PROJECT SUMMARY The major goal of the proposed work is to create, characterize, and optimize de novo binuclear mini- metalloenzymes for hydrolase activity. Hydrolytic cleavage, or the breaking apart of a molecule by the addition of water, is an essential biochemical reaction that governs cellular processes across all life kingdoms. Several rare genetic diseases come from hydrolase malfunctions, making them important to understand. Metal ions are frequently used to catalyze these reactions due to their ability to strongly polarize the O–H bond in water even at neutral pH. A small artificial enzyme called DFsc, which contains two metal ions in the active site, is capable of hydrolyzing small molecules and larger DNA substrates. Activity was observed with zinc, which is commonly used by nature, and titanium, which has no known natural function. This project seeks to (1) understand the structural and electronic factors that contribute to hydrolase activity in the DFsc system and (2) develop additional mini-metalloenzymes with metals underutilized by nature for environmentally-friendly and catalytically-efficient hydrolytic cleavage. To accomplish these goals, we will design, produce, and comprehensively characterize a series of DFsc proteins whose metal-coordinating amino acids are systematically varied. This library of proteins will be screened for hydrolase activity using DNAse and phosphatase assays and a variety of metal ions. Successful completion of these aims will result in a robust model system that will provide an understanding of the structure-activity relationships in natural and artificial metallohydrolases.