Effects of Genetic Background and Gene Sharing on the Evolvability of a Promiscuous Enzyme Activity

该奖项是根据2009年《美国复苏和再投资法案》(公法111-5)提供资金的。新的酶可以通过招募具有催化新需要的反应的混杂能力的酶来进化。该项目介绍了利用大肠杆菌谷氨酰磷酸(GP)还原酶的混合N-乙酰谷氨酰磷酸(NAGP)还原酶活性来进化新活性的早期阶段。这些实验将结合体外进化方法来产生和鉴定具有增强的NAGP还原酶活性的酶，以及经典的酶学方法来表征改进的酶的催化能力。第一个目标是研究混杂活动的进化潜力如何取决于混杂活动的水平及其进化性--即是否存在一种改进的酶的进化轨迹，以避免中间体降低有机体的适合性。第二个目标是探索在基因复制提供编码酶的基因拷贝之前，保持GP还原酶活性的要求如何限制NAGP还原酶活性的出现，该基因拷贝可以进化为专门化新的功能。第三个目标是使用动力学方法剖析进化变体中NAGP还原酶活性改善的机制原因，该方法允许测量整个反应中各个步骤的速率常数。这项工作将为自生命起源以来支配微生物中新酶进化的过程提供新的见解，并继续影响暴露在新环境压力下的微生物中新酶的进化，例如有毒污染物或抗生素的存在。此外，对同源酶中混杂活性的可进化性的研究将引起蛋白质工程师的极大兴趣，他们的目标是为工业目的进化新的绿色催化剂。这项工作的其他更广泛的影响包括为高中教师举办分子进化研讨会，为少数族裔初中和高中女生夏令营开发微生物进化实验室，以及培训本科生。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). New enzymes can evolve by recruitment of enzymes that have a promiscuous ability to catalyze a newly needed reaction. This project addresses the early stages of evolution of a new activity using the promiscuous N-acetyl glutamyl phosphate (NAGP) reductase activity of E. coli glutamyl phosphate (GP) reductase. The experiments will combine in vitro evolution methods for generating and identifying enzymes with enhanced NAGP reductase activities with classical enzymological approaches for characterizing the catalytic abilities of the improved enzymes. The first objective is to examine how the potential for evolution of promiscuous activities depends on both the level of the promiscuous activity and its evolvability - i.e. whether there is an evolutionary trajectory to an improved enzyme that avoids intermediates that decrease the fitness of the organism. The second objective is to explore how a requirement to maintain GP reductase activity constrains the emergence of NAGP reductase activity before gene duplication provides a gene copy encoding an enzyme that can evolve to specialize in the new function. The third objective is to dissect the mechanistic reasons for improvements in NAGP reductase activity in evolved variants using kinetic approaches that allow measurement of rate constants for individual steps in the overall reaction.Broader Impact. This work will provide new insights into the processes that have governed the evolution of new enzymes in microbes since the origin of life and continue to affect the evolution of new enzymes in microbes exposed to novel environmental stresses such as the presence of toxic pollutants or antibiotics. In addition, work on the evolvability of promiscuous activities in homologous enzymes will be of great interest to protein engineers who aim to evolve new green catalysts for industrial purposes. Other broader impacts of this work include development of a workshop for high school teachers on molecular evolution, development of a lab on microbial evolution for a summer camp for minority middle school and high school girls, and training of undergraduates.