Fairchild C D, Zhao J, Zhou J, Colson S E, Bryant D A, Glazer A N
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):7017-21. doi: 10.1073/pnas.89.15.7017.
Phycobiliproteins, unlike other light-harvesting proteins involved in photosynthesis, bear covalently attached chromophores. The bilin chromophores are attached through thioether bonds to cysteine residues. The cyanobacterium Synechococcus sp. PCC 7002 has eight distinct bilin attachment sites on seven polypeptides, all of which carry the same chromophore, phycocyanobilin. When two genes in the phycocyanin operon of this organism, cpcE and cpcF, are inactivated by insertion, together or separately, the surprising result is elimination of correct bilin attachment at only one site, that on the alpha subunit of phycocyanin. We have overproduced CpcE and CpcF in Escherichia coli. In vitro, these proteins catalyze the attachment of phycocyanobilin to the alpha subunit of apophycocyanin at the appropriate site, alpha-Cys-84, to form the correct adduct. CpcE and CpcF also efficiently catalyze the reverse reaction, in which the bilin from holo-alpha subunit is transferred either to the apo-alpha subunit of the same C-phycocyanin or to the apo-alpha subunit of a heterologous C-phycocyanin. The forward and reverse reactions each require both CpcE and CpcF and are specific for the alpha-Cys-84 position. Phycocyanobilin is the immediate precursor of the protein-bound bilin.
藻胆蛋白与参与光合作用的其他捕光蛋白不同，其带有共价连接的发色团。胆色素发色团通过硫醚键连接到半胱氨酸残基上。蓝藻聚球藻属（Synechococcus sp.）PCC 7002在7种多肽上有8个不同的胆色素附着位点，所有这些位点都携带相同的发色团——藻蓝胆素。当该生物体藻蓝蛋白操纵子中的两个基因cpcE和cpcF一起或分别通过插入而失活时，令人惊讶的结果是仅藻蓝蛋白α亚基上的一个位点的胆色素正确附着被消除。我们在大肠杆菌中过量表达了CpcE和CpcF。在体外，这些蛋白质催化藻蓝胆素在合适的位点α-Cys-84处与脱辅基藻蓝蛋白的α亚基结合，形成正确的加合物。CpcE和CpcF也能高效催化逆反应，即全α亚基中的胆色素被转移到同一C-藻蓝蛋白的脱辅基α亚基或异源C-藻蓝蛋白的脱辅基α亚基上。正向和逆向反应都需要CpcE和CpcF，并且对α-Cys-84位点具有特异性。藻蓝胆素是蛋白质结合胆色素的直接前体。
