Biogenesis of Phycobiliproteins

Shen, Gaozhong; Schluchter, Wendy M.; Bryant, Donald A.

doi:10.1074/jbc.m708164200

Cited by 88 publications

(52 citation statements)

References 45 publications

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“…Phylogenetic analyses of cyanobacteria based on their PCB lyases suggested that the heterodimeric lyases CpcS-1 and CpcU are also responsible for PCB attachment in Synechocystis sp. PCC 6803 (Shen et al 2008). These findings complement our knowledge of the biosynthesis pathway of the APC trimer.…”

Section: Introductionsupporting

confidence: 69%

“…Tryptic digestion was performed as described with modifications (Arciero et al 1988;Shen et al 2008). Briefly, rAPCs in 50 mM potassium phosphate (pH 7.0) were acidified with 1 N HCl to pH 2.0.…”

Section: Tryptic Digestion Of Apcmentioning

confidence: 99%

“…Tryptic peptide analysis was performed as described with modifications (Arciero et al 1988;Shen et al 2008). Digested samples were analyzed by a C18 reverse-phase HPLC column (4.6 9 150 mm, Aglient, USA), using 50 mM phosphate buffer (pH 3.0) as solvent A and methanol as solvent B, with a ratio of A:B = 45:55.…”

Section: High Performance Liquid Chromatography (Hplc)mentioning

confidence: 99%

“…PCC 7002 and Anabaena sp. PCC 7120 (Saunee et al 2008;Shen et al 2008;Zhao et al 2007). Phylogenetic analyses of cyanobacteria based on their PCB lyases suggested that the heterodimeric lyases CpcS-1 and CpcU are also responsible for PCB attachment in Synechocystis sp.…”

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Liu

Chen

et al. 2010

Photosynth Res

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Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC a and b subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

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Section: Introductionsupporting

confidence: 69%

Section: Tryptic Digestion Of Apcmentioning

confidence: 99%

Section: High Performance Liquid Chromatography (Hplc)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Liu

Chen

et al. 2010

Photosynth Res

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“…The lyase is ubiquitous among cyanobacteria and has been subdivided into several classes (9). Some classes including the enzyme from Synechococcus sp.…”

Section: Discussionmentioning

confidence: 99%

Catalytic Mechanism of S-type Phycobiliprotein Lyase

Kupka¹,

Zhang

et al. 2009

Journal of Biological Chemistry

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We now show that CpcS1 binds PCB and PEB rapidly with bi-exponential kinetics (38/119 and 12/8300 ms, respectively). Chromophore binding to the lyase is reversible and much faster than the spontaneous, but low fidelity chromophore addition to the apo-protein in the absence of the lyase. This indicates kinetic control by the enzyme, which then transfers the chromophore to the apo-protein in a slow (tens of minutes) but stereo-and regioselectively corrects the reaction. This mode of action is reminiscent of chaperones but does not require ATP. The amino acid residues Arg-18 and Arg-149 of the lyase are essential for chromophore attachment in vitro and in Escherichia coli, mutations of His-21, His-22, Trp-75, Trp-140, and Arg-147 result in reduced activity (<30% of wild type in vitro). Mutants R147Q and W69M were active but had reduced capacity for PCB binding; additionally, with W69M there was loss of fidelity in chromophore attachment. Imidazole is a noncompetitive inhibitor, supporting a bilin-binding function of histidine. Evidence was obtained that CpcS1 also catalyzes exchange of C-␤84-bound PCB in biliproteins by PEB.Phycobiliproteins are a homologous family of light-harvesting proteins present in cyanobacteria, red algae, and cryptophytes that absorb light in the spectral region between the chlorophyll absorption maxima at ϳ430 and ϳ680 nm: they contain linear tetrapyrroles (phycobilins) of which 1-4 are covalently attached to the subunits by thioether bonds to conserved cysteines (1-4). The chromophores are derived from heme by oxidative ring-opening and subsequent reduction (5) and then attached post-translationally. The correct attachment of most chromophores is catalyzed by lyases. Three phylogenetically unrelated types of lyases have been characterized in cyanobacteria (6). They are specific for certain binding sites and chromophores (7-16), but the reaction mechanisms are still unclear. A chaperone-like action has been proposed for E/Ftype lyases that catalyze chromophore attachment to Cys-84 of ␤-subunits of phyco(erythro)cyanins (17, 18). A more highly evolved function is suggested, however, by concomitant isomerizing reactions catalyzed by certain lyases (12,19,20), and by the finding that most lyases can bind the chromophore and then transfer it to the acceptor protein (15,21).Chromophore binding is particularly pronounced with the S-type lyases. CpcS1 from Nostoc PCC7120 6 rapidly forms an adduct with phycocyanobilin (PCB), 7 and the latter can be transferred in a much slower reaction to cysteine 84 of the ␤-subunits of phycocyanin (CpcB) or phycoerythrocyanin (PecB), suggesting that PCB-CpcS1 is an intermediate of the enzymatic reaction (21). Model reactions of PCB with nucleophiles resulted in the formation of addition products in which the chromophore is isomerized and which are also substrates for a CpcS1-catalzed chromophore transfer to the apo-proteins (20). We now report chromophore binding kinetics obtained by stopped-flow techniques, and identification of functional amino acid residues f...

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Phycobiliprotein Biosynthesis in Cyanobacteria: Structure and Function of Enzymes Involved in Post-translational Modification

Schluchter

Shen

Alvey

et al. 2010

Recent Advances in Phototrophic Prokaryotes

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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 beta-72, which occurs on the beta-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 beta-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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Biogenesis of Phycobiliproteins

Cited by 88 publications

References 45 publications

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Catalytic Mechanism of S-type Phycobiliprotein Lyase

Phycobiliprotein Biosynthesis in Cyanobacteria: Structure and Function of Enzymes Involved in Post-translational Modification

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