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Donald A Bryant - One of the best experts on this subject based on the ideXlab platform.
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far red light allophycocyanin subunits play a role in chlorophyll d accumulation in far red light
Photosynthesis Research, 2020Co-Authors: Nathan Soulier, Gavin M Turner, Ming Yang Ho, Tatiana N Laremore, Gaozhong Shen, Donald A BryantAbstract:Some terrestrial cyanobacteria acclimate to and utilize far-red light (FRL; λ = 700–800 nm) for oxygenic photosynthesis, a process known as far-red light photoacclimation (FaRLiP). A conserved, 20-gene FaRLiP cluster encodes core subunits of Photosystem I (PSI) and Photosystem II (PSII), five Phycobiliprotein subunits of FRL-bicylindrical cores, and enzymes for synthesis of chlorophyll (Chl) f and possibly Chl d. Deletion mutants for each of the five apc genes of the FaRLiP cluster were constructed in Synechococcus sp. PCC 7335, and all had similar phenotypes. When the mutants were grown in white (WL) or red (RL) light, the cells closely resembled the wild-type (WT) strain grown under the same conditions. However, the WT and mutant strains were very different when grown under FRL. Mutants grown in FRL were unable to assemble FRL-bicylindrical cores, were essentially devoid of FRL-specific Phycobiliproteins, but retained RL-type phycobilisomes and WL-PSII. The transcript levels for genes of the FaRLiP cluster in the mutants were similar to those in WT. Surprisingly, the Chl d contents of the mutant strains were greatly reduced (~ 60–99%) compared to WT and so were the levels of FRL-PSII. We infer that Chl d may be essential for the assembly of FRL-PSII, which does not accumulate to normal levels in the mutants. We further infer that the cysteine-rich subunits of FRL allophycocyanin may either directly participate in the synthesis of Chl d or that FRL bicylindrical cores stabilize FRL-PSII to prevent loss of Chl d.
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Phycobiliprotein biosynthesis in cyanobacteria structure and function of enzymes involved in post translational modification
Advances in Experimental Medicine and Biology, 2010Co-Authors: Wendy M Schluchter, Avijit Biswas, Gaozhong Shen, Richard M Alvey, Nicolle A Saunee, Shervonda R Williams, Crystal A Mille, Donald A BryantAbstract:Cyanobacterial Phycobiliproteins are brilliantly colored due to the presence of covalently attached chromophores called bilins, linear tetrapyrroles derived from heme. For most Phycobiliproteins, these post-translational modifications are catalyzed by enzymes called bilin lyases; these enzymes ensure that the appropriate bilins are attached to the correct cysteine residues with the proper stereochemistry on each Phycobiliprotein subunit. Phycobiliproteins also contain a unique, post-translational modification, the methylation of a conserved asparagine (Asn) present at β-72, which occurs on the β-subunits of all Phycobiliproteins. We have identified and characterized several new families of bilin lyases, which are responsible for attaching PCB to Phycobiliproteins as well as the Asn methyl transferase for β-subunits in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. All of the enzymes responsible for synthesis of holo-Phycobiliproteins are now known for this cyanobacterium, and a brief discussion of each enzyme family and its role in the biosynthesis of Phycobiliproteins is presented here. In addition, the first structure of a bilin lyase has recently been solved (PDB ID: 3BDR). This structure shows that the bilin lyases are most similar to the lipocalin protein structural family, which also includes the bilin-binding protein found in some butterflies.
