Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment.
Characterization of phycocyanin produced by cpcE and cpcF mutants and identification of an intergenic suppressor of the defect in bilin attachment.
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DOI：
10.1016/s0021-9258(18)41979-5
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发表时间：
1992-08
期刊：
影响因子：
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通讯作者：
R. Swanson;J. Zhou;J. Leary;T. Williams;R. de Lorimier;D. Bryant;A. Glazer
中科院分区：
文献类型：
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作者：
R. Swanson;J. Zhou;J. Leary;T. Williams;R. de Lorimier;D. Bryant;A. Glazer
通过cpcE或cpcF基因的插入失活构建的蓝细菌聚球藻属PCC 7002的突变体产生低水平的光谱可检测的藻蓝蛋白。在这些菌株中产生的大多数藻蓝蛋白似乎缺乏α亚基藻蓝胆素（PCB）发色团（Zhou，J.，加斯帕里奇湾E、Stirewalt，V.L.，de Lorimier，R.，和Bryant，D. A.（1992）J.Biol.Chem.267，16138-16145）。在突变体中产生的藻蓝蛋白的纯化揭示了两个馏分，每个具有异常吸收光谱。主要组分的胰蛋白酶肽图谱显示不存在α-84 PCB肽。来源于β亚基的两种PCB肽是正常的。不太丰富的藻蓝蛋白部分的胰蛋白酶消化物含有来自α亚基的胆色素肽家族。存在几种不同的胆色素加合物。一个主要的组成部分是中胆绿素加合物，以前描述的产品在体外反应的PCB和脱藻蓝蛋白。用cpcE突变体和cpcF突变体获得了相同的结果。与PCB和含有载脂蛋白α亚基的组分的体外反应表明，α-84胆色素附着位点未被修饰，并且能够形成加合物。观察到两种菌株的假回复突变体以高频率出现。对来自cpcE假回复突变体的藻蓝蛋白进行分析，其产生了接近野生型水平的具有携带PCB的α亚基的藻蓝蛋白，揭示了单个氨基酸取代，α-Tyr 129-Cys。该残基在迄今为止测序的所有藻蓝蛋白中是保守的，形成α-84胆色素结合位点的一部分，并且位于α-Cys 84的5 A内。通过定点诱变构建含有该取代的突变cpcA基因，并与cpcB一起沿着转化到含有插入失活cpcE的cpcBAC缺失菌株中。该菌株产生高水平的藻蓝蛋白，并且大多数α亚基在α-Cys 84处携带PCB。
Mutants of the cyanobacterium Synechococcus sp. PCC 7002 constructed by the insertional inactivation of either the cpcE or cpcF gene produce low levels of spectroscopically detectable phycocyanin. The majority of the phycocyanin produced in these strains appears to lack the alpha subunit phycocyanobilin (PCB) chromophore (Zhou, J., Gasparich, G. E., Stirewalt, V. L., de Lorimier, R., and Bryant, D. A. (1992) J. Biol. Chem. 267, 16138-16145). Purification of the phycocyanin produced in the mutants revealed two fractions each with an aberrant absorption spectrum. Tryptic peptide maps of the major fraction showed that the alpha-84 PCB peptide was absent. The two PCB peptides derived from the beta subunit were normal. Tryptic digests of the less abundant phycocyanin fraction contained a family of bilin peptides derived from the alpha subunit. Several distinct bilin adducts were present. A major component was a mesobiliverdin adduct, a previously described product of the in vitro reaction of PCB and apophycocyanin. The same results were obtained with both the cpcE mutant and the cpcF mutant. In vitro reactions with PCB and the fractions containing apo alpha subunit showed that the alpha-84 bilin attachment site was unmodified and competent for adduct formation. Pseudo-revertants of both strains were observed to arise at high frequency. Analysis of the phycocyanin from a cpcE pseudo-revertant, which produced a near wild-type level of phycocyanin with alpha subunit carrying PCB, revealed a single amino acid substitution, alpha-Tyr129—-Cys. This residue, which is conserved in all phycocyanins sequenced to date, forms part of the alpha-84 bilin binding site and lies within 5 A of alpha-Cys84. A mutated cpcA gene containing this substitution was constructed by site-directed mutagenesis and transformed, along with cpcB, into a cpcBAC deletion strain containing an insertionally inactivated cpcE. This strain produces high levels of phycocyanin and the majority of the alpha subunit carries PCB at alpha-Cys84.