Virginia W. Cornish, PhD

Our approach to creating novel enzymes is unique in that it strives to be a direct selection for catalysis and yet remain general and applicable to a wide array of reactions. The selection strategy involves two stages: (1) engineering a cell line that is capable of discriminating between the substrate and product of a reaction, and (2) introducing large libraries of >106 proteins into this cell line and identifying the proteins that can convert the substrate to the product-based on their ability to confer a selective advantage to the individual cells. The projects follow three main directions: methodology development, mechanistic studies of the basis for enzyme catalysis, and the generation of proteins that catalyze commercially important, demanding transformations. The methodology hinges on the construction of a cell line in which transcriptional activation of a gene essential for the survival of a cell is dependent on the product, but not the substrate, of a reaction. For example, in E. coli the dimerization of the DNA-binding protein 1repressor will be modified to be dependent on the small-molecule product. These projects will combine synthetic chemistry with protein design to provide small molecules that can enter the cell and regulate transcription efficiently. The ability to compare the catalytic efficiencies of large libraries of protein variants will provide a new tool for understanding the basis for enzyme catalysis. Initially, the selection strategy will be applied to P99 cephalosporinase variants not only to test existing theories of catalysis such as the role of critical active-site residues-but also to provide new insights into the basis for the impressive specifications and rate enhancements achieved by enzymes. The catalysis of chemically difficult transformations remains an important goal in organic chemistry. Natural enzymes carry out a diverse range of reactions, often both enantio-and regio-selectively. Our aim is to use the genetic selection to create synthetically useful protein catalysts for novel transformations.