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cpcm posttranslationally methylates asparagine 71 72 of Phycobiliprotein beta subunits in synechococcus sp strain pcc 7002 and synechocystis sp strain pcc 6803
Journal of Bacteriology, 2008Co-Authors: Gaozhong Shen, Wendy M Schluchter, Heidi S Leonard, Donald A BryantAbstract:Cyanobacteria produce phycobilisomes, which are macromolecular light-harvesting complexes mostly assembled from Phycobiliproteins. Phycobiliprotein beta subunits contain a highly conserved gamma-N-methylasparagine residue, which results from the posttranslational modification of Asn71/72. Through comparative genomic analyses, we identified a gene, denoted cpcM, that (i) encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases, (ii) is found in all sequenced cyanobacterial genomes, and (iii) often occurs near genes encoding Phycobiliproteins in cyanobacterial genomes. The cpcM genes of Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 were insertionally inactivated. Mass spectrometric analyses of Phycobiliproteins isolated from the mutants confirmed that the CpcB, ApcB, and ApcF were 14 Da lighter than their wild-type counterparts. Trypsin digestion and mass analyses of Phycobiliproteins isolated from the mutants showed that tryptic peptides from phycocyanin that included Asn72 were also 14 Da lighter than the equivalent peptides from wild-type strains. Thus, CpcM is the methyltransferase that modifies the amide nitrogen of Asn71/72 of CpcB, ApcB, and ApcF. When cells were grown at low light intensity, the cpcM mutants were phenotypically similar to the wild-type strains. However, the mutants were sensitive to high-light stress, and the cpcM mutant of Synechocystis sp. strain PCC 6803 was unable to grow at moderately high light intensities. Fluorescence emission measurements showed that the ability to perform state transitions was impaired in the cpcM mutants and suggested that energy transfer from Phycobiliproteins to the photosystems was also less efficient. The possible functions of asparagine N methylation of Phycobiliproteins are discussed.
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biogenesis of Phycobiliproteins iii cpcm is the asparagine methyltransferase for Phycobiliprotein β subunits in cyanobacteria
Journal of Biological Chemistry, 2008Co-Authors: Crystal A Miller, Gaozhong Shen, Donald A Bryant, Shervonda R Williams, Heidi S Leonard, Ivan G Pinsky, Brandy M Turner, Leon Harrison, Ariane F Fletcher, Wendy M SchluchterAbstract:All Phycobiliproteins contain a conserved, post-translational modification on asparagine 72 of their β-subunits. Methylation of this Asn to produce γ-N-methylasparagine has been shown to increase energy transfer efficiency within the phycobilisome and to prevent photoinhibition. We report here the biochemical characterization of the product of sll0487, which we have named cpcM, from the cyanobacterium Synechocystis sp. PCC 6803. Recombinant apo-phycocyanin and apo-allophycocyanin subunits were used as the substrates for assays with [methyl-3H]S-adenosylmethionine and recombinant CpcM. CpcM methylated the β-subunits of Phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding α-subunits (CpcA, ApcA, and ApcD), although they are similar in primary and tertiary structure. CpcM preferentially methylated its CpcB substrate after chromophorylation had occurred at Cys82. CpcM exhibited lower activity on trimeric phycocyanin after complete chromophorylation and oligomerization had occurred. Based upon these in vitro studies, we conclude that this post-translational modification probably occurs after chromophorylation but before trimer assembly in vivo.
Gaozhong Shen - One of the best experts on this subject based on the ideXlab platform.
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far red light allophycocyanin subunits play a role in chlorophyll d accumulation in far red light
Photosynthesis Research, 2020Co-Authors: Nathan Soulier, Gavin M Turner, Ming Yang Ho, Tatiana N Laremore, Gaozhong Shen, Donald A BryantAbstract:Some terrestrial cyanobacteria acclimate to and utilize far-red light (FRL; λ = 700–800 nm) for oxygenic photosynthesis, a process known as far-red light photoacclimation (FaRLiP). A conserved, 20-gene FaRLiP cluster encodes core subunits of Photosystem I (PSI) and Photosystem II (PSII), five Phycobiliprotein subunits of FRL-bicylindrical cores, and enzymes for synthesis of chlorophyll (Chl) f and possibly Chl d. Deletion mutants for each of the five apc genes of the FaRLiP cluster were constructed in Synechococcus sp. PCC 7335, and all had similar phenotypes. When the mutants were grown in white (WL) or red (RL) light, the cells closely resembled the wild-type (WT) strain grown under the same conditions. However, the WT and mutant strains were very different when grown under FRL. Mutants grown in FRL were unable to assemble FRL-bicylindrical cores, were essentially devoid of FRL-specific Phycobiliproteins, but retained RL-type phycobilisomes and WL-PSII. The transcript levels for genes of the FaRLiP cluster in the mutants were similar to those in WT. Surprisingly, the Chl d contents of the mutant strains were greatly reduced (~ 60–99%) compared to WT and so were the levels of FRL-PSII. We infer that Chl d may be essential for the assembly of FRL-PSII, which does not accumulate to normal levels in the mutants. We further infer that the cysteine-rich subunits of FRL allophycocyanin may either directly participate in the synthesis of Chl d or that FRL bicylindrical cores stabilize FRL-PSII to prevent loss of Chl d.
