Nonenzymatic chromophore attachment in biliproteins: conformational control by the detergent Triton X-100

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Biliproteins are post-translationally modified by chromophore addition. In phycoerythrocyanin, the heterodimeric lyase PecE/F covalently attaches phycocyanobilin (PCB) to cysteine-␣84 of the apoprotein PecA, with concomitant isomerization to phycoviolobilin. We found that: (a) PecA adds autocatalytically PCB, yielding a low absorbance, low fluorescence PCB⅐PecA adduct, termed P645 according to its absorption maximum; (b) In the presence of PecE, a high absorbance, high fluorescence PCB⅐PecA adduct is formed, termed P641; (c) PecE is capable of transforming P645 to P641; (d) When in stop-flow experiments, PecA and PecE were preincubated before chromophore addition, a red-shifted intermediate (P650, ‫؍‬ 32 ms) was observed followed by a second, which was blue-shifted (P605, ‫؍‬ 0.5 s), and finally a third (P638, ‫؍‬ 14 s) that yielded the adduct (P641, ‫؍‬ 20 min); (e) The reaction was slower, and P605 was missing, if PecA and PecE were not preincubated; (f) Gel filtration gave no evidence of a stable complex between PecA and PecE; however, complex formation is induced by adding PCB; and (g) A red-shifted intermediate was also formed, but more slowly, with phycoerythrobilin, and denaturation showed that this is not yet covalently bound. We conclude, therefore, that PecA and PecE form a weak complex that is stabilized by PCB, that the first reaction step involves a conformational change and/or protonation of PCB, and that PecE has a chaperone-like function on the chromoprotein.Phycobiliproteins are a family of light-harvesting proteins of cyanobacteria, rhodophytes, and cryptophytes (1-4). The apoproteins are post-translationally modified by regio-and stereoselective chromophore attachment to cysteines. Addition to the cysteine-␣84 of cyanobacterial phycocyanins and phycoerythrins is catalyzed by the heterodimeric E/F-type lyases (5). The first enzyme of this type studied, phycocyanobilin:cysteine-␣84-phycocyanin lyase, from the cyanobacterium, Synechococcus sp. PCC 7002, attaches phycocyanobilin (PCB) 3 to apo-␣-phycocyanin (CpcA) (6). It is encoded by two genes, cpcE and cpcF, located on the phycocyanin (CPC) operon downstream of the two apo-phycocyanin and two linker genes. A similar operon structure has been found in several other organisms, but the respective genes can also be located in other positions (5, 7). The functions of the two subunits are still unclear. Chromophore attachment to the phylogenetically related site on the ␤-subunit, cysteine-␤82, is catalyzed by a single-subunit protein, or mixtures of similar proteins (8, 9), whereas the chromophores are attached autocatalytically in phytochromes (10, 11), allophycocyanin, ApcA (12), and the core-membrane linker, ApcE (13). The situation is complicated by the capacity of apo-phycobiliproteins like CpcA/B to also add chromophores autocatalytically but only slowly and in an error-prone fashion (14 -17).The isomerizing phycoviolobilin:cysteine-␣84-phycoerythrocyanin lyase is a member of the E/F-type lyases, which, in addition to chromophore attachment, ha...

Section: Assignment Of Intermediatesmentioning

confidence: 99%

Section: Protein-protein Interactionsmentioning

confidence: 99%

Biliprotein Chromophore Attachment

Böhm

Endres

Scheer

et al. 2007

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“…S2), seems critical for the bilin lyases (see Scheer and Zhao (6)). Indirectly, conformational control as a determinant for chromophore binding is also supported by the effect of Triton X-100 on the spontaneous chromophore attachment (39). Although phycobilins are prone to oxidation (59), direct redox control seems unlikely, because CpcS1, in vitro, does not require reducing agents and is even inhibited by mercaptoethanol (14).…”

Section: Discussionmentioning

confidence: 99%

“…Studies of bilin lyases are complicated by a background of spontaneous, but faulty, attachment of chromophores to the same binding sites (6): for example, PCB is bound to CpcB(C155I) in the absence of a lyase, but the reaction results in a non-natural product mixture that has a much lower extinction coefficient than the proper chromoprotein obtained with the lyase (37)(38)(39). We also studied this process by rapid mixing and time-resolved absorption spectroscopy.…”

Section: Cpcs1mentioning

confidence: 99%

Catalytic Mechanism of S-type Phycobiliprotein Lyase

Kupka¹,

Zhang

et al. 2009

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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...

“…The genes cpcA, cpcE, cpcF, pecA, pecE, and pecF were PCR-amplified as described previously from Nostoc PCC7120 and Mastigocladus laminosus (Fischerella PCC7603) (15,18,26,29), and nblB, cpcS1, ho1, pcyA, pecB, and cpcB were from Nostoc PCC7120 (26,29). Mutants pecB(C155I), pecB(C84A), cpcB(C155I), and cpcB(C84S), were generated from pecB or cpcB of M. laminosus (30).…”

Section: Methodsmentioning

confidence: 99%

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

Zhao

Zhang

Tu³

et al. 2007

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Genes all5292 (cpcS2) and alr0617 (cpcS1) in the cyanobacterium Nostoc PCC7120 are homologous to the biliprotein lyase cpcS, and genes all5339 (cpcT1) and alr0647 (cpcT2) are homologous to the lyase cpcT. The functions of the encoded proteins were screened in vitro and in a heterologous Escherichia coli system with plasmids conferring biosynthesis of the phycocyanobilin chromophore and of the acceptor proteins ␤-phycoerythrocyanin (PecB) or ␤-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine ␤155 but does not discriminate between CpcB and PecB. The in vitro reconstitutions required no cofactors, and kinetic constants were determined for CpcT1 under in vitro conditions. No lyase activity was found for the lyase homologues CpcS2 and CpcT2, but complexes are formed in vitro between CpcT1 and CpcS1, CpcT2, or PecE (subunit of phycoviolobilin:␣-phycoerythrocyanin isomerase lyase). The genes coding the inactive homologues, cpcS2 and cpcT2, are transcribed in N-starved Nostoc. In sequential binding experiments with CpcT1 and CpcS1, a chromophore at cysteine 84 inhibited the subsequent attachment to cysteine 155, whereas the inverse sequence generates subunits carrying both chromophores.