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

Kai-Hong Zhao,Juan Zhang,Jun-Ming Tu,Stephan Böhm,Matthias Plöscher,Lutz Eichacker,Claudia Bubenzer,Hugo Scheer,Xing Wang,Ming Zhou

doi:10.1074/jbc.m703038200

Kai-Hong Zhao, Juan Zhang + Show 8 more

Open Access

https://doi.org/10.1074/jbc.m703038200

Copy DOI

Abstract

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 beta-phycoerythrocyanin (PecB) or beta-phycocyanin (CpcB). CpcT1 is a regioselective biliprotein lyase attaching phycocyanobilin exclusively to cysteine beta155 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:alpha-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.

Highlights

Phycobilisomes, the light-harvesting antennas in Cyanobacteria and red algae, are supramolecular complexes of phycobiliproteins and linkers [1,2,3]
Estimation of chromoproteins produced in E. coli with enzymes from Nostoc PCC7120 and apoproteins from M. laminosus in the absence of any lyase and under catalysis of one or two of CpcS1, CpcT1, CpcS2, and CpcT2
Compared with enzymatic chromophore attachment to the ␣-subunits of phycobiliproteins, studies have only recently begun on the catalytic attachment of chromophores to the ␤-subunits, where the process is complicated by the presence of two or even more binding sites [1, 2, 27, 49]

Summary

EXPERIMENTAL PROCEDURES

Proteins—Cloning and expression generally followed standard procedures (Sambrook et al [28]). Lyase Activity Assay—For optimization of reconstitution the desired wild-type or mutant apoprotein PecB or CpcB (10 –50 ␮M) was mixed with one or more of CpcS1, CpcT1, NblB, CpcS2, CpcT2, CpcE/F, and PecE/F (10 –50 ␮M) in KPB (20 – 1000 mM, pH 6.0 –11.0) containing one or more of Tris1⁄7HCl, NaCl, mercaptoethanol, various metal ions, EDTA, Triton X-100, and glycerol and assayed as published previously [26]. Chromophore Cleavage Assay—One or two of CpcS1, CpcT1, NblB, CpcS2, CpcT2, CpcE/F, and PecE/F (2 ␮M each) were incubated with donor chromoprotein (reconstituted and purified PCB-PecB(C84A) or PCB-CpcB(C84S) (2 ␮M)), PecA (4 ␮M), and PecE/F (4 ␮M) at 37 °C for 3 h. After purification [27], the isolated chromopeptides were analyzed by mass spectrometry in positive ion mode using a Q-Tof Premier mass spectrometer (Waters Micromass Technologies, Manchester, NH) with a nanoelectrospray ionization source

RESULTS

Sequential Addition of PCB to

Chromopeptide sequence

DISCUSSION