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Phycobiliprotein biosynthesis in cyanobacteria structure and function of enzymes involved in post translational modification
Advances in Experimental Medicine and Biology, 2010Co-Authors: Wendy M Schluchter, Avijit Biswas, Gaozhong Shen, Richard M Alvey, Nicolle A Saunee, Shervonda R Williams, Crystal A Mille, Donald A BryantAbstract:Cyanobacterial Phycobiliproteins are brilliantly colored due to the presence of covalently attached chromophores called bilins, linear tetrapyrroles derived from heme. For most Phycobiliproteins, these post-translational modifications are catalyzed by enzymes called bilin lyases; these enzymes ensure that the appropriate bilins are attached to the correct cysteine residues with the proper stereochemistry on each Phycobiliprotein subunit. Phycobiliproteins also contain a unique, post-translational modification, the methylation of a conserved asparagine (Asn) present at β-72, which occurs on the β-subunits of all Phycobiliproteins. We have identified and characterized several new families of bilin lyases, which are responsible for attaching PCB to Phycobiliproteins as well as the Asn methyl transferase for β-subunits in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. All of the enzymes responsible for synthesis of holo-Phycobiliproteins are now known for this cyanobacterium, and a brief discussion of each enzyme family and its role in the biosynthesis of Phycobiliproteins is presented here. In addition, the first structure of a bilin lyase has recently been solved (PDB ID: 3BDR). This structure shows that the bilin lyases are most similar to the lipocalin protein structural family, which also includes the bilin-binding protein found in some butterflies.
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cpcm posttranslationally methylates asparagine 71 72 of Phycobiliprotein beta subunits in synechococcus sp strain pcc 7002 and synechocystis sp strain pcc 6803
Journal of Bacteriology, 2008Co-Authors: Gaozhong Shen, Wendy M Schluchter, Heidi S Leonard, Donald A BryantAbstract:Cyanobacteria produce phycobilisomes, which are macromolecular light-harvesting complexes mostly assembled from Phycobiliproteins. Phycobiliprotein beta subunits contain a highly conserved gamma-N-methylasparagine residue, which results from the posttranslational modification of Asn71/72. Through comparative genomic analyses, we identified a gene, denoted cpcM, that (i) encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases, (ii) is found in all sequenced cyanobacterial genomes, and (iii) often occurs near genes encoding Phycobiliproteins in cyanobacterial genomes. The cpcM genes of Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 were insertionally inactivated. Mass spectrometric analyses of Phycobiliproteins isolated from the mutants confirmed that the CpcB, ApcB, and ApcF were 14 Da lighter than their wild-type counterparts. Trypsin digestion and mass analyses of Phycobiliproteins isolated from the mutants showed that tryptic peptides from phycocyanin that included Asn72 were also 14 Da lighter than the equivalent peptides from wild-type strains. Thus, CpcM is the methyltransferase that modifies the amide nitrogen of Asn71/72 of CpcB, ApcB, and ApcF. When cells were grown at low light intensity, the cpcM mutants were phenotypically similar to the wild-type strains. However, the mutants were sensitive to high-light stress, and the cpcM mutant of Synechocystis sp. strain PCC 6803 was unable to grow at moderately high light intensities. Fluorescence emission measurements showed that the ability to perform state transitions was impaired in the cpcM mutants and suggested that energy transfer from Phycobiliproteins to the photosystems was also less efficient. The possible functions of asparagine N methylation of Phycobiliproteins are discussed.
