The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1
The biliverdin chromophore binds covalently to a conserved cysteine residue in the N-terminus of Agrobacterium phytochrome Agp1
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DOI：
10.1021/bi035693l
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发表时间：
2004-03-30
期刊：
影响因子：
2.9
通讯作者：
Abian, J
中科院分区：
文献类型：
--
作者：
Lamparter, T;Carrascal, M;Abian, J
光敏色素是广泛分布的胆蛋白光感受器。典型地，发色团在自催化裂解酶反应期间变成共价连接到蛋白质。植物和蓝细菌光敏色素将胆色素与环A亚乙基侧链结合，而其他细菌光敏色素利用胆绿素作为发色团，其具有乙烯基环A侧链。对于农杆菌光敏色素Agp 1，定点诱变提供的证据表明，胆绿素是绑定到半胱氨酸20。这种半胱氨酸在细菌同系物中高度保守，但其作为附着位点的作用尚未得到证实。因此，我们对蛋白水解全肽片段进行了质谱研究。为此，Agp 1表达载体被重新工程化以产生具有N-末端亲和标签的蛋白质。在蛋白水解之后，发色团与ca. 5 kDa的片段，显示附着位点位于靠近N-末端。用纯化的色肽进行的质谱分析证实了半胱氨酸20作为胆绿素附着位点的作用。我们还通过定点突变分析了高度保守的组氨酸250的作用。同源氨基酸在植物光敏色素中起着重要但尚未确定的作用，并已被提议作为异常球菌光敏色素的发色团附着位点。我们发现，在Agp 1，这个氨基酸是共价连接，但需要紧密的发色团-蛋白质相互作用。
Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.