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The Journal of Biological Chemistry Mar 2019Phycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due...
Phycoerythrin (PE) is a green light-absorbing protein present in the light-harvesting complex of cyanobacteria and red algae. The spectral characteristics of PE are due to its prosthetic groups, or phycoerythrobilins (PEBs), that are covalently attached to the protein chain by specific bilin lyases. Only two PE lyases have been identified and characterized so far, and the other bilin lyases are unknown. Here, using analyses, markerless deletion, biochemical assays with purified and recombinant proteins, and site-directed mutagenesis, we examined the role of a putative lyase-encoding gene, , in the cyanobacterium Analyzing the phenotype of the deletion, we found that is required for proper PE biogenesis, specifically for ligation of the doubly linked PEB to Cys-48/Cys-59 residues of the CpeB subunit of PE. We also show that in a heterologous host, CpeF can attach PEB to Cys-48/Cys-59 of CpeB, but only in the presence of the chaperone-like protein CpeZ. Additionally, we report that CpeF likely ligates the A ring of PEB to Cys-48 prior to the attachment of the D ring to Cys-59. We conclude that CpeF is the bilin lyase responsible for attachment of the doubly ligated PEB to Cys-48/Cys-59 of CpeB and together with other specific bilin lyases contributes to the post-translational modification and assembly of PE into mature light-harvesting complexes.
Topics: Cyanobacteria; Phycobilins; Phycoerythrin; Recombinant Proteins
PubMed: 30670589
DOI: 10.1074/jbc.RA118.007221 -
Physical Chemistry Chemical Physics :... Sep 2021Cyanobacteriochromes (CBCRs) are bi-stable photoreceptor proteins with high potential for biotechnological applications. Most of these proteins utilize phycocyanobilin...
Cyanobacteriochromes (CBCRs) are bi-stable photoreceptor proteins with high potential for biotechnological applications. Most of these proteins utilize phycocyanobilin (PCB) as a light-sensing co-factor, which is unique to cyanobacteria, but some variants also incorporate biliverdin (BV). The latter are of particular interest for biotechnology due to the natural abundance and red-shifted absorption of BV. Here, AmI-g2 was investigated, a CBCR capable of binding both PCB and BV. The assembly kinetics and primary photochemistry of AmI-g2 with both chromophores were studied . The assembly reaction with PCB is roughly 10× faster than BV, and the formation of a non-covalent intermediate was identified as the rate-limiting step in the case of BV. This step is fast for PCB, where the formation of the covalent thioether bond between AmI-g2 and PCB becomes rate-limiting. The photochemical quantum yields of the forward and backward reactions of AmI-g2 were estimated and discussed in the context of homologous CBCRs.
Topics: Biliverdine; Cyanobacteria; Kinetics; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Protein Binding; Quantum Theory; Spectrophotometry
PubMed: 34374395
DOI: 10.1039/d1cp02696h -
Acta Crystallographica. Section F,... Aug 2015The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE...
The crystallographic analysis of a marine cyanobacterium (Phormidium sp. A09DM) phycoerythrin (PE) that shows distinct sequence features compared with known PE structures from cyanobacteria and red algae is reported. Phormidium PE was crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant. Diffraction data were collected on the protein crystallography beamline at the Indus-2 synchrotron. The crystals diffracted to about 2.1 Å resolution at 100 K. The crystals, with an apparent hexagonal morphology, belonged to space group P1, with unit-cell parameters a = 108.3, b = 108.4 Å, c = 116.6 Å, α = 78.94, β = 82.50, γ = 60.34°. The molecular-replacement solution confirmed the presence of 12 αβ monomers in the P1 cell. The Phormidium PE elutes as an (αβ)3 trimer of αβ monomers from a molecular-sieve column and exists as [(αβ)3]2 hexamers in the crystal lattice. Unlike red algal PE proteins, the hexamers of Phormidium PE do not form higher-order structures in the crystals. The existence of only one characteristic visual absorption band at 564 nm suggests the presence of phycoerythrobilin chromophores, and the absence of any other types of bilins, in the Phormidium PE assembly.