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biogenesis of Phycobiliproteins iii cpcm is the asparagine methyltransferase for Phycobiliprotein β subunits in cyanobacteria
Journal of Biological Chemistry, 2008Co-Authors: Crystal A Miller, Gaozhong Shen, Donald A Bryant, Shervonda R Williams, Heidi S Leonard, Ivan G Pinsky, Brandy M Turner, Leon Harrison, Ariane F Fletcher, Wendy M SchluchterAbstract:All Phycobiliproteins contain a conserved, post-translational modification on asparagine 72 of their β-subunits. Methylation of this Asn to produce γ-N-methylasparagine has been shown to increase energy transfer efficiency within the phycobilisome and to prevent photoinhibition. We report here the biochemical characterization of the product of sll0487, which we have named cpcM, from the cyanobacterium Synechocystis sp. PCC 6803. Recombinant apo-phycocyanin and apo-allophycocyanin subunits were used as the substrates for assays with [methyl-3H]S-adenosylmethionine and recombinant CpcM. CpcM methylated the β-subunits of Phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding α-subunits (CpcA, ApcA, and ApcD), although they are similar in primary and tertiary structure. CpcM preferentially methylated its CpcB substrate after chromophorylation had occurred at Cys82. CpcM exhibited lower activity on trimeric phycocyanin after complete chromophorylation and oligomerization had occurred. Based upon these in vitro studies, we conclude that this post-translational modification probably occurs after chromophorylation but before trimer assembly in vivo.
Hiroki Saeki - One of the best experts on this subject based on the ideXlab platform.
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anti inflammatory effects of dulse palmaria palmata resulting from the simultaneous water extraction of Phycobiliproteins and chlorophyll a
Food Research International, 2017Co-Authors: Mizuho Nishizawa, Yutaka Shimizu, Hiroki SaekiAbstract:The use of dulse (Palmaria palmata) as a source of edible anti-inflammatory products was evaluated in this study. Phycobiliproteins and chlorophyll a were simultaneously extracted from lyophilized dulse leaves via water-extraction, and subjected to thermolysin digestion to produce thermolysin-digested water-extract (d-DWE). d-DWE significantly reduced tumor necrosis factor-α, interleukin-6, and nitric oxide in LPS-stimulated murine macrophages (RAW 264.7 cells), and orally administered d-DWE mitigated acute inflammation in carrageenan-induced paw edema of mice. Mass spectrometry revealed d-DWE contained peptide LRDGEIILRY (derived from phycoerythrin β-chain) and chlorophyll a decomposition products, and they individually reduced the secretion of the proinflammatory mediators in LPS-stimulated RAW 264.7 cells. These results indicate the anti-inflammatory activity could be from a combined effect of Phycobiliprotein and chlorophyll a decomposition products prepared from the water-extract of dulse. Thus, inexpensive and safe water-extraction method is effective for the extraction of anti-inflammatory components from dulse.
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anti inflammatory effects of dulse palmaria palmata resulting from the simultaneous water extraction of Phycobiliproteins and chlorophyll a
Food Research International, 2017Co-Authors: Mizuho Nishizawa, Yutaka Shimizu, Hiroki SaekiAbstract:The use of dulse (Palmaria palmata) as a source of edible anti-inflammatory products was evaluated in this study. Phycobiliproteins and chlorophyll a were simultaneously extracted from lyophilized dulse leaves via water-extraction, and subjected to thermolysin digestion to produce thermolysin-digested water-extract (d-DWE). d-DWE significantly reduced tumor necrosis factor-α, interleukin-6, and nitric oxide in LPS-stimulated murine macrophages (RAW 264.7 cells), and orally administered d-DWE mitigated acute inflammation in carrageenan-induced paw edema of mice. Mass spectrometry revealed d-DWE contained peptide LRDGEIILRY (derived from phycoerythrin β-chain) and chlorophyll a decomposition products, and they individually reduced the secretion of the proinflammatory mediators in LPS-stimulated RAW 264.7 cells. These results indicate the anti-inflammatory activity could be from a combined effect of Phycobiliprotein and chlorophyll a decomposition products prepared from the water-extract of dulse. Thus, inexpensive and safe water-extraction method is effective for the extraction of anti-inflammatory components from dulse.
Wendy M Schluchter - One of the best experts on this subject based on the ideXlab platform.
