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The FEBS Journal Jan 2018Ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXα (BV) to form open-chain tetrapyrroles used for...
UNLABELLED
Ferredoxin-dependent bilin reductases (FDBRs) are a class of enzymes reducing the heme metabolite biliverdin IXα (BV) to form open-chain tetrapyrroles used for light-perception and light-harvesting in photosynthetic organisms. Thus far, seven FDBR families have been identified, each catalysing a distinct reaction and either transferring two or four electrons from ferredoxin onto the substrate. The newest addition to the family is PcyX, originally identified from metagenomics data derived from phage. Phylogenetically, PcyA is the closest relative catalysing the reduction of BV to phycocyanobilin. PcyX, however, converts the same substrate to phycoerythrobilin, resembling the reaction catalysed by cyanophage PebS. Within this study, we aimed at understanding the evolution of catalytic activities within FDBRs using PcyX as an example. Additional members of the PcyX clade and a remote member of the PcyA family were investigated to gain insights into catalysis. Biochemical data in combination with the PcyX crystal structure revealed that a conserved aspartate-histidine pair is critical for activity. Interestingly, the same residues are part of a catalytic Asp-His-Glu triad in PcyA, including an additional Glu. While this Glu residue is replaced by Asp in PcyX, it is not involved in catalysis. Substitution back to a Glu failed to convert PcyX to a PcyA. Therefore, the change in regiospecificity is not only caused by individual catalytic amino acid residues. Rather the combination of the architecture of the active site with the positioning of the substrate triggers specific proton transfer yielding the individual phycobilin products.
ENZYMES
Suggested EC number for PcyX: 1.3.7.6 DATABASES: The PcyX X-ray structure was deposited in the PDB with the accession code 5OWG.
Topics: Asparagine; Bacteriophages; Bile Pigments; Catalysis; Crystallography, X-Ray; Evolution, Molecular; Ferredoxins; Methionine; Mutagenesis, Site-Directed; Oceans and Seas; Oxidoreductases; Phylogeny; Protein Conformation; Substrate Specificity
PubMed: 29156487
DOI: 10.1111/febs.14341 -
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 -
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 -
Proceedings of the National Academy of... Sep 2017Phycobilisomes are highly organized pigment-protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and...
Phycobilisomes are highly organized pigment-protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment-protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.
Topics: Cyanobacteria; Energy Transfer; Light-Harvesting Protein Complexes; Macromolecular Substances; Models, Biological; Photosynthesis; Phycobilins; Phycobilisomes; Phycocyanin; Spectrometry, Fluorescence
PubMed: 28847963
DOI: 10.1073/pnas.1705435114 -
Photosynthesis Research Mar 2018Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel...
Plants and algae have developed various light-harvesting mechanisms for optimal delivery of excitation energy to the photosystems. Cryptophyte algae have evolved a novel soluble light-harvesting antenna utilizing phycobilin pigments to complement the membrane-intrinsic Chl a/c-binding LHC antenna. This new antenna consists of the plastid-encoded β-subunit, a relic of the ancestral phycobilisome, and a novel nuclear-encoded α-subunit unique to cryptophytes. Together, these proteins form the active αβ·αβ-tetramer. In all cryptophyte algae investigated so far, the α-subunits have duplicated and diversified into a large gene family. Although there is transcriptional evidence for expression of all these genes, the X-ray structures determined to date suggest that only two of the α-subunit genes might be significantly expressed at the protein level. Using proteomics, we show that in phycoerythrin 545 (PE545) of Guillardia theta, the only cryptophyte with a sequenced genome, all 20 α-subunits are expressed when the algae grow under white light. The expression level of each protein depends on the intensity of the growth light, but there is no evidence for a specific light-dependent regulation of individual members of the α-subunit family under the growth conditions applied. GtcpeA10 seems to be a special member of the α-subunit family, because it consists of two similar N- and C-terminal domains, which likely are the result of a partial tandem gene duplication. The proteomics data of this study have been deposited to the ProteomeXchange Consortium and have the dataset identifiers PXD006301 and 10.6019/PXD006301.
Topics: Acclimatization; Amino Acid Sequence; Cells, Cultured; Cryptophyta; Light; Light-Harvesting Protein Complexes; Models, Genetic; Models, Molecular; Photosynthesis; Phycobiliproteins; Plant Proteins; Protein Subunits; Proteomics; Spectrometry, Fluorescence; Temperature
PubMed: 28540588
DOI: 10.1007/s11120-017-0400-0 -
Biological Research May 2017Acaryochloris marina is an oxygenic cyanobacterium that utilizes far-red light for photosynthesis. It has an expanded genome, which helps in its adaptability to the... (Review)
Review
Acaryochloris marina is an oxygenic cyanobacterium that utilizes far-red light for photosynthesis. It has an expanded genome, which helps in its adaptability to the environment, where it can survive on low energy photons. Its major light absorbing pigment is chlorophyll d and it has α-carotene as a major carotenoid. Light harvesting antenna includes the external phycobilin binding proteins, which are hexameric rods made of phycocyanin and allophycocyanins, while the small integral membrane bound chlorophyll binding proteins are also present. There is specific chlorophyll a molecule in both the reaction center of Photosystem I (PSI) and PSII, but majority of the reaction center consists of chlorophyll d. The composition of the PSII reaction center is debatable especially the role and position of chlorophyll a in it. Here we discuss the photosystems of this bacterium and its related biology.
Topics: Adaptation, Physiological; Chlorophyll; Cyanobacteria; Genome, Bacterial; Photosynthesis
PubMed: 28526061
DOI: 10.1186/s40659-017-0120-0 -
Nature Communications May 2017Marine chromophoric dissolved organic matter (CDOM) and its related fluorescent components (FDOM), which are widely distributed but highly photobleached in the surface...
