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

Abstract

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...

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

“…This elution order could be estimated from the calculated mass of the tryptic peptides, with a-Cys-81 at 2,337.6 Da and b-Cys-81 at 852.0 Da. This was consistent with results from Zhao et al (2007). The identical retention time of the rAPC and native APC digestions suggested that the binding site of PCB in rAPC was identical to the native protein.…”

“…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.…”

“…Their pioneering work used a dual plasmid system for PCB biosynthesis and attachment to apo-phycobiliprotein subunits using various lyases, and established the feasibility of producing holo-phycobiliproteins in a heterologous host. Using this strategy, Zhao et al (2007) expressed APC a and b subunits (ApcA and ApcB) with covalently bound bilins in E. coli. Native phycobiliproteins usually form an ab heterodimer primary assembly unit, and then assemble into a higher structure.…”

Catalytic Mechanism of S-type Phycobiliprotein Lyase

“…The formation of chromophorylated phycobiliprotein was detected by the emission at 645 nm for PCB-containing chromoproteins (15), and at 575 nm for PEB-containing chromoproteins (13). Complete spectra in the 350 to 700 nm range were recorded to verify correct attachment and the absence of spontaneous addition products (6).…”

“…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).…”