Defining the bilin lyase domain: lessons from the extended phytochrome superfamily.
Defining the bilin lyase domain: lessons from the extended phytochrome superfamily.
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DOI：
10.1021/bi001123z
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发表时间：
2000-10
期刊：
影响因子：
2.9
通讯作者：
Shu-Hsing Wu;J. Lagarias
中科院分区：
文献类型：
--
作者：
Shu-Hsing Wu;J. Lagarias
通过对基因组数据库的模式搜索，发现了光敏色素相关基因家族的新成员，并将其用于构建一个130-180个氨基酸基序，该基序界定了Blin裂解酶结构域，该结构域是扩展的光敏色素家族的一个亚域，足以与线性四吡咯(Bilins)共价连接。为了验证这一假说，我们选择了聚球藻的一个新的光敏色素相关基因sll0821的部分基因。扩增出编码含两个潜在胆红素结合位点的大蛋白PCC6803，并对重组蛋白进行了胆红素结合活性和光敏色素活性检测。我们的实验表明，该蛋白的两个位点，即蓝藻光敏色素2的Cph2，都具有胆碱裂解酶活性，显示出两类不同的胆林裂解酶结构域--其胆林加合物是红色的，远红光可逆的，而第二类的胆林加合物是非光致变色的。对Cph2的24 kDa片段的光致变色藻蓝胆素和荧光藻红胆素加合物的光谱分析确定，通过模式搜索鉴定的基序代表真正的胆林裂解酶结构域。对几乎所有扩展光敏色素超家族成员的Blin裂解酶结构域中高度保守的带电残基进行定点突变，发现了一个对Blin结合至关重要的谷氨酸残基。
Through pattern searches of genomic databases, new members of the growing family of phytochrome-related genes were identified and used to construct a 130-180 amino acid motif that delimits the bilin lyase domain, a subdomain of the extended phytochrome family that is sufficient for covalent attachment of linear tetrapyrroles (bilins). To test this hypothesis, portions of locus sll0821, a novel phytochrome-related gene from Synechocystis sp. PCC6803 that encodes a large protein with two potential bilin binding sites, were amplified, and the recombinant apoproteins were tested for bilin binding and phytochrome photoactivity. Our experiments indicated that both sites of this protein, termed Cph2 for cyanobacterial phytochrome 2, possessed bilin lyase activity, revealing two distinct classes of bilin lyase domains--those whose bilin adducts are red, far-red reversible and a second class whose bilin adducts are nonphotochromic. Spectroscopic analysis of photochromic phycocyanobilin and fluorescent phycoerythrobilin adducts of a 24-kDa fragment of Cph2 definitively established that the motif identified by pattern searches represents a bona fide bilin lyase domain. Site-directed mutagenesis of highly conserved charged residues within bilin lyase domains of nearly all members of the extended phytochrome superfamily has identified a glutamate residue critical for bilin binding.