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首页> 外文期刊>Journal of Molecular Biology >In vitro evolution of beta-glucuronidase into a beta-galactosidaseproceeds through non-specific intermediates
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In vitro evolution of beta-glucuronidase into a beta-galactosidaseproceeds through non-specific intermediates

机译：β-葡糖醛酸糖苷酶通过非特异性中间体在体外进化为β-半乳糖苷酶

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摘要

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.
机译：大肠杆菌β-葡萄糖醛酸苷酶（GUS）在体外进化为催化β-半乳糖苷底物水解的效率（k（cat）/ Km）是野生型的500倍，特异性转化为5200万倍。在三轮DNA改组和筛选中分离出的32个克隆中重复出现的氨基酸置换被定位到活性位点。通过将这些突变单独或组合引入野生型gusA基因中，研究了这些突变的功能后果。纯化的突变蛋白在与一系列底物类似物的反应中的动力学行为表明，四个突变说明了底物特异性的变化，并且它们是协同的。与野生型和进化形式不同，进化中间体对与葡糖醛酸苷或半乳糖苷不同的底物表现出广泛的特异性。这些结果与“补缀品”假说是一致的，该假说假定现代酶与祖先具有广泛的特异性。

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