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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 -
The FEBS Journal Oct 2009In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor-5A (eIF-5A) in Plasmodium. Here, we...
In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor-5A (eIF-5A) in Plasmodium. Here, we describe the cloning and expression of deoxyhypusine hydroxylase (DOHH), which completes the modification of eIF-5A through hydroxylation of deoxyhypusine. The dohh cDNA sequence revealed an ORF of 1236 bp encoding a protein of 412 amino acids with a calculated molecular mass of 46.45 kDa and an isoelectric point of 4.96. Interestingly, DOHH from Plasmodium has a FASTA SCORE of only 27 compared with its human ortholog and contains several matches similar to E-Z-type HEAT-like repeat proteins (IPR004155 (InterPro), PF03130 (Pfam), SM00567 (SMART) present in the phycocyanin lyase subunits of cyanobacteria. Purified DOHH protein displayed hydroxylase activity in a novel in vitro DOHH assay, but phycocyanin lyase activity was absent. dohh is present as a single-copy gene and is transcribed in the asexual blood stages of the parasite. A signal peptide at the N-terminus might direct the protein to a different cellular compartment. During evolution, Plasmodium falciparum acquired an apicoplast that lost its photosynthetic function. It is possible that plasmodial DOHH arose from an E/F-type phycobilin lyase that gained a new role in hydroxylation. Structured digital abstract: * MINT-7255047: DHS (uniprotkb:P49366) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415) * MINT-7255326: DOHH (uniprotkb:Q8I701) enzymaticly reacts (MI:0414) with eIF-5A (uniprotkb:Q710D1) by enzymatic studies (MI:0415).
Topics: Amino Acid Sequence; Animals; Blotting, Western; Gas Chromatography-Mass Spectrometry; Lysine; Mixed Function Oxygenases; Molecular Sequence Data; Molecular Structure; Plasmodium; Polymerase Chain Reaction; Protozoan Proteins; Sequence Homology, Amino Acid
PubMed: 19740108
DOI: 10.1111/j.1742-4658.2009.07272.x -
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 -
Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress.Biochimica Et Biophysica Acta Feb 2012Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction...
Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.
Topics: Cyanobacteria; Electron Transport; Fluorescence Recovery After Photobleaching; Light; Microscopy, Confocal; Models, Biological; Phycobilisomes; Protein Multimerization; Protein Structure, Quaternary; Spectrometry, Fluorescence; Stress, Physiological; Synechocystis; Temperature
PubMed: 22138629
DOI: 10.1016/j.bbabio.2011.11.008 -
Proceedings of the National Academy of... Sep 2001The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme....
The bilin prosthetic groups of the phytochrome photoreceptors and the light-harvesting phycobiliprotein antennae arise from the oxygen-dependent ring opening of heme. Two ferredoxin-dependent enzymes contribute to this conversion: a heme oxygenase and a bilin reductase with discrete double-bond specificity. Using a dual plasmid system, one expressing a truncated cyanobacterial apophytochrome 1, Cph1(N514), and the other expressing a two-gene operon consisting of a heme oxygenase and a bilin reductase, these studies establish the feasibility of producing photoactive phytochromes in any heme-containing cell. Heterologous expression systems for phytochromes not only will facilitate genetic analysis of their assembly, spectrophotometric activity, and biological function, but also might afford the means to regulate gene expression by light in nonplant cells.
Topics: Apoproteins; Bacterial Proteins; Biliverdine; Cyanobacteria; Genetic Engineering; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Phytochrome; Protein Kinases; Pyrroles; Tetrapyrroles
PubMed: 11553807
DOI: 10.1073/pnas.191375198 -
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 -
The Journal of Biological Chemistry Nov 2007Genes all5292 (cpcS2) and alr0617 (cpcS1) in the cyanobacterium Nostoc PCC7120 are homologous to the biliprotein lyase cpcS, and genes all5339 (cpcT1) and alr0647...
Genes all5292 (cpcS2) and alr0617 (cpcS1) in the cyanobacterium Nostoc PCC7120 are homologous to the biliprotein lyase cpcS, and genes all5339 (cpcT1) and alr0647 (cpcT2) are homologous to the lyase cpcT. The functions of the encoded proteins were screened in vitro and in a heterologous Escherichia coli system with plasmids conferring biosynthesis of the phycocyanobilin chromophore and of the acceptor proteins beta-phycoerythrocyanin (PecB) or beta-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine beta155 but does not discriminate between CpcB and PecB. The in vitro reconstitutions required no cofactors, and kinetic constants were determined for CpcT1 under in vitro conditions. No lyase activity was found for the lyase homologues CpcS2 and CpcT2, but complexes are formed in vitro between CpcT1 and CpcS1, CpcT2, or PecE (subunit of phycoviolobilin:alpha-phycoerythrocyanin isomerase lyase). The genes coding the inactive homologues, cpcS2 and cpcT2, are transcribed in N-starved Nostoc. In sequential binding experiments with CpcT1 and CpcS1, a chromophore at cysteine 84 inhibited the subsequent attachment to cysteine 155, whereas the inverse sequence generates subunits carrying both chromophores.