Topics: Amino Acid Sequence; Ammonium Sulfate; Crystallization; Crystallography, X-Ray; Cyanobacteria; Gene Expression; Models, Molecular; Molecular Sequence Data; Phycobilins; Phycoerythrin; Protein Binding; Protein Multimerization; Protein Structure, Secondary; Protein Structure, Tertiary; Protein Subunits; Sequence Alignment; Structural Homology, Protein
PubMed: 26249689
DOI: 10.1107/S2053230X15010134 -
The Journal of Biological Chemistry Nov 2018Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible...
Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible spectrum of light, but the regulation of the chromophorylation of CBCRs is unknown. Slr1393 from sp. PCC 6803 is a CBCR containing three consecutive GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains, of which only the third one (Slr1393g3) can be phycocyanobilin-chromophorylated. The protein Slr2111 from sp. PCC 6803 includes a cystathionine β-synthase (CBS) domain pair of an as yet unknown function at its N terminus. CBS domains are often characterized as sensors of cellular energy status by binding nucleotides. In this work, we demonstrate that Slr2111 strongly interacts with Slr1393 and , which generates a complex in a 1:1 molar ratio. This tight interaction inhibits the chromophorylation of Slr1393g3, even if the chromophore is present. Instead, the complex stability and thereby the chromophorylation of Slr1393 are regulated by the binding of nucleotides (ATP, ADP, AMP) to the CBS domains of Slr2111 with varying affinities. It is demonstrated that residues Asp-53 and Arg-97 of Slr2111 are involved in nucleotide binding. While ATP binds to Slr2111, the association between the two proteins gets weaker and chromophorylation of Slr1393 are enabled. In contrast, AMP binding to Slr2111 leads to a stronger association, thereby inhibiting the chromophorylation. It is concluded that Slr2111 acts as a sensor of the cellular energy status that regulates the chromophorylation of Slr1393 and thereby its function as a light-driven histidine kinase.
Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Bacterial Proteins; Carrier Proteins; Histidine Kinase; Kinetics; Molecular Docking Simulation; Mutation; Photoreceptors, Microbial; Phycobilins; Phycocyanin; Protein Binding; Protein Conformation; Sequence Alignment; Synechocystis
PubMed: 30242127
DOI: 10.1074/jbc.RA118.003830 -
Missouri Medicine 2015Nicotine and soluble semi-stable aldehydes and ketones in cigarette smoke are key mediators of the elevated risks for vascular disease, cancer, and chronic obstructive...
Nicotine and soluble semi-stable aldehydes and ketones in cigarette smoke are key mediators of the elevated risks for vascular disease, cancer, and chronic obstructive pulmonary disease observed in smokers. Nicotine, via sympathetic stimulation, increases risk for both vascular disease and cancer. Comprehensive suppression of sympathetic activity with the well-tolerated drug carvedilol, which inhibits betal 1, beta2 and alphal adrenergic receptors, may be protective to smokers and other nicotine addicts. The soluble aldehydes and ketones in tobacco smoke appear to exert their adverse effects through activation of NADPH oxidase complexes in vascular tissues and in the lungs. The phytochemical phycocyanobilin (PhyCB), richly supplied by the edible cyanobacterium spirulina, in studies on rodents and in human cell cultures has shown the ability to safely mimic intracellular bilirubin's physiological role as an inhibitor ofNADPH oxidase activity. It therefore may have potential for mitigating the pro-oxidative effects of tobacco smoke aldehydes and ketones. Joint administration of carvedilol and spirulina merits exploration as a strategy for moderating the pathogenic impact of smoking in chronic tobacco users who either fail to quit or refuse to try cessation of tobacco. Carvedilol may be appropriate for those who manage a nicotine addiction in other ways (smokeless tobacco, e-cigarettes, nicotine gum). Further clinical studies to evaluate the impact of carvedilol on cardiovascular risk factors in nicotine addicts, and rodent studies to assess markers of lung inflammation in smoke- exposed rodents fed PhyCB, are recommended.
