Biogenesis of Phycobiliproteins

Abstract

The Synechococcus sp. PCC 7002 genome encodes three genes, denoted cpcS-I, cpcU, cpcV, with sequence similarity to cpeS. CpcS-I copurified with His 6 -tagged (HT) CpcU as a heterodimer, CpcSU. When CpcSU was assayed for bilin lyase activity in vitro with phycocyanobilin (PCB) and apophycocyanin, the reaction product had an absorbance maximum of 622 nm and was highly fluorescent ( max ‫؍‬ 643 nm). In control reactions with PCB and apophycocyanin, the products had absorption maxima at 635 nm and very low fluorescence yields, indicating they contained the more oxidized mesobiliverdin (Arciero, D. M., Bryant, D. A., and Glazer, A. N. (1988) J. Biol. Chem. 263, 18343-18349). Tryptic peptide mapping showed that the CpcSU-dependent reaction product had one major PCB-containing peptide that contained the PCB binding site Cys-82. The CpcSU lyase was also tested with recombinant apoHT-allophycocyanin (aporHT-AP) and PCB in vitro. AporHT-AP formed an ApcA/ApcB heterodimer with an apparent mass of ϳ27 kDa. When aporHT-AP was incubated with PCB and CpcSU, the product had an absorbance maximum of 614 nm and a fluorescence emission maximum at 636 nm, the expected maxima for monomeric holo-AP. When no enzyme or CpcS-I or CpcU was added alone, the products had absorbance maxima between 645 and 647 nm and were not fluorescent. When these reaction products were analyzed by gel electrophoresis and zinc-enhanced fluorescence emission, only the reaction products from CpcSU had PCB attached to both AP subunits. Therefore, CpcSU is the bilin lyase-responsible for attachment of PCB to Cys-82 of CpcB and Cys-81 of ApcA and ApcB.

CpeS Is a Lyase Specific for Attachment of 3Z-PEB to Cys82 of β-phycoerythrin from Prochlorococcus marinus MED4

“…are ␤Cys 82 -DHBV and most likely a ␤Cys 82 -PEB with an altered, incorrect configuration or conformation. Indeed, nonenzymatic addition of bilins to other phycobiliproteins was shown to be inefficient and to produce oxidized side products (11,35,38,39,46,53). In the case of CpcA, CpcB, PecA, and PecB from Mastigocladus laminosus, PCB adds spontaneously to the correct Cys 84 binding site but in a non-native configuration (54).…”

“…Until now, only members of groups CpcS-I, CpcS-III, and CpcU were well (38,39,46). 4 While the function of CpcV group lyases is enigmatic, S-type lyases of groups CpeS and CpeU were speculated to attach PEB to PE subunits, due to their encoding in gene clusters associated to PE maturation (45).…”

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

“…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.…”

Modular generation of fluorescent phycobiliproteins

“…9,10 This latter approach required the multi-plasmid-based production of: (i) the apoprotein, (ii) heme oxygenase, (iii) one or more reductases required to generate the chromophore from biliverdin, and (iv) one or more lyase(s). [19][20][21] A somewhat simplified approach reducing the number of plasmids required for transformants capable of producing biliproteins has recently been demonstrated. 22 It is based on expressing the structural gene fused to genes coding for chromophore biosynthesis from endogenous heme.…”