Topics: Bacterial Proteins; Binding Sites; Escherichia coli; Lyases; Multiprotein Complexes; Nostoc; Phycobilins; Phycocyanin; Protein Binding; Protein Subunits; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 17895251
DOI: 10.1074/jbc.M703038200 -
PloS One 2009Photosynthetic light-harvesting proteins are the mechanism by which energy enters the marine ecosystem. The dominant prokaryotic photoautotrophs are the cyanobacterial...
BACKGROUND
Photosynthetic light-harvesting proteins are the mechanism by which energy enters the marine ecosystem. The dominant prokaryotic photoautotrophs are the cyanobacterial genera Prochlorococcus and Synechococcus that are defined by two distinct light-harvesting systems, chlorophyll-bound protein complexes or phycobilin-bound protein complexes, respectively. Here, we use the Global Ocean Sampling (GOS) Project as a unique and powerful tool to analyze the environmental diversity of photosynthetic light-harvesting genes in relation to available metadata including geographical location and physical and chemical environmental parameters.
METHODS
All light-harvesting gene fragments and their metadata were obtained from the GOS database, aligned using ClustalX and classified phylogenetically. Each sequence has a name indicative of its geographic location; subsequent biogeographical analysis was performed by correlating light-harvesting gene budgets for each GOS station with surface chlorophyll concentration.
CONCLUSION/SIGNIFICANCE
Using the GOS data, we have mapped the biogeography of light-harvesting genes in marine cyanobacteria on ocean-basin scales and show that an environmental gradient exists in which chlorophyll concentration is correlated to diversity of light-harvesting systems. Three functionally distinct types of light-harvesting genes are defined: (1) the phycobilisome (PBS) genes of Synechococcus; (2) the pcb genes of Prochlorococcus; and (3) the iron-stress-induced (isiA) genes present in some marine Synechococcus. At low chlorophyll concentrations, where nutrients are limited, the Pcb-type light-harvesting system shows greater genetic diversity; whereas at high chlorophyll concentrations, where nutrients are abundant, the PBS-type light-harvesting system shows higher genetic diversity. We interpret this as an environmental selection of specific photosynthetic strategy. Importantly, the unique light-harvesting system isiA is found in the iron-limited, high-nutrient low-chlorophyll region of the equatorial Pacific. This observation demonstrates the ecological importance of isiA genes in enabling marine Synechococcus to acclimate to iron limitation and suggests that the presence of this gene can be a natural biomarker for iron limitation in oceanic environments.
Topics: Animals; Bacterial Proteins; Base Sequence; Chlorophyll; Computational Biology; Cyanobacteria; Databases, Nucleic Acid; Ecosystem; Environment; Genes, Bacterial; Iron; Light-Harvesting Protein Complexes; Photosynthesis; Phycobilins; Phycobilisomes; Phytoplankton; Seawater
PubMed: 19240807
DOI: 10.1371/journal.pone.0004601 -
FEBS Letters Nov 2001We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism...
We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXalpha which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies.
Topics: Animals; Bacterial Proteins; Bile Pigments; Cyanobacteria; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Escherichia coli Proteins; Heme Oxygenase (Decyclizing); Hydrogen-Ion Concentration; Oxidoreductases; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Phytochrome; Protein Kinases; Pyrroles; Rats; Recombinant Proteins; Spectrum Analysis; Temperature; Tetrapyrroles; Transformation, Bacterial
PubMed: 11728472
DOI: 10.1016/s0014-5793(01)02988-x -
The Journal of General Physiology Sep 1957The phycobilin pigments were freshly extracted from Porphyra naiadum in the cold. At least two types of phycoerythrin (I and II) can be distinguished by electrophoresis,...
The phycobilin pigments were freshly extracted from Porphyra naiadum in the cold. At least two types of phycoerythrin (I and II) can be distinguished by electrophoresis, chromatography, and spectral characteristics. At pH 5.0 phycoerythrin II has a relatively large negative charge, while phycoerythrin I is nearly iso-electric. At pH 7.0, however, phycoerythrin I has the larger negative charge. Mobilities have been calculated by visual measurement of electrophoresis. Phycoerythrin II can be converted to phycoerythrin I by storing at pH 7.0. Chromatography indicates at least two types of phycocyanin as well.
Topics: Eukaryota; Phycobilins; Phycocyanin; Phycoerythrin; Pigments, Biological; Porphyra
PubMed: 13463270
DOI: 10.1085/jgp.41.1.77