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Phycobiliprotein biosynthesis in cyanobacteria structure and function of enzymes involved in post translational modification
Advances in Experimental Medicine and Biology, 2010Co-Authors: Wendy M Schluchter, Avijit Biswas, Gaozhong Shen, Richard M Alvey, Nicolle A Saunee, Shervonda R Williams, Crystal A Mille, Donald A BryantAbstract:Cyanobacterial Phycobiliproteins are brilliantly colored due to the presence of covalently attached chromophores called bilins, linear tetrapyrroles derived from heme. For most Phycobiliproteins, these post-translational modifications are catalyzed by enzymes called bilin lyases; these enzymes ensure that the appropriate bilins are attached to the correct cysteine residues with the proper stereochemistry on each Phycobiliprotein subunit. Phycobiliproteins also contain a unique, post-translational modification, the methylation of a conserved asparagine (Asn) present at β-72, which occurs on the β-subunits of all Phycobiliproteins. We have identified and characterized several new families of bilin lyases, which are responsible for attaching PCB to Phycobiliproteins as well as the Asn methyl transferase for β-subunits in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. All of the enzymes responsible for synthesis of holo-Phycobiliproteins are now known for this cyanobacterium, and a brief discussion of each enzyme family and its role in the biosynthesis of Phycobiliproteins is presented here. In addition, the first structure of a bilin lyase has recently been solved (PDB ID: 3BDR). This structure shows that the bilin lyases are most similar to the lipocalin protein structural family, which also includes the bilin-binding protein found in some butterflies.
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cpcm posttranslationally methylates asparagine 71 72 of Phycobiliprotein beta subunits in synechococcus sp strain pcc 7002 and synechocystis sp strain pcc 6803
Journal of Bacteriology, 2008Co-Authors: Gaozhong Shen, Wendy M Schluchter, Heidi S Leonard, Donald A BryantAbstract:Cyanobacteria produce phycobilisomes, which are macromolecular light-harvesting complexes mostly assembled from Phycobiliproteins. Phycobiliprotein beta subunits contain a highly conserved gamma-N-methylasparagine residue, which results from the posttranslational modification of Asn71/72. Through comparative genomic analyses, we identified a gene, denoted cpcM, that (i) encodes a protein with sequence similarity to other S-adenosylmethionine-dependent methyltransferases, (ii) is found in all sequenced cyanobacterial genomes, and (iii) often occurs near genes encoding Phycobiliproteins in cyanobacterial genomes. The cpcM genes of Synechococcus sp. strain PCC 7002 and Synechocystis sp. strain PCC 6803 were insertionally inactivated. Mass spectrometric analyses of Phycobiliproteins isolated from the mutants confirmed that the CpcB, ApcB, and ApcF were 14 Da lighter than their wild-type counterparts. Trypsin digestion and mass analyses of Phycobiliproteins isolated from the mutants showed that tryptic peptides from phycocyanin that included Asn72 were also 14 Da lighter than the equivalent peptides from wild-type strains. Thus, CpcM is the methyltransferase that modifies the amide nitrogen of Asn71/72 of CpcB, ApcB, and ApcF. When cells were grown at low light intensity, the cpcM mutants were phenotypically similar to the wild-type strains. However, the mutants were sensitive to high-light stress, and the cpcM mutant of Synechocystis sp. strain PCC 6803 was unable to grow at moderately high light intensities. Fluorescence emission measurements showed that the ability to perform state transitions was impaired in the cpcM mutants and suggested that energy transfer from Phycobiliproteins to the photosystems was also less efficient. The possible functions of asparagine N methylation of Phycobiliproteins are discussed.
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biogenesis of Phycobiliproteins iii cpcm is the asparagine methyltransferase for Phycobiliprotein β subunits in cyanobacteria
Journal of Biological Chemistry, 2008Co-Authors: Crystal A Miller, Gaozhong Shen, Donald A Bryant, Shervonda R Williams, Heidi S Leonard, Ivan G Pinsky, Brandy M Turner, Leon Harrison, Ariane F Fletcher, Wendy M SchluchterAbstract:All Phycobiliproteins contain a conserved, post-translational modification on asparagine 72 of their β-subunits. Methylation of this Asn to produce γ-N-methylasparagine has been shown to increase energy transfer efficiency within the phycobilisome and to prevent photoinhibition. We report here the biochemical characterization of the product of sll0487, which we have named cpcM, from the cyanobacterium Synechocystis sp. PCC 6803. Recombinant apo-phycocyanin and apo-allophycocyanin subunits were used as the substrates for assays with [methyl-3H]S-adenosylmethionine and recombinant CpcM. CpcM methylated the β-subunits of Phycobiliproteins (CpcB, ApcB, and ApcF) and did not methylate the corresponding α-subunits (CpcA, ApcA, and ApcD), although they are similar in primary and tertiary structure. CpcM preferentially methylated its CpcB substrate after chromophorylation had occurred at Cys82. CpcM exhibited lower activity on trimeric phycocyanin after complete chromophorylation and oligomerization had occurred. Based upon these in vitro studies, we conclude that this post-translational modification probably occurs after chromophorylation but before trimer assembly in vivo.
