In Vitro Evolution to Diversify an Enzyme's Specificity

体外进化使酶的特异性多样化

• 批准号: 0109668
• 负责人: Ichiro Matsumura
• 金额: $ 33万
• 依托单位: Emory University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-10-01 至 2005-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0109668&HistoricalAwards=false
• 关键词: Vitro; Evolution; Diversify; Enzyme; Specificity

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.

该项目的目标是了解酶如何进化以识别新的底物。对适应性进化的更好理解将解释生命的复杂分子机制是如何产生的，并使工业或医学应用中新型催化剂的人工选择成为可能。理论上，酶可以通过以下有序机制适应新的底物：1）破坏活性位点构象稳定性的突变最初会扩大底物特异性； 2）活性位点的灵活性使得突变的发生能够引入与新底物的新相互作用； 3）稳定新的生产活性位点构象的突变进一步提高了催化活性。这一假设将通过指导一种称为 β-葡萄糖醛酸酶 (GUS) 的模型酶的进化来检验。编码GUS的基因会随机突变。所得文库将在大肠杆菌群体中表达，并筛选在与新型底物类似物的反应中表现出增强的催化活性的变体酶。相应的等位基因将被分离并随机突变以进行另一轮筛选。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52,000,000倍。该过程将持续进行，直到与野生型 lacZ β-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途径发生的结构和功能变化，并推断出适应的规则。