My goal is to understand how new catalytic mechanisms evolve. Current biochemistry textbooks emphasize the specificity and efficiency of extant enzymes. The changes in protein structure that create new catalytic activities are difficult to understand, even in retrospect, and nearly impossible to predict or model. My research team used functional genomics tools to identify 41 multi-copy suppressors that rescue 21 Escherichia coli auxotrophs from starvation on minimal media. Most of the selected proteins are unrelated in structure to those missing in the rescued auxotrophs, but some evince broad similarities in catalytic function. These surprising preliminary results suggest that protein multifunctionality is common and biologically relevant. Here I propose to evolve selected promiscuous enzymes in vitro, and to study the resulting changes in active-site structure and catalytic mechanism. These experiments will establish a simple structural model that will facilitate the directed evolution of new protein pharmaceuticals ("biologics") and diagnostic reagents. Public Health Relevance: Protein engineers emulate molecular evolution in the laboratory to create new protein pharmaceuticals and diagnostic reagents. The experiments described in this grant proposal will help us to better understand adaptive evolution at the molecular level, thereby accelerating the artificial evolution of clinically useful biomolecules.