Marine chromophoric dissolved organic matter (CDOM) and its related fluorescent components (FDOM), which are widely distributed but highly photobleached in the surface ocean, are critical in regulating light attenuation in the ocean. However, the origins of marine FDOM are still under investigation. Here we show that cultured picocyanobacteria, Synechococcus and Prochlorococcus, release FDOM that closely match the typical fluorescent signals found in oceanic environments. Picocyanobacterial FDOM also shows comparable apparent fluorescent quantum yields and undergoes similar photo-degradation behaviour when compared with deep-ocean FDOM, further strengthening the similarity between them. Ultrahigh-resolution mass spectrometry (MS) and nuclear magnetic resonance spectroscopy reveal abundant nitrogen-containing compounds in Synechococcus DOM, which may originate from degradation products of the fluorescent phycobilin pigments. Given the importance of picocyanobacteria in the global carbon cycle, our results indicate that picocyanobacteria are likely to be important sources of marine autochthonous FDOM, which may accumulate in the deep ocean.
Topics: Aquatic Organisms; Carbon Cycle; Fluorescent Dyes; Magnetic Resonance Spectroscopy; Nitrogen; Oceans and Seas; Photobleaching; Phycobilins; Prochlorococcus; Seawater; Synechococcus
PubMed: 28513605
DOI: 10.1038/ncomms15284 -
Bioengineered Jan 2018The fusion protein (SLA) of streptavidin and allophycocyanin α subunit (holo-ApcA) was biosynthesized in Escherichia coli by a dual plasmid system. The recombinant SLA,...
The fusion protein (SLA) of streptavidin and allophycocyanin α subunit (holo-ApcA) was biosynthesized in Escherichia coli by a dual plasmid system. The recombinant SLA, purified by affinity chromatography, showed spectral properties similar to natural allophycocyanin α subunit (ApcA). Spectral and Zinc staining analysis indicated that the recombinant SLA covalently bound phycocyanobilin (PCB). To improve chromophorylation rate of recombinant SLA, an in vitro chromophore attachment reaction system was established, which contained partially chromophylated SLA, PCB and lyase CpcS. Spectral analysis showed that PCB bound to the recombinant SLA rapidly during the reaction. The chromophorylation rate of SLA was improved from 21.1% to 86.5%. Immunofluorescence assay showed that SLA with high chromophorylation rate had higher detection signal. Thus, in vitro chromophore attachment is an effective way to improve the chromophorylation rate of recombinant phycobiliprotein.
Topics: Cloning, Molecular; Escherichia coli; Fluorescent Antibody Technique, Direct; Gene Expression; Genetic Vectors; Hydrogen-Ion Concentration; Phycobilins; Phycocyanin; Protein Subunits; Recombinant Fusion Proteins; Streptavidin; Temperature
PubMed: 28448741
DOI: 10.1080/21655979.2017.1321282 -
Frontiers in Microbiology 2017Marine has successfully adapted to environments with different light colors, which likely contributes to this genus being the second most abundant group of...
Marine has successfully adapted to environments with different light colors, which likely contributes to this genus being the second most abundant group of microorganisms worldwide. Populations of that grow in deep, blue ocean waters contain large amounts of the blue-light absorbing chromophore phycourobilin (PUB) in their light harvesting complexes (phycobilisomes). Here, we show that all strains adapted to blue light possess a gene called . MpeU is structurally similar to phycobilin lyases, enzymes that ligate chromophores to phycobiliproteins. Interruption of caused a reduction in PUB content, impaired phycobilisome assembly and reduced growth rate more strongly in blue than green light. When was reintroduced in the mutant background, the less phenotype was complemented in terms of PUB content and phycobilisome content. Fluorescence spectra of mutant cells and purified phycobilisomes revealed red-shifted phycoerythrin emission peaks, likely indicating a defect in chromophore ligation to phycoerythrin-I (PE-I) or phycoerythrin-II (PE-II). Our results suggest that MpeU is a lyase-isomerase that attaches a phycoerythrobilin to a PEI or PEII subunit and isomerizes it to PUB. MpeU is therefore an important determinant in adaptation of spp. to capture photons in blue light environments throughout the world's oceans.
PubMed: 28270800
DOI: 10.3389/fmicb.2017.00243 -
Plant, Cell & Environment Nov 2017Anoxygenic photosynthetic prokaryotes arose in ancient oceans ~3.5 billion years ago. The evolution of oxygenic photosynthesis by cyanobacteria followed soon after,... (Review)
Review
Anoxygenic photosynthetic prokaryotes arose in ancient oceans ~3.5 billion years ago. The evolution of oxygenic photosynthesis by cyanobacteria followed soon after, enabling eukaryogenesis and the evolution of complex life. The Archaeplastida lineage dates back ~1.5 billion years to the domestication of a cyanobacterium. Eukaryotic algae have subsequently radiated throughout oceanic/freshwater/terrestrial environments, adopting distinctive morphological and developmental strategies for adaptation to diverse light environments. Descendants of the ancestral photosynthetic alga remain challenged by a typical diurnally fluctuating light supply ranging from ~0 to ~2000 μE m s . Such extreme changes in light intensity and variations in light quality have driven the evolution of novel photoreceptors, light-harvesting complexes and photoprotective mechanisms in photosynthetic eukaryotes. This minireview focuses on algal light sensors, highlighting the unexpected roles for linear tetrapyrroles (bilins) in the maintenance of functional chloroplasts in chlorophytes, sister species to streptophyte algae and land plants.
Topics: Acclimatization; Aquatic Organisms; Eukaryota; Light; Models, Biological; Photosynthesis
PubMed: 28245058
DOI: 10.1111/pce.12943