The conversion of genetically encoded adenosine (A) to inosine (I) at the wobble position of tRNA anticodons is essential for the flow of biological information. This modification allows for a single tRNA species to recognize multiple synonymous codons containing A, C or U in the last (3'-most) position and ensures that all sense codons are appropriately decoded. While these enzymes catalyze one of the most critical and widely conserved reactions in biology, a detailed understanding has been hampered by the lack of direct high resolution structural information and the inability to perform quantitative kinetic analyses. The overall goal of the proposed work is to develop the tools and infrastructure to define the structure-function relationships of the prokaryotic and eukaryotic adenosine deaminases that act on tRNA (ADATs). In order to achieve these goals, three specific aims will be addressed: 1) High resolution structural analysis of a liganded and unliganded prokaryotic wobble-specific deaminase, 2) Detailed structural and mechanistic analysis of eukaryotic wobble-specific deaminases, and 3) Identification of structural determinants in a different substrate recognition between bacterial and eukaryotic ADAT.