Biosynthesis of Fluorescent Allophycocyanin α-Subunits by Autocatalytic Bilin Attachment

Hu, I-Chen; Lee, Tian-Ren; Lin, Huifen; Chiueh, Chuang-Chun; Lyu, Ping‐Chiang

doi:10.1021/bi052067a

Cited by 26 publications

(28 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…This E. coli system was superior to in vitro studies for poorly soluble proteins like CpeA, CpeB, ApcA2, and ApcB, and, furthermore, autocatalytic chromophore addition (17)(18)(19)(20) was suppressed; it is generally Ͻ10% ( Table 1). Possibly the most striking example was ApcA1, which is known to attach PCB autocatalytically in good yield (19), but, in the E. coli system, this background autocatalytic binding, in the absence of the lyase, was strongly reduced (Table 1). Moreover, the fluorescence emission of the autocatalytically bound product was red-shifted compared with that of the product of lyase-catalyzed binding, which indicates some chromophore oxidation to mesobiliverdin during autocatalytic binding.…”

Section: Resultsmentioning

confidence: 99%

“…Possibly, lyases with similarly broad specificity as CpeS1 can be found for the other binding sites and for the occasional secondary attachment to ring D in phycoerythrins. Currently, very little is known about the mode of action of any of these lyases; it also has to be separated from the autocatalytic capacities, albeit of low fidelity, of many of the apoproteins (17)(18)(19). Consequently, we emphasize the advantages of the multiplasmidic E. coli system for these studies.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

Zhao¹,

Tu³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

106

137

View full text Add to dashboard Cite

Phycobilisomes, the light-harvesting complexes of cyanobacteria and red algae, contain two to four types of chromophores that are attached covalently to seven or more members of a family of homologous proteins, each carrying one to four binding sites. Chromophore binding to apoproteins is catalyzed by lyases, of which only few have been characterized in detail. The situation is complicated by nonenzymatic background binding to some apoproteins. Using a modular multiplasmidic expression-reconstitution assay in Escherichia coli with low background binding, phycobilin:cystein-84 biliprotein lyase (CpeS1) from Anabaena PCC7120, has been characterized as a nearly universal lyase for the cysteine-84-binding site that is conserved in all biliproteins. It catalyzes covalent attachment of phycocyanobilin to all allophycocyanin subunits and to cysteine-84 in the ␤-subunits of C-phycocyanin and phycoerythrocyanin. Together with the known lyases, it can thereby account for chromophore binding to all binding sites of the phycobiliproteins of Anabaena PCC7120. Moreover, it catalyzes the attachment of phycoerythrobilin to cysteine-84 of both subunits of C-phycoerythrin. The only exceptions not served by CpeS1 among the cysteine-84 sites are the ␣-subunits from phycocyanin and phycoerythrocyanin, which, by sequence analyses, have been defined as members of a subclass that is served by the more specialized E/F type lyases.biliprotein biosynthesis ͉ light-harvesting ͉ photosynthesis ͉ phycobilisome P hycobilisomes, the extramembraneous light-harvesting antennas in cyanobacteria and red algae, use four different types of linear tetrapyrrole chromophores to harvest light in the green gap of chlorophyll absorption (1-6). These phycobilins are covalently bound to seven or more proteins, each carrying one to four binding sites. The chromophores are biosynthesized from the cyclic irontetrapyrrole, heme, by ring opening at C-5, followed by reduction and, sometimes, also by isomerization (7-9). In the last step, these phycobilins are covalently attached to cysteines of the apoprotein via a thioether bond to C-3 1 on ring A ( Fig. 1) and in some cases by an additional thioether bond to C-18 1 on ring D (6, 10-12). This step, the binding to the apoprotein, is presently only poorly understood; it involves a considerable number of binding sites and chromophores, as well as the proper regulation and coordination of events. ¶ An increasing number of lyases has recently been identified that catalyze the chromophore addition and are specific not only for the chromophore but also for the apoprotein and the binding site (12-16). Based on the capacity of several of the respective apoproteins to also bind the chromophores autocatalytically (17-21), a chaperone-like function has been suggested (12). It enhances and guides the autocatalytic binding, which is generally of low fidelity, possibly by conformational control of the chromophore (18). At the same time, this autocatalytic binding interferes with the lyase analyses (22). The situation is some...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

Zhao¹,

Tu³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

106

137

View full text Add to dashboard Cite

show abstract

“…2D) (32) and that holo-ApcA can be produced in E. coli (1 g liter Ϫ1 ) (68). Although one report claimed that holo-ApcA could be produced without any bilin lyase (33), the results presented here and in other studies show that either the heterologous CpcS-I/CpcU-type bilin lyase or the CpcS-III single subunit-type bilin lyase (also called CpeS1 by Zhao et al [75]) is required for maximal and correct addition of PCB to ApcA (25,46,72,75). The results of analyses of bilin lyase mutants also strongly support the conclusion that bilin lyases are essential for AP biogenesis (53).…”

Section: Discussionmentioning

confidence: 99%

“…IPTG (0.5 mM) was then added, and the cultures were incubated for an additional 4 h at 30°C. Cells were harvested and homogenized with 35 ml of acetone (33). The supernatant was vacuum dried to remove the acetone, and the dried pellet was dissolved in methanol (1.0 ml).…”

mentioning

confidence: 99%

Biosynthesis of Cyanobacterial Phycobiliproteins in Escherichia coli : Chromophorylation Efficiency and Specificity of All Bilin Lyases from Synechococcus sp. Strain PCC 7002

Biswas

Vasquez

Dragomani

et al. 2010

Appl Environ Microbiol

View full text Add to dashboard Cite

Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and ␣-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter ؊1 of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (␣ AP-B ) and ApcF (␤ 18 ). The N-terminal, allophycocyanin-like domain of ApcE (L CM 99 ) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of ␤-phycocyanin.

show abstract

“…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).…”

mentioning

confidence: 99%

Biliprotein Chromophore Attachment

Böhm

Endres

Scheer

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Biosynthesis of Fluorescent Allophycocyanin α-Subunits by Autocatalytic Bilin Attachment

Cited by 26 publications

References 38 publications

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

Phycobilin:cystein-84 biliprotein lyase, a near-universal lyase for cysteine-84-binding sites in cyanobacterial phycobiliproteins

Biosynthesis of Cyanobacterial Phycobiliproteins in Escherichia coli : Chromophorylation Efficiency and Specificity of All Bilin Lyases from Synechococcus sp. Strain PCC 7002

Biliprotein Chromophore Attachment

Contact Info

Product

Resources

About