Molecular cloning and expression analysis of a new bilin lyase: the cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHS314
Molecular cloning and expression analysis of a new bilin lyase: the cpcT gene encoding a bilin lyase responsible for attachment of phycocyanobilin to Cys-153 on the β-subunit of phycocyanin in Arthrospira platensis FACHS314
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DOI：
10.1016/j.gene.2014.04.050
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发表时间：
2014-07-10
期刊：
影响因子：
3.5
通讯作者：
Zhao, Bing-Ran
中科院分区：
文献类型：
--
作者：
Zhang, Ran;Feng, Xiao-Ting;Zhao, Bing-Ran
为了研究藻蓝蛋白13亚基的组装，首次从钝顶节旋藻FACHB 314中克隆了cpcT基因。为探讨cpcT的功能，将藻蓝蛋白13亚基和cpcT的DNA分别转化到大肠杆菌BL 21中，构建了含藻蓝胆素合成所需的血红素加氧酶1（Hox 1）和铁氧还蛋白氧化还原酶（PcyA）基因的质粒pET-hox 1-pcyA。转化菌株显示出特异的藻蓝蛋白荧光，且荧光强度比仅含藻蓝蛋白β亚基的菌株强，表明CpcT能够促进藻蓝蛋白组装产生荧光。为了研究脱辅基藻蓝蛋白和藻蓝胆素可能的结合位点，分别对β亚基的Cys-82和Cys-153进行突变，得到两种突变体。结果表明，Cys-153可能是A. Platensis FACHB 314。(C)2014爱思唯尔有限公司版权所有。
To study the assembly of phycocyanin 13 subunit, the gene cpcT was first cloned from Arthrospira platensis FACHB314. To explore the function of cpcT, the DNA of phycocyanin 13 subunit and cpcT were transformed into Escherichia coli BL21 with the plasmid pET-hox1-pcyA, which contained the genes hemeoxygenase 1 (Hox1) and ferredoxin oxidoreductase (PcyA) needed to produce phycocyanobilin. The transformed strains showed specific phycocyanin fluorescence, and the fluorescence intensity was stronger than the strains with only phycocyanin beta subunit, indicating that CpcT can promote the assembly of phycocyanin to generate fluorescence. To study the possible binding sites of apo-phycocyanin and phycocyanobilin, the Cys-82 and Cys-153 of the beta subunit were individually mutated, giving two kinds of mutants. The results show that Cys-153 maybe the active site for beta subunit binding to phycocyanobilins, which is catalyzed by CpcT in A. platensis FACHB314. (C) 2014 Elsevier B.V. All rights reserved.