Attachment of Noncognate Chromophores to CpcA of Synechocystis sp PCC 6803 and Synechococcus sp PCC 7002 by Heterologous Expression in Escherichia coli
Attachment of Noncognate Chromophores to CpcA of Synechocystis sp PCC 6803 and Synechococcus sp PCC 7002 by Heterologous Expression in Escherichia coli
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DOI：
10.1021/bi200307s
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发表时间：
2011-06-07
期刊：
影响因子：
2.9
通讯作者：
Bryant, Donald A.
中科院分区：
文献类型：
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作者：
Alvey, Richard M.;Biswas, Avijit;Bryant, Donald A.
许多蓝细菌使用颜色鲜艳的多亚基大分子结构（称为藻胆体）作为天线来增强光合作用的光捕获。最近的研究已经定义了合成藻胆蛋白发色团的酶以及将这些发色团附着到其同源脱辅基蛋白上的许多藻胆蛋白裂解酶。通过使用大肠杆菌中的异源表达系统检查了藻蓝蛋白 a 亚基 (CpcA) 结合替代线性四吡咯发色团的能力。当大肠杆菌菌株表达来自拟南芥的 3Z-藻蓝蛋白：铁氧还蛋白氧化还原酶 (PcyA)、3Z-藻蓝蛋白：铁氧还蛋白氧化还原酶 (HY2) 或来自肌病毒的藻红蛋白合酶 (PebS) 时，会产生藻蓝蛋白、植物色素或藻红蛋白。分别为P-SSM4。来自集胞藻属的 CpcA。 PCC 6803 或聚球藻属 sp。 PCC 7002 在这些菌株中与来自 Noctoc sp 的藻蓝蛋白 a 亚基藻蓝蛋白裂解酶 CpcE/CpcF 或藻红蓝蛋白 a 亚基藻蓝蛋白异构化裂解酶 PecE/PecF 共表达。 PCC 7120。两种裂解酶都能够将三种不同的线性四吡咯发色团连接至 CpcA；因此，使用该系统可以产生多达六种不同的 CpcA 变体，每种变体都具有独特的发色团。其中一种发色团，称为植物紫胆素，尚未在自然状态下观察到。重组蛋白具有意想不到的和潜在有用的特性，其中包括非常高的荧光量子产率和光化学活性。使用嵌合裂解酶PecE/CpcF和CpcE/PecF来表明将藻蓝蛋白转化为藻紫红蛋白的异构化活性存在于PecF而不是PecE。最后，描述了重组藻蓝蛋白 R-PCIII 的光谱特性，其中 CpcA 亚基携带藻红蛋白发色团。
Many cyanobacteria use brilliantly pigmented, multisubunit macromolecular structures known as phycobilisomes as antenna to enhance light harvesting for photosynthesis. Recent studies have defined the enzymes that synthesize phycobilin chromophores as well as many of the phycobilin lyase enzymes that attach these chromophores to their cognate apoproteins. The ability of the phycocyanin a-subunit (CpcA) to bind alternative linear tetrapyrrole chromophores was examined through the use of a heterologous expression system in Escherichia coli. E. coli strains produced phycocyanobilin, phytochromobilin, or phycoerythrobilin when they expressed 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), 3Z-phytochromobilin:ferredoxin oxidoreductase (HY2) from Arabidopsis thaliana, or phycoerythrobilin synthase (PebS) from the myovirus P-SSM4, respectively. CpcA from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 was coexpressed in these strains with the phycocyanin a-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin a-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Both lyases were capable of attaching three different linear tetrapyrrole chromophores to CpcA; thus, up to six different CpcA variants, each with a unique chromophore, could be produced with this system. One of these chromophores, denoted phytoviolobilin, has not yet been observed naturally. The recombinant proteins had unexpected and potentially useful properties, which included very high fluorescence quantum yields and photochemical activity. Chimeric lyases PecE/CpcF and CpcE/PecF were used to show that the isomerizing activity that converts phycocyanobilin to phycoviolobilin resides with PecF and not PecE. Finally, spectroscopic properties of recombinant phycocyanin R-PCIII, in which the CpcA subunits carry a phycoerythrobilin chromophore, are described.