used this gradient to test the hypothesis that benthic autotroph ecophysiology is enhanced on nutrient-enriched reefs. We measured metabolism and photophysiology of common benthic taxa, including the algae Porolithon, Avrainvillea, and Halimeda, and the corals Pocillopora and Montipora. We found that temperature (27.2-28.7°C) was inversely related to dissolved inorganic nitrogen (0.46-4.63 μM) and surface chlorophyll a concentrations (0.108-0.147 mg m-3), which increased near the equator. Contrary to our prediction, ecophysiology did not consistently track these patterns in all taxa. Though metabolic rates were generally variable, Porolithon and Avrainvillea photosynthesis was highest at the most productive and equatorial island (northernmost). Porolithon photosynthetic rates also generally increased with proximity to the equator. Photophysiology (maximum quantum yield) increased near the equator and was highest at northern islands in all taxa. Photosynthetic pigments also were variable, but chlorophyll a and carotenoids in Avrainvillea and Montipora were highest at the northern islands. Phycobilin pigments of Porolithon responded most consistently across the upwelling gradient, with higher phycoerythrin concentrations closer to the equator. Our findings demonstrate that the effects of in situ nutrient enrichment on benthic autotrophs may be more complex than laboratory experiments indicate. While upwelling is an important feature in some reef ecosystems, ancillary factors may regulate the associated consequences of nutrient enrichment on benthic reef organisms.
Topics: Animals; Anthozoa; Autotrophic Processes; Chlorophyta; Coral Reefs; Ecosystem; Nitrogen; Pacific Islands; Photosynthesis; Rhodophyta; Temperature
PubMed: 32017799
DOI: 10.1371/journal.pone.0228448 -
Proceedings of the National Academy of... Feb 2020The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 ( PCC6803) carrying a phycocyanobilin chromophore could be solved in both...
The three-dimensional (3D) crystal structures of the GAF3 domain of cyanobacteriochrome Slr1393 ( PCC6803) carrying a phycocyanobilin chromophore could be solved in both 15- dark-adapted state, Pr, λ = 649 nm, and 15- photoproduct, Pg, λ = 536 nm (resolution, 1.6 and 1.86 Å, respectively). The structural data allowed identifying the large spectral shift of the Pr-to-Pg conversion as resulting from an out-of-plane rotation of the chromophore's peripheral rings and an outward movement of a short helix formed from a formerly unstructured loop. In addition, a third structure (2.1-Å resolution) starting from the photoproduct crystals allowed identification of elements that regulate the absorption maxima. In this peculiar form, generated during X-ray exposition, protein and chromophore conformation still resemble the photoproduct state, except for the D-ring already in 15- configuration and tilted out of plane akin the dark state. Due to its formation from the photoproduct, it might be considered an early conformational change initiating the parental state-recovering photocycle. The high quality and the distinct features of the three forms allowed for applying quantum-chemical calculations in the framework of multiscale modeling to rationalize the absorption maxima changes. A systematic analysis of the PCB chromophore in the presence and absence of the protein environment showed that the direct electrostatic effect is negligible on the spectral tuning. However, the protein forces the outer pyrrole rings of the chromophore to deviate from coplanarity, which is identified as the dominating factor for the color regulation.
Topics: Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Light; Models, Molecular; Photochemical Processes; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Protein Conformation; Protein Domains; Structure-Activity Relationship; Synechocystis
PubMed: 31964827
DOI: 10.1073/pnas.1910208117 -
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 -
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 -
FEBS Letters Apr 2020The phycobilisome (PBS) is the cyanobacterial antenna complex which transfers absorbed light energy to the photosystem II (PSII), while the excess energy is...
The phycobilisome (PBS) is the cyanobacterial antenna complex which transfers absorbed light energy to the photosystem II (PSII), while the excess energy is nonphotochemically quenched by interaction of the PBS with the orange carotenoid protein (OCP). Here, the molecular model of the PBS-PSII-OCP supercomplex was utilized to assess the resonance energy transfer from PBS to PSII and, using the excitonic theory, the transfer from PBS to OCP. Our estimates show that the effective energy migration from PBS to PSII is realized due to the existence of several transfer pathways from phycobilin chromophores of the PBS to the neighboring antennal chlorophyll molecules of the PSII. At the same time, the single binding site of photoactivated OCP and the PBS is sufficient to realize the quenching.
Topics: Bacterial Proteins; Carotenoids; Energy Transfer; Fluorescence; Fluorescence Resonance Energy Transfer; Photosystem II Protein Complex; Phycobilisomes; Synechocystis
PubMed: 31799708
DOI: 10.1002/1873-3468.13709 -
The Journal of Biological Chemistry Sep 2019Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent...
Phycobilins are light-harvesting pigments of cyanobacteria, red algae, and cryptophytes. The biosynthesis of phycoerythrobilin (PEB) is catalyzed by the subsequent action of two ferredoxin-dependent bilin reductases (FDBRs). Although 15,16-dihydrobiliverdin (DHBV):ferredoxin oxidoreductase (PebA) catalyzes the two-electron reduction of biliverdin IXα to 15,16-DHBV, PEB:ferredoxin oxidoreductase (PebB) reduces this intermediate further to PEB. Interestingly, marine viruses encode the FDBR PebS combining both activities within one enzyme. Although PebA and PebS share a canonical fold with similar substrate-binding pockets, the structural determinants for the stereo- and regiospecific modification of their tetrapyrrole substrates are incompletely understood, also because of the lack of a PebB structure. Here, we solved the X-ray crystal structures of both substrate-free and -bound PEBB from the cryptophyte at 1.90 and 1.65 Å, respectively. The structures of PEBB exhibit the typical α/β/α-sandwich fold. Interestingly, the open-chain tetrapyrrole substrate DHBV is bound in an unexpected flipped orientation within the canonical FDBR active site. Biochemical analyses of the WT enzyme and active site variants identified two central aspartate residues Asp-99 and Asp-219 as essential for catalytic activity. In addition, the conserved Arg-215 plays a critical role in substrate specificity, binding orientation, and active site integrity. Because these critical residues are conserved within certain FDBRs displaying A-ring reduction activity, we propose that they present a conserved mechanism for this reaction. The flipped substrate-binding mode indicates that two-electron reducing FDBRs utilize the same primary site within the binding pocket and that substrate orientation is the determinant for A- or D-ring regiospecificity.
Topics: Bacteriophages; Bile Pigments; Biliverdine; Catalysis; Catalytic Domain; Cryptophyta; Cyanobacteria; Eukaryota; Oxidation-Reduction; Oxidoreductases; Phycobilins; Phycoerythrin; Protein Conformation; Substrate Specificity; Tetrapyrroles
PubMed: 31366727
DOI: 10.1074/jbc.RA119.009306