Limits...
Biliprotein maturation: the chromophore attachment.

Scheer H, Zhao KH - Mol. Microbiol. (2008)

Bottom Line: The discovery of new activities for the latter lyases, and of new types of lyases, have reinvigorated research activities in the subject.So far, work has mainly concentrated on cyanobacterial phycobiliproteins.Methodological advances in the process, however, as well as the finding of often large numbers of homologues, opens new possibilities for research on the subsequent assembly/disassembly of the phycobilisome in cyanobacteria and red algae, on the assembly and organization of the cryptophyte light-harvesting system, on applications in basic research such as protein folding, and on the use of phycobiliproteins for labelling.

View Article: PubMed Central - PubMed

Affiliation: Department Biologie I, Universität München, Menzinger Strasse 67, D-80638 München, Germany.

ABSTRACT
Biliproteins are a widespread group of brilliantly coloured photoreceptors characterized by linear tetrapyrrolic chromophores, bilins, which are covalently bound to the apoproteins via relatively stable thioether bonds. Covalent binding stabilizes the chromoproteins and is mandatory for phycobilisome assembly; and, it is also important in biliprotein applications such as fluorescence labelling. Covalent binding has, on the other hand, also considerably hindered biliprotein research because autocatalytic chromophore additions are rare, and information on enzymatic addition by lyases was limited to a single example, an EF-type lyase attaching phycocyanobilin to cysteine-alpha84 of C-phycocyanin. The discovery of new activities for the latter lyases, and of new types of lyases, have reinvigorated research activities in the subject. So far, work has mainly concentrated on cyanobacterial phycobiliproteins. Methodological advances in the process, however, as well as the finding of often large numbers of homologues, opens new possibilities for research on the subsequent assembly/disassembly of the phycobilisome in cyanobacteria and red algae, on the assembly and organization of the cryptophyte light-harvesting system, on applications in basic research such as protein folding, and on the use of phycobiliproteins for labelling.

Show MeSH

Related in: MedlinePlus

Reaction schemes of the S-type (top) and isomerizing E/F-type lyases (bottom). Non-covalent chromophore binding is indicated by broken lines, covalent binding by solid lines and colouration of both the chromophore and the protein. Intermediates (Pxxx) are named according to their absorption maxima at xxx nm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2327270&req=5

fig03: Reaction schemes of the S-type (top) and isomerizing E/F-type lyases (bottom). Non-covalent chromophore binding is indicated by broken lines, covalent binding by solid lines and colouration of both the chromophore and the protein. Intermediates (Pxxx) are named according to their absorption maxima at xxx nm.

Mentions: CpcS1 is a relatively simple system. It is active as a monomer, and does not require cofactors. A more complex situation has been found with S-type lyases from other organisms classified as group I, where the concerted action of two subunits (CpcS and CpcU) is required (Saunée et al., 2008; Shen et al., 2008). CpcS1 from Nostoc binds the chromophore rapidly (∼0.1 s) and reversibly, and then transfers it in a much slower, irreversible reaction to the apoproteins (∼15 min) (Tu et al., 2008) (Fig. 3A). The optical properties of the CpcS1-PCB adduct are intermediate between free bilins and native phycocyanins: it fluoresces weakly, and the absorption is increased only moderately (i.e. about twofold). PCB is boundrelatively weakly, as might be expected for an intermediate: it is retained during Ni2+-affinity chromatography, but mostly lost during SDS-PAGE, tryptic digestion or mass-spectral analysis. It is unclear, therefore, if a weak, covalent bond or a relatively stable non-covalent bond is formed during the catalytic cycle. In the latter case, the small amounts of covalently bound PCB seen on SDS-PAGE would constitute a side product. The strongest evidence so far for a genuine covalent bond comes from the spectrophotometric absorption of the urea-denatured intermediate: it matches that of denatured PC and is at much shorter wavelengths than that of free PCB. CpcS1 also catalyses the addition of small molecules to PCB (J.M. Tu, S. Böhm, K.H. Zhao, H. Scheer, unpublished). Adducts with thiols and imidazole were isolated after incubation with PCB and CpcS1, and mercaptoethanol is also able to cleave PCB from the CpcS1 adduct. Thiols can add spontaneously to the 3-ethylidene group (Köst et al., 1975) or, at much higher thiol concentrations, to the central methine bridge of PCB (Kufer and Scheer, 1982). To our knowledge, spontaneous formation of an imidazole adduct has not been reported, but its formation by the lyase is intriguing, because conserved histidines have been found in several lyases (Zhao et al., 2005; 2006). Furthermore, a histidine is frequently found next to the binding cysteine of plant-type phytochromes, and even conserved in several bacterial phytochromes with N-terminal binding site (Wu and Lagarias, 2000; Lamparter, 2004). Chromophorylation by CpcS1 might then involve a histidine-bound intermediate. Because, as pointed out to us by L. Moroder (Martinsried), imidiazolides are relatively labile and can be cleaved by thiols (Shaltiel, 1967), this could be a model for the reaction catalysed by CpcS1.


