Evolution of highly active enzymes by homology-independent recombination
Evolution of highly active enzymes by homology-independent recombination
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DOI：
10.1073/pnas.0504556102
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发表时间：
2005-07-19
影响因子：
11.1
通讯作者：
Georgiou, G
中科院分区：
文献类型：
--
作者：
Griswold, KE;Kawarasaki, Y;Georgiou, G
Theta类GST酶hGSTT1-1(人GST theta-1-1)和rGSTT2-2(大鼠GST theta-2-2)具有54.3%的氨基酸同源性，并表现出不同的底物特异性。使用与同源无关的技术[用于产生杂交酶的增量截断(瘙痒)和抓挠]和低同源性技术(重组依赖的指数放大PCR)来创建包含交叉(C/O)的嵌合酶文库，这些嵌合酶位于DNA家族改组无法到达的位置。使用荧光rGSTT2-2特异底物7-氨基-4-氯甲基香豆素的高通量流式细胞仪筛选导致分离出具有一个或两个C/O的活性变异体。其中一种酶SCR23(与hGSTT1-1的同源性为83%)是由一个基因编码的，该基因与rGSTT2-2的相应序列交换了hGSTT1-1的螺旋4和5。与亲本相比，该变异体的选择底物具有更好的k(CAT)，并且还显示了谷胱甘肽与乙氰酸的偶联活性，这是一种两种亲本酶都不识别的化合物。这些结果突出了组合同源非依赖和低同源重组方法在产生独特的、高活性的酶方面的力量，并提出了一种可能的酶“人性化”的方法。
The theta-class GST enzymes hGSTT1-1 (human GST theta-1-1) and rGSTT2-2 (rat GST theta-2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C/Os) at positions not accessible by DNA family shuffling. High-throughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C/Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k(cat) with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and low-homology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme "humanization."