The Cyanobacteriochrome, TePixJ, Isomerizes Its Own Chromophore by Converting Phycocyanobilin to Phycoviolobilin
The Cyanobacteriochrome, TePixJ, Isomerizes Its Own Chromophore by Converting Phycocyanobilin to Phycoviolobilin
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DOI：
10.1021/bi101626t
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发表时间：
2011-02-15
期刊：
影响因子：
2.9
通讯作者：
Ikeuchi, Masahiko
中科院分区：
文献类型：
--
作者：
Ishizuka, Takami;Kamiya, Ayumi;Ikeuchi, Masahiko
蓝藻趋光性调节蛋白TePixJ是蓝藻色素亚家族的一员，它结合紫藻素（PVB）作为发色团，并在蓝光吸收（Pb）和绿光吸收（Pg）形式之间表现出可逆的光转换。我们在体内和体外重建了pvb结合光活性全配合物。载脂蛋白和藻蓝胆素（PCB）在大肠杆菌中的共表达（体内重组）产生PCB结合和pvb结合的全蛋白混合物。载子蛋白与合成的PCB在体外重组后迅速生成光活性复合物，该复合物与PCB共价结合，并在紫外-可见光谱（λ (max)值分别为430和545 nm）下表现出两种物质之间的部分可逆光转化。进一步的孵育产生了PCB到PVB的缓慢异构化，并伴随着光反应性的改善。定点突变证实，Cys522和第二个保守的Cys494对于光活性复合物的组装都是必不可少的。傅里叶变换红外光谱（FTIR）显示了绿光诱导交联和蓝光诱导释放的巯基，可能是Cys494。这些结果表明，Pb/ pg型蓝藻色素TePixJ的组装至少需要三个步骤：(i) PCB的快速稳定的色磷酸化，（ii）额外的光可逆的色磷酸化，以及（iii）随后PCB到PVB的缓慢异构化。除了已知的Cys522自溶酶活性和光可逆异构酶活性（PCB的C15和C16的Z和E异构体）外，TePixJ的GAF结构域似乎还有其他作用：作为异构酶（将PCB转化为PVB）和光可逆自溶酶，具有第二个保守的Cys残基。
The cyanobacterial phototaxis regulator protein, TePixJ, is a member of the subfamily of cyanobacteriochromes that binds phycoviolobilin (PVB) as a chromophore and exhibits reversible photoconversion between blue light-absorbing (Pb) and green light-absorbing (Pg) forms. We reconstituted the PVB-binding photoactive holocomplex in vivo and in vitro. Coexpression of the apoprotein and phycocyanobilin (PCB) in Escherichia coli (in vivo reconstitution) produced a mixture of the PCB-bound and PVB-bound holoproteins. Reconstitution in vitro of the apoprotein and synthetic PCB quickly generated a photoactive complex, which covalently bound PCB and exhibited partially reversible photoconversion between two species by UV-vis spectroscopy (with a lambda(max) values of 430 and 545 nm). Further incubation produced slow isomerization of PCB to PVB with concomitant improvement of photoreactivity. Site-directed mutagenesis confirmed that Cys522, and a second conserved Cys (Cys494), are both essential for the assembly of the photoactive complex. Fourier transform infrared (FTIR) spectroscopy revealed green light-induced cross-linking, and blue light-induced release, of a thiol group, possibly that of Cys494. These results suggest that the Pb/Pg-type cyanobacteriochrome TePixJ is assembled in at least three steps: (i) rapid and stable chromophorylation of PCB, (ii) additional photoreversible chromophorylation, and (iii) subsequent slow isomerization of PCB to PVB. In addition to its known autolyase activity with Cys522 and photoreversible isomerase activity (of the Z and E isomers at C15 and C16 of PCB), the GAF domain of TePixJ therefore appears to have other roles: as an isomerase (converting PCB to PVB) and as a photoreversible autolyase with a second conserved Cys residue.