Creating new enzymes (proteins that speed up chemical reactions) is a formidable challenge for which few effective design strategies exist. However, nature frequently creates new enzymes, for example when enzymes are needed to degrade man-made chemicals and to use these as a nutrient source. This project will develop a strategy for creating new enzymes based on a fundamental understanding of both chemistry and of the natural evolution of enzymes. The project will probe the degree to which the range of naturally occurring enzymes can be expanded to deliver novel chemistries and has significant potential to contribute to the competitiveness of the chemical and pharmaceutical industries. The project will provide interdisciplinary training and preparation that will help graduate students and postdoctoral fellows to develop careers in a rapidly changing academic and professional environment.
The project extends a proof-of-concept demonstration that the catalytic functions of cytochrome P450s can accommodate synthetic precursors to new reactive intermediates as a result of the enzymes' versatile iron-heme prosthetic group. These new reactive intermediates open up the possibility of an array of enzyme-catalyzed reactions that are not known in nature. The project will explore how the non-natural reaction kinetics can be fully optimized and how the new reactions can be incorporated into metabolic pathways using a combination of in vivo and in vitro approaches. Directed evolution will be used to improve the expression of mutated cytochrome P450s, improve reaction kinetics, and perturb the rate of electron transfer for improved nitrene-transfer reaction selectivity. Detailed kinetic, structural and biochemical studies of the 'fossil record' of all evolutionary intermediates will provide fundamental insights into the molecular pathways by which new amination activities can be realized. The specific focus on intermolecular alkene aziridination and C-H amination draws on expertise in directed evolution and understanding of mechanistic similarities between P450's native chemistry and the desired new enzyme activities.
This award is funded jointly by the Systems and Synthetic Biology Program, Division of Molecular and Cellular Biosciences; the Chemistry of Life Processes Program, Division of Chemistry, and the Biocatalysis Program of the Chemical, Bioengineering, Environmental, and Transport Systems Division.