Biliprotein maturation: the chromophore attachment.

Scheer H, Zhao KH - Mol. Microbiol. (2008)

Reaction schemes of the S-type (top) and isomerizing E/F-type lyases (bottom). Non-covalent chromophore binding is indicated by broken lines, covalent binding by solid lines and colouration of both the chromophore and the protein. Intermediates (Pxxx) are named according to their absorption maxima at xxx nm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2327270&req=5

fig03: Reaction schemes of the S-type (top) and isomerizing E/F-type lyases (bottom). Non-covalent chromophore binding is indicated by broken lines, covalent binding by solid lines and colouration of both the chromophore and the protein. Intermediates (Pxxx) are named according to their absorption maxima at xxx nm.
Mentions: CpcS1 is a relatively simple system. It is active as a monomer, and does not require cofactors. A more complex situation has been found with S-type lyases from other organisms classified as group I, where the concerted action of two subunits (CpcS and CpcU) is required (Saunée et al., 2008; Shen et al., 2008). CpcS1 from Nostoc binds the chromophore rapidly (∼0.1 s) and reversibly, and then transfers it in a much slower, irreversible reaction to the apoproteins (∼15 min) (Tu et al., 2008) (Fig. 3A). The optical properties of the CpcS1-PCB adduct are intermediate between free bilins and native phycocyanins: it fluoresces weakly, and the absorption is increased only moderately (i.e. about twofold). PCB is boundrelatively weakly, as might be expected for an intermediate: it is retained during Ni2+-affinity chromatography, but mostly lost during SDS-PAGE, tryptic digestion or mass-spectral analysis. It is unclear, therefore, if a weak, covalent bond or a relatively stable non-covalent bond is formed during the catalytic cycle. In the latter case, the small amounts of covalently bound PCB seen on SDS-PAGE would constitute a side product. The strongest evidence so far for a genuine covalent bond comes from the spectrophotometric absorption of the urea-denatured intermediate: it matches that of denatured PC and is at much shorter wavelengths than that of free PCB. CpcS1 also catalyses the addition of small molecules to PCB (J.M. Tu, S. Böhm, K.H. Zhao, H. Scheer, unpublished). Adducts with thiols and imidazole were isolated after incubation with PCB and CpcS1, and mercaptoethanol is also able to cleave PCB from the CpcS1 adduct. Thiols can add spontaneously to the 3-ethylidene group (Köst et al., 1975) or, at much higher thiol concentrations, to the central methine bridge of PCB (Kufer and Scheer, 1982). To our knowledge, spontaneous formation of an imidazole adduct has not been reported, but its formation by the lyase is intriguing, because conserved histidines have been found in several lyases (Zhao et al., 2005; 2006). Furthermore, a histidine is frequently found next to the binding cysteine of plant-type phytochromes, and even conserved in several bacterial phytochromes with N-terminal binding site (Wu and Lagarias, 2000; Lamparter, 2004). Chromophorylation by CpcS1 might then involve a histidine-bound intermediate. Because, as pointed out to us by L. Moroder (Martinsried), imidiazolides are relatively labile and can be cleaved by thiols (Shaltiel, 1967), this could be a model for the reaction catalysed by CpcS1.

Bottom Line: The discovery of new activities for the latter lyases, and of new types of lyases, have reinvigorated research activities in the subject.So far, work has mainly concentrated on cyanobacterial phycobiliproteins.Methodological advances in the process, however, as well as the finding of often large numbers of homologues, opens new possibilities for research on the subsequent assembly/disassembly of the phycobilisome in cyanobacteria and red algae, on the assembly and organization of the cryptophyte light-harvesting system, on applications in basic research such as protein folding, and on the use of phycobiliproteins for labelling.

View Article: PubMed Central - PubMed

Affiliation: Department Biologie I, Universität München, Menzinger Strasse 67, D-80638 München, Germany.

ABSTRACT
Biliproteins are a widespread group of brilliantly coloured photoreceptors characterized by linear tetrapyrrolic chromophores, bilins, which are covalently bound to the apoproteins via relatively stable thioether bonds. Covalent binding stabilizes the chromoproteins and is mandatory for phycobilisome assembly; and, it is also important in biliprotein applications such as fluorescence labelling. Covalent binding has, on the other hand, also considerably hindered biliprotein research because autocatalytic chromophore additions are rare, and information on enzymatic addition by lyases was limited to a single example, an EF-type lyase attaching phycocyanobilin to cysteine-alpha84 of C-phycocyanin. The discovery of new activities for the latter lyases, and of new types of lyases, have reinvigorated research activities in the subject. So far, work has mainly concentrated on cyanobacterial phycobiliproteins. Methodological advances in the process, however, as well as the finding of often large numbers of homologues, opens new possibilities for research on the subsequent assembly/disassembly of the phycobilisome in cyanobacteria and red algae, on the assembly and organization of the cryptophyte light-harvesting system, on applications in basic research such as protein folding, and on the use of phycobiliproteins for labelling.

Show MeSH
Related in: MedlinePlus