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.
