The enolase superfamily is the paradigm for understanding how new reactions result from divergent evolution from a common ancestor. Four subgroups can be recognized in the superfamily on the basis of conservation of active site residues; three are the focus of this experimental project: the 13- methylaspartate ammonia lyase (MAL) subgroup, the muconate lactonizing enzyme (MLE), and the mandelate racemase (MR) subgroup. These subgroups contain approximately 500 members, 250 of which have unknown functions. The goal is to develop an integrated sequence-structure-computation strategy for predicting the substrate specificities of the unknown members that will allow their functions to be assigned. Three Specific Aims are developed in this component project: 1) The o-succinylbenzoate synthase reaction is catalyzed by >16 different sequence motifs. The liganded structures will be determined and used by the conformational project to both evaluate and refine the modeling and docking algorithms as well as predict functional promiscuity. 2) We have identified homologues of D-glucarate dehydratase and MAL that do not catalyze these reactions. We will structurally characterize the homologues to facilitate efforts by the computational