Ming Zhou - One of the best experts on this subject based on the ideXlab platform.
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energy transfer between fusion biliproteins co expressed with Phycobiliprotein in escherichia coli
Protein Expression and Purification, 2016Co-Authors: Nan Zhou, Ming ZhouAbstract:In cyanobacteria, Phycobiliproteins (PBS) show excellent energy transfer among the chromophores absorbing over most of the visible. The energy transfers are used to study phycobilisome assembly and bioimaging. Using All4261GAF2(C81L) as energy donor, ApcE(1-240/Δ87-130) as energy acceptor, we co-expressed fusion protein ApcE(1-240/Δ87-130)::All4261GAF2(C81L) with Phycobiliprotein in Escherichia Coli and studied the energy transfer between two protein domains. With N-terminal His6 tag, ApcE(1-240/Δ87-130)::All4261GAF2(C81L) cannot be purified by nickel-affinity column. We added six histidines in the C-terminal of ApcE(1-240/Δ87-130)::All4261GAF2(C81L) and co-expressed it with Phycobiliprotein. ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was purified successfully and only singly chromophorylated at All4261GAF2(C81L)His6 domain. The singly chromophorylate ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was incubated with fresh PCB and the doubly chromophorylated PCB-ApcE(1-240/Δ87-130)::PCB-All4261GAF2(C81L)His6 was obtained. The double chromophored fusion protein absorbed light in the range of 615-660 nm, and fluoresced only at 668 nm. Photochemistry analysis showed that excitation energy transfer from the short-wavelength absorbing at All4261GAF2(C81L) domain was achieved successfully to the long-wavelength absorbing at the ApcE(1-240/Δ87-130) domain.
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structure and mechanism of the Phycobiliprotein lyase cpct
Journal of Biological Chemistry, 2014Co-Authors: Wei Zhou, Xiaoli Zeng, Ming Zhou, Hugo Scheer, Kai-hong Zhao, Wen Long Ding, Liang Liang Dong, Bin Zhao, Xiaojing YangAbstract:Pigmentation of light-harvesting Phycobiliproteins of cyanobacteria requires covalent attachment of open-chain tetrapyrroles, bilins, to the apoproteins. Thioether formation via addition of a cysteine residue to the 3-ethylidene substituent of bilins is mediated by lyases. T-type lyases are responsible for attachment to Cys-155 of Phycobiliprotein β-subunits. We present crystal structures of CpcT (All5339) from Nostoc (Anabaena) sp. PCC 7120 and its complex with phycocyanobilin at 1.95 and 2.50 Å resolution, respectively. CpcT forms a dimer and adopts a calyx-shaped β-barrel fold. Although the overall structure of CpcT is largely retained upon chromophore binding, arginine residues at the opening of the binding pocket undergo major rotameric rearrangements anchoring the propionate groups of phycocyanobilin. Based on the structure and mutational analysis, a reaction mechanism is proposed that accounts for chromophore stabilization and regio- and stereospecificity of the addition reaction. At the dimer interface, a loop extending from one subunit partially shields the opening of the phycocyanobilin binding pocket in the other subunit. Deletion of the loop or disruptions of the dimer interface significantly reduce CpcT lyase activity, suggesting functional relevance of the dimer. Dimerization is further enhanced by chromophore binding. The chromophore is largely buried in the dimer, but in the monomer, the 3-ethylidene group is accessible for the apoPhycobiliprotein, preferentially from the chromophore α-side. Asp-163 and Tyr-65 at the β- and α-face near the E-configured ethylidene group, respectively, support the acid-catalyzed nucleophilic Michael addition of cysteine 155 of the apoprotein to an N-acylimmonium intermediate proposed by Grubmayr and Wagner (Grubmayr, K., and Wagner, U. G. (1988) Monatsh. Chem. 119, 965–983).
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Modular generation of fluorescent Phycobiliproteins
Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology, 2013Co-Authors: Kun Chang, Ming Zhou, Hugo Scheer, Juan Luo, Kai-hong ZhaoAbstract:Phycobiliproteins are brightly-fluorescent light-harvesting pigments for photosynthesis in cyanobacteria and red algae. They are also of interest as fluorescent biomarkers, but their heterologous generation in vivo has previously required multiple transformations. We report here a modular approach that requires only two DNA segments. The first codes for the apo-protein. The second codes for fusions capable of chromophore biosynthesis and its covalent attachment to the apo-protein; it contains the genes of heme oxygenase, a bilin reductase, and a chromophore lyase. Phycobiliproteins containing phycoerythrobilin (λfluor ∼ 560 nm), phycourobilin (λfluor ∼ 500 nm), phycocyanobilin (λfluor ∼ 630 nm) or phycoviolobilin (λfluor ∼ 580 nm) were obtained in high yield in E. coli. This approach facilitates chromophorylation studies of Phycobiliproteins, as well as their use for fluorescence labeling based on their high fluorescence.
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a minimal phycobilisome fusion and chromophorylation of the truncated core membrane linker and phycocyanin
Biochimica et Biophysica Acta, 2012Co-Authors: Kun Tang, Hugo Scheer, Xiaoli Zeng, Yi Yang, Zhibin Wang, Ming Zhou, Dror Noy, Kaihong Zhao, Ming Zhou, Hugo Scheer, Kai-hong ZhaoAbstract:Abstract Phycobilisomes, the light-harvesting antennas in cyanobacteria and red algae, consist of an allophycocyanin core that is attached to the membrane via a core-membrane linker, and rods comprised of phycocyanin and often also phycoerythrin or phycoerythrocyanin. Phycobiliproteins show excellent energy transfer among the chromophores that renders them biomarkers with large Stokes-shifts absorbing over most of the visible spectrum and into the near infrared. Their application is limited, however, due to covalent binding of the chromophores and by solubility problems. We report construction of a water-soluble minimal chromophore-binding unit of the red-absorbing and fluorescing core-membrane linker. This was fused to minimal chromophore-binding units of phycocyanin. After double chromophorylation with phycocyanobilin, in E. coli , the fused Phycobiliproteins absorbed light in the range of 610–660 nm, and fluoresced at ~ 670 nm, similar to phycobilisomes devoid of phycoerythr(ocyan)in. The fused Phycobiliprotein could also be doubly chromophorylated with phycoerythrobilin, resulting in a chromoprotein absorbing around 540–575 nm, and fluorescing at ~ 585 nm. The broad absorptions and the large Stokes shifts render these chromoproteins candidates for imaging; they may also be helpful in studying phycobilisome assembly.
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heterogenous expression of synechocystis sp pcc 6803 deg proteases and their possible roles on Phycobiliprotein degradation in vitro
Journal of Wuhan University of Technology-materials Science Edition, 2011Co-Authors: Ming Zhou, Kai-hong Zhao, Hongli Diao, Ting Zhou, Juang Zhang, Cheng YangAbstract:The Synechocystis sp. PCC 6803 genome harbours a Deg gene family consisting of three members, htrA (degP, slr1204), hhoA (degQ, sll1679) and hhoB (degS, sll1427). This work provided biochemical characterization of HhoA, HtrA and HhoB from Synechocystis sp. PCC 6803. Firstly mature HhoA, HhoB and HtrA from Synechocystis sp. PCC 6803 were cloned and expressed as soluble recombinant his-tagged fusion protein in Escherichia coli. Then the proteolytic activity of HhoA, HhoB and HtrA was tested using casein, bovine serum albumin, five recombinant chromoproteins and cyanobacterial phycocyanin as substrates in vitro. The experimental results showed that HhoA and HtrA had proteolytic activity on casein, five recombinant chromoproteins and cyanobacterial phycocyanin. No proteolytic activity of HhoB was found using all substrates in vitro, indicating functional difference among Deg proteases from Synechocystis sp. PCC 6803. Therefore, the results indicated the biochemical properties of HhoA and HtrA on hydrolysis of proteins and Phycobiliproteins in vitro, which implicated that they were proteases possibly involved in Phycobiliprotein degradation in vivo.