Topics: Adrenergic beta-Antagonists; Carbazoles; Cardiovascular Diseases; Carvedilol; Humans; Neoplasms; Phycobilins; Phycocyanin; Propanolamines; Smoking Cessation; Spirulina; Tobacco Use Disorder
PubMed: 25812281
DOI: No ID Found -
Biochimica Et Biophysica Acta Jun 2016Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to...
Adapting photosynthesis to the near-infrared: non-covalent binding of phycocyanobilin provides an extreme spectral red-shift to phycobilisome core-membrane linker from Synechococcus sp. PCC7335.
Phycobiliproteins that bind bilins are organized as light-harvesting complexes, phycobilisomes, in cyanobacteria and red algae. The harvested light energy is funneled to reaction centers via two energy traps, allophycocyanin B and the core-membrane linker, ApcE1 (conventional ApcE). The covalently bound phycocyanobilin (PCB) of ApcE1 absorbs near 660 nm and fluoresces near 675 nm. In cyanobacteria capable of near infrared photoacclimation, such as Synechococcus sp. PCC7335, there exist even further spectrally red shifted components absorbing >700 nm and fluorescing >710 nm. We expressed the chromophore domain of the extra core-membrane linker from Synechococcus sp. PCC7335, ApcE2, in E. coli together with enzymes generating the chromophore, PCB. The resulting chromoproteins, PCB-ApcE2(1-273) and the more truncated PCB-ApcE2(24-245), absorb at 700 nm and fluoresce at 714 nm. The red shift of ~40 nm compared with canonical ApcE1 results from non-covalent binding of the chromophore by which its full conjugation length including the Δ3,3(1) double bond is preserved. The extreme spectral red-shift could not be ascribed to exciton coupling: dimeric PCB-ApcE2(1-273) and monomeric-ApcE2(24-245) absorbed and fluoresced similarly. Chromophorylation of ApcE2 with phycoerythrobilin- or phytochromobilin resulted in similar red shifts (absorption at 615 and 711 nm, fluorescence at 628 or 726 nm, respectively), compared to the covalently bound chromophores. The self-assembled non-covalent chromophorylation demonstrates a novel access to red and near-infrared emitting fluorophores. Brightly fluorescent biomarking was exemplified in E. coli by single-plasmid transformation.
Topics: Bacterial Proteins; Escherichia coli; Microscopy, Fluorescence; Models, Molecular; Photosynthesis; Phycobilins; Phycobilisomes; Phycocyanin; Phycoerythrin; Protein Multimerization; Protein Structure, Tertiary; Recombinant Proteins; Spectrometry, Fluorescence; Synechococcus
PubMed: 27045046
DOI: 10.1016/j.bbabio.2016.03.033 -
Cells Sep 2020It is estimated that the genus is responsible for about 17% of net primary production in the Global Ocean. Blooms of these organisms are observed in tropical,...
It is estimated that the genus is responsible for about 17% of net primary production in the Global Ocean. Blooms of these organisms are observed in tropical, subtropical and even temperate zones, and they have been recorded recently even beyond the polar circle. The long-term scenarios forecast a growing expansion of sp. and its area of dominance. This is, among others, due to their high physiological plasticity in relation to changing environmental conditions. Three phenotypes of the genus sp. (Type 1, Type 2, and Type 3a) were tested in controlled laboratory conditions in order to identify their response to various irradiance (10, 55, 100 and 145 µmol photons m s) and temperature (15, 22.5 and 30 °C) conditions. The highest total pigment content per cell was recorded at 10 μmol photons m s at all temperature variants with the clear dominance of phycobilins among all the pigments. In almost every variant the highest growth rate was recorded for the Type 1. The lowest growth rates were observed, in general, for the Type 3a. However, it was recognized to be less temperature sensitive in comparison to the other two types and rather light-driven with the highest plasticity and adaptation potential. The highest amounts of carotenoids were produced by Type 2 which also showed signs of the cell stress even around 55 μmol photons m s at 15 °C and 22.5 °C. This may imply that the Type 2 is the most susceptible to higher irradiances. Picocyanobacteria sp. require less light intensity to achieve the maximum rate of photosynthesis than larger algae. They also tolerate a wide range of temperatures which combined together make them gain a powerful competitive advantage. Our results will provide key information for the ecohydrodynamical model development. Thus, this work would be an important link in forecasting future changes in the occurrence of these organisms in the context of global warming.
Topics: Humans; Phenotype; Photosynthesis; Synechococcus; Temperature
PubMed: 32899279
DOI: 10.3390/cells9092030 -
PloS One 2018Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to...
Phycobilisomes (PBS) are accessory light harvesting protein complexes formed mainly by phycobiliproteins (PBPs). The PBPs absorb light that is efficiently transferred to Photosystems due to chromophores covalently bound to specific cysteine residues. Besides phycobiliproteins (PE), the PBS contains linker proteins responsible for assembly and stabilization of the whole complex and the tuning of energy transfer steps between chromophores. The linker (γ33) from Gracilaria chilensis, is a chromophorylated rod linker associated to (αβ)6 hexamers of R-phycoerythrin (R-PE). Its role in the energy transfer process is not clear yet. Structural studies as well as the composition and location of the chromophores are essential to understand their involvement in the energy transfer process in PBS. To achieve this, the coding gene of γ33 was cloned and sequenced. The sequence was analyzed by informatics tools, to obtain preliminary information which leaded the next experiments. The protein was purified from R-phycoerythrin, and the sequence confirmed by mass spectrometry. The coding sequence analysis revealed a protein of 318 aminoacid residues containing a chloroplastidial transit peptide (cTP) of 39 aminoacids at the N-terminus. The conservation of cysteines revealed possible chromophorylation sites. Using α and β R-PE subunits as spectroscopic probes in denaturation assays, we deduced a double bonded phycourobilin (PUB) on γ33 subunit that were confirmed between Cys62 and Cys73 (DL-PUB62/73) by mass spectrometry. The cysteines involved in the double link are located in a helical region, in a conformation that reminds the position of the DL-PUB50/61 in the β subunit of R-PE. The position of single linked PUB at Cys95 and a single linked PEB at Cys172 were also confirmed. Spectroscopic studies show the presence of both types of chromophores and that there are not energy transfer by FRET among them.
Topics: Amino Acid Sequence; Gracilaria; Phycobilins; Phycoerythrin; Plant Proteins; Protein Subunits; Sequence Analysis; Urobilin
PubMed: 29634783
DOI: 10.1371/journal.pone.0195656 -
Molecules (Basel, Switzerland) Mar 2016A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained...
Molecular Cloning of cpcU and Heterodimeric Bilin Lyase Activity Analysis of CpcU and CpcS for Attachment of Phycocyanobilin to Cys-82 on the β-Subunit of Phycocyanin in Arthrospira platensis FACHB314.
A new bilin lyase gene cpcU was cloned from Arthrospira platensis FACHB314 to study the assembly of the phycocyanin β-Subunit. Two recombinant plasmids, one contained the phycocyanobilin (PCB) producing genes (hoxI and pcyA), while the other contained the gene of the β-Subunit of phycobiliprotein (cpcB) and the lyase gene (cpcU, cpcS, or cpcU/S) were constructed and separately transferred into Escherichia coli in order to test the activities of relevant lyases for catalyzing PCB addition to CpcB during synthesizing fluorescent β-PC of A. platensis FACHB314. The fluorescence intensity examination showed that Cys-82 maybe the active site for the β-Subunit binding to PCBs and the attachment could be carried out by CpcU, CpcS, or co-expressed cpcU/S in A. platensis FACHB314.
Topics: Amino Acid Sequence; Bile Pigments; Cloning, Molecular; Cyanobacteria; Gene Expression; Lyases; Phycobilins; Phycocyanin; Phylogeny; Protein Binding; Protein Multimerization; Recombinant Proteins
PubMed: 26999083
DOI: 10.3390/molecules21030357 -
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