In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates
In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates
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DOI：
10.1006/jmbi.2000.4259
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发表时间：
2001-01-12
影响因子：
5.6
通讯作者：
Ellington, AD
中科院分区：
文献类型：
--
作者：
Matsumura, I;Ellington, AD
大肠杆菌β-葡萄糖醛酸酶（GUS）在体外进化为催化β-半乳糖苷底物水解的效率比野生型高500倍（k（cat）/K-m），特异性反转5200万倍。通过3轮DNA改组和筛选，将32个克隆中重复出现的氨基酸替换定位到活性位点。这些突变的功能后果进行了研究，通过将它们单独或组合到其他野生型gusA基因。纯化的突变蛋白在与一系列底物类似物反应中的动力学行为表明，四个突变占底物特异性的变化，并且它们是协同的。进化中间体，不像野生型和进化形式，表现出扩大的特异性底物不同的葡萄糖醛酸苷或半乳糖苷。这些结果与“拼凑”假说一致，该假说假定现代酶与祖先有广泛的特异性。(C)北京：科学出版社.
The Escherichia coli beta-glucuronidase (GUS) was evolved in vitro to catalyze the hydrolysis of a beta-galactoside substrate 500 times more efficiently (k(cat)/K-m) than the wild-type, with a 52 million-fold inversion in specificity. The amino acid substitutions that recurred among 32 clones isolated in three rounds of DNA shuffling and screening were mapped to the active site. The functional consequences of these mutations were investigated by introducing them individually or in combination into otherwise wild-type gusA genes. The kinetic behavior of the purified mutant proteins in reactions with a series of substrate analogues show that four mutations account for the changes in substrate specificity, and that they are synergistic. An evolutionary intermediate, unlike the wildtype and evolved forms, exhibits broadened specificity for substrates dissimilar to either glucuronides or galactosides. These results are consistent with the "patchwork" hypothesis, which postulates that modern enzymes diverged from ancestors with broad specificity. (C) 2001 Academic Press.