In Vitro Evolution to Diversify an Enzyme's Specificity

这个项目的目标是了解酶如何进化来识别新的底物。更好地理解适应性进化将解释复杂的生命分子机制是如何产生的，并使人工选择用于工业或医疗应用的新型催化剂成为可能。酶理论上可以通过以下有序机制适应新的底物：1)破坏活性部位构象不稳定的突变最初扩大了底物专一性；2)活性部位的灵活性使突变的发生引入了与新底物的新的相互作用；3)稳定新的生产活性部位构象的突变进一步提高了催化活性。这一假说将通过指导一种名为β-葡萄糖醛酸苷酶(GUS)的模型酶的进化来检验。编码GUS的基因将随机突变。所得到的文库将在大肠杆菌群体中表达，并筛选在与新底物类似物反应中表现出更高催化活性的变异酶。相应的等位基因将被分离并随机突变，用于下一轮筛查。经过三轮进化，GUS的β-半乳糖苷酶活性已经提高了500倍，特异性倒置了5200万倍。这一过程将继续进行，直到与野生型LacZβ-半乳糖苷酶的催化效率和特异性相匹配。整个进化过程也将用不同的底物类似物重复。将研究和比较每条进化路径上发生的结构和功能变化，并推断出适应的规则。

The objective of this project is to learn how enzymes evolve to recognize new substrates. A better understanding of adaptive evolution will explain how the complex molecular machinery of life arose, and enable the artificial selection of novel catalysts for industrial or medical applications. Enzymes could theoretically adapt to new substrates by the following ordered mechanism: 1) mutations that destabilize the conformation of the active-site initially broaden substrate specificity; 2) active-site flexibility enables the occurrence of mutations that introduce novel interactions with the new substrate; 3) mutations that stabilize the new productive active-site conformation further improve catalytic activity. This hypothesis will be tested by directing the evolution of a model enzyme called beta-glucuronidase (GUS). The gene encoding GUS will be randomly mutated. The resulting library will be expressed in populations of Escherichia coli, and screened for variant enzymes exhibiting increased catalytic activity in reactions with a novel substrate analogue. The corresponding alleles will be isolated and randomly mutated for another round of screening. After three rounds of evolution, the beta- galactosidase activity of GUS has already increased 500-fold, with a 52,000,000-fold inversion of specificity. The process will be continued until the catalytic efficiency and specificity of the wild-type lacZ beta-galactosidase has been matched. The entire evolutionary process will also be repeated with different substrate analogues. The structural and functional changes that occur along each evolutionary pathway will be studied and compared, and the rules governing adaptation will be inferred.