Lyase Activities of CpcS- and CpcT-like Proteins from Nostoc PCC7120 and Sequential Reconstitution of Binding Sites of Phycoerythrocyanin and Phycocyanin β-Subunits

Zhao, Kai‐Hong; Zhang, Juan; Tu, Jing; Böhm, Stephan; Plöscher, Matthias; Eichacker, Lutz A.; Bubenzer, Claudia; Scheer, Hugo; Wang, Xing; Zhou, Ming

doi:10.1074/jbc.m703038200

Cited by 65 publications

(68 citation statements)

References 54 publications

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“…PCC 7120 (Nostoc), a single protein, CpcT (All5339), is responsible for ␤-subunit chromophorylation with PCB at Cys-␤155 in CPC and phycoerythrocyanin. Sequential binding studies indicated that this step is hindered by a preceding chromophorylation at Cys-␤84 by CpcS (25).…”

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confidence: 99%

“…The T-type lyases constitute a third class of lyases that is distantly related to CpcS (23). They catalyze the covalent attachment of PCB or phycoerythrobilin to Cys-␤155 sites (consensus numbering) in ␤-subunits of cyanobacterial (25,34) and cryptophyte biliproteins (35), and with a different stereochemistry than CpcS. In Nostoc (Anabaena) sp.…”

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confidence: 99%

“…S-type lyases are more universal, among which CpcS is the best studied. Alone or in combination with CpcU (22)(23)(24)(25), it catalyzes PCB attachment to conserved Cys-␤84 sites in various biliprotein ␤-subunits and to the related Cys-82 sites (consensus numbering) in both subunits of allophycocyanin (22,26). CpcS transiently binds bilins such as PCB (14).…”

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Structure and Mechanism of the Phycobiliprotein Lyase CpcT

Zhou

Ding

Zeng

et al. 2014

Journal of Biological Chemistry

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Background: Phycobiliprotein lyases catalyze covalent attachment of chromophores to cyanobacterial antenna proteins. Results: We present crystal structures of a T-type lyase and its complex with phycocyanobilin. Conclusion: The proposed reaction mechanism accounts for chromophore stabilization and regio-and stereospecificity. Significance: This work sheds light on a crucial step in phycobiliprotein maturation and provides a model for other types of phycobiliprotein lyases.

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Structure and Mechanism of the Phycobiliprotein Lyase CpcT

Zhou

Ding

Zeng

et al. 2014

Journal of Biological Chemistry

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“…fied from Mastigocladus laminosus, subsequently producing cpcB(C155I) via site-directed mutation (24), and apcA and cpcS1 were obtained from Nostoc PCC 7120 (14,25). They were cloned first into pBluescript (Stratagene) and then subcloned into pET-30 (Novagen, Munich).…”

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“…Lyase Activity Assay-Chromophore reconstitution with apo-phycobiliprotein was assayed as described before (13,15,25) using the following standard reaction conditions: KPB (500 mM, pH 7.5) containing NaCl (150 -200 mM), CpcS1 (or mutants, 10 -30 M), and apo-phycobiliprotein (5-30 M). PCB (final concentration ϭ 2-15 M) was added as a concentrated DMSO solution where the final concentration of DMSO in the reaction mixture was 1% (v/v).…”

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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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A Single‐Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome**

2023

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Cyanobacteriochrome (CBCR) cGMP‐specific phosphodiesterase, adenylyl cyclase, and FhlA (GAF) domains bind bilin cofactors to confer sensory wavelengths important for various cyanobacterial photosensory processes. Many isolated GAF domains autocatalytically bind bilins, including the third GAF domain of CBCR Slr1393 from Synechocystis sp. PCC6803, which binds phycoerythrobilin (PEB) to yield a bright orange fluorescent protein. Compared to green fluorescent proteins, the smaller size and lack of an oxygen requirement for fluorescence make Slr1393g3 a promising platform for new genetically encoded fluorescent tools. Slr1393g3, however, shows low PEB binding efficiency (chromophorylation) at ~3% compared to total Slr1393g3 expressed in E. coli. Here we used site‐directed mutagenesis and plasmid redesign methods to improve Slr1393g3‐PEB binding and demonstrate its utility as a fluorescent marker in live cells. Mutation at a single site, Trp496, tuned the emission over ~30 nm, likely by shifting autoisomerization of PEB to phycourobilin (PUB). Plasmid modifications for tuning relative expression of Slr1393g3 and PEB synthesis enzymes also improved chromophorylation and moving from a dual to single plasmid system facilitated exploration of a range of mutants via site saturation mutagenesis and sequence truncation. Collectively, the PEB/PUB chromophorylation was raised up to a total of 23% with combined sequence truncation and W496H mutation.

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Lyase Activities of CpcS- and CpcT-like Proteins from Nostoc PCC7120 and Sequential Reconstitution of Binding Sites of Phycoerythrocyanin and Phycocyanin β-Subunits

Cited by 65 publications

References 54 publications

Structure and Mechanism of the Phycobiliprotein Lyase CpcT

Structure and Mechanism of the Phycobiliprotein Lyase CpcT

Catalytic Mechanism of S-type Phycobiliprotein Lyase

A Single‐Site Mutation Tunes Fluorescence and Chromophorylation of an Orange Fluorescent Cyanobacteriochrome**

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