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）资助的。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。 本项目利用大肠杆菌的N-乙酰谷氨酰磷酸（NAGP）还原酶活性研究了一种新活性进化的早期阶段。大肠杆菌谷氨酰磷酸（GP）还原酶。 实验将结合联合收割机在体外进化的方法来产生和识别酶与增强NAGP还原酶活性的经典酶学方法来表征的催化能力的改进的酶。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可进化性-即是否存在进化轨迹以改进酶，从而避免降低生物体适应性的中间体。第二个目标是探索如何保持GP还原酶活性的要求约束NAGP还原酶活性的出现之前，基因复制提供了一个基因拷贝编码的酶，可以进化到专门的新功能。第三个目标是剖析NAGP还原酶活性改善的机制的原因，在进化的变体使用动力学方法，允许测量速率常数的个别步骤在整个反应。这项工作将为自生命起源以来控制微生物中新酶进化的过程提供新的见解，并继续影响暴露于新环境压力（如有毒污染物或抗生素的存在）的微生物中新酶的进化。此外，同源酶中混杂活性的进化性工作将对旨在为工业目的进化新的绿色催化剂的蛋白质工程师产生极大的兴趣。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讲习班，为少数民族中学和高中女生的夏令营建立微生物进化实验室，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.