Using traditional chemistry, the production and purification of high value molecules (fragrances, flavors, dyes, antibiotics) often uses a lot of energy, has low yield of product, and generates significant quantities of hazardous wastes. The goal of this project is to make commercially important molecules using strategies that minimize waste and maximize yield, so that industry can employ these approaches and thereby improve chemical processes. Enzymes - Nature's protein catalysts - will be used instead of traditional methods, since they are produced from renewable resources, they work in water instead of organic solvents, and they are completely biodegradable. In addition to developing new reaction strategies, the three-dimensional structures of the enzymes will be determined. Computer modeling will indicate how their shapes change, which can dramatically impact their behavior. This knowledge will be put to use to improve the enzymes, making them better-suited to the needs of chemical synthesis. If successful, these efforts could result in a much more sustainable and environmentally-friendly chemical industry. The PI's active involvement with the Science for Life (S4L) program at the University of Florida will contribute directly to the development of a highly capable STEM workforce.
The first part of this project focuses on modifying enzymes to catalyze thio-Claisen condensation and beta-keto acid decarboxylation, in order to create a chiral alpha-amino-beta-hydroxy ketone in a simple, single-reactor system. Enantioselective functional group interconversion is the subject of the second part. This focuses on a family of enzymes (Old Yellow Enzymes, OYEs) that stereoselectively reduce electron-deficient alkenes. Members of this family have highly conserved amino acid sequences and essentially identical x-ray crystal structures, yet exhibit widely varying stereoselectivities that appears to be related to a protein loop that changes structure during the catalytic cycle. Molecular dynamics calculations revealed that the loops have very different dynamic properties from enzyme to enzyme, which may be isolated to a single amino acid in the loop. Site-saturation mutagenesis at this position will generate mutants that will be examined both experimentally (regarding substrate acceptance and stereoselectivity) and computationally (to identify differences in dynamic properties). Additional positions in this loop will be characterized by both experiment and computation to better define how this important structural feature constrols substrate binding. A fundamental understanding of the role that structural dynamics plays in the regulation of enzyme activity would dramatically expand the ability to design proteins with desired functionality, contributing to a sustainable biomanufacturing industry.
