State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
Gene. 2012 Dec 10;511(1):88-95. doi: 10.1016/j.gene.2012.09.004. Epub 2012 Sep 11.
The phycocyanin lyase CpcT1 (encoded by gene all5339) and lyase CpcS1 (encoded by gene alr0617) are capable of catalyzing the phycocyanobilin (PCB) covalently bound to the different sites of phycocyanin's and phycoerythrocyanin's β subunits, respectively. Lyase CpcS1, whose catalytic mechanism had been researched clearly, participates in the covalent coupling of phycobilin and apoprotein in the form of chaperone, and its important amino acids have been confirmed. In order to identify the functional amino acid residues of CpcT1, chemical modification was conducted to arginine, histidine, tryptophan, lysine and amino acid carboxyl of CpcT1. The results indicated that the catalytic activity of the CpcT1 was changed. After the modification of arginine, tryptophan and histidine, site-directed mutations were performed to those highly conserved amino acids which were selected by means of homologous comparison. The mutated lyase, apoprotein and the enzymes that synthesize the phycobilins were recombined in Escherichia coli (E. coli) and in vitro, yielding chromoproteins, which were detected by fluorescence and UV absorption spectrometry. The spectra were compared with that of the chromoprotein catalyzed by wild type lyase CpcT1, achieving relative specific activities of the various mutants. Meanwhile, the mutants were expressed in E. coli, and then circular dichroism structure of near-UV region was determined. The results demonstrated that H33F, W175S, R97A, C137S and C116S influence the catalytic activity of CpcT1. Being different from wild CpcT1, a great deal of α helix was involved in the structure of circular dichroism of R97A and W13S. CpcT1 or its mutants and the enzymes that synthesize the phycobilins, were reconstituted in E. coli and detected by spectra to check the bounding of lyases and PCB. The results of spectra and SDS-PAGE confirm that CpcT1 and its mutants cannot bind phycobilin, differing from the catalytic mechanism of CpcS1.
藻蓝胆素裂合酶 CpcT1（由基因 all5339 编码）和裂合酶 CpcS1（由基因 alr0617 编码）能够分别催化藻蓝胆素（PCB）与藻蓝蛋白和藻红蛋白β亚基不同位点的共价结合。裂合酶 CpcS1 的催化机制已经得到了深入研究，它以伴侣的形式参与藻胆素和脱辅基蛋白的共价偶联，并且其重要的氨基酸已经得到了确认。为了鉴定 CpcT1 的功能氨基酸残基，我们对 CpcT1 的精氨酸、组氨酸、色氨酸、赖氨酸和氨基酸羧基进行了化学修饰。结果表明，CpcT1 的催化活性发生了变化。在修饰了精氨酸、色氨酸和组氨酸后，我们对通过同源比较选择的高度保守氨基酸进行了定点突变。突变后的裂合酶、脱辅基蛋白和合成藻胆素的酶在大肠杆菌（E. coli）中重组，并在体外生成了色蛋白，通过荧光和紫外吸收光谱进行检测。将这些光谱与野生型裂合酶 CpcT1 催化的色蛋白的光谱进行比较，得出了各种突变体的相对比活性。同时，突变体在大肠杆菌中表达，然后通过近紫外区圆二色性结构确定。结果表明，H33F、W175S、R97A、C137S 和 C116S 影响 CpcT1 的催化活性。与野生型 CpcT1 不同，R97A 和 W13S 的圆二色性近紫外区结构中涉及大量的α螺旋。将 CpcT1 或其突变体与合成藻胆素的酶在大肠杆菌中重组，并通过光谱检测来检查裂合酶与 PCB 的结合情况。光谱和 SDS-PAGE 的结果证实，CpcT1 及其突变体不能结合藻胆素，这与 CpcS1 的催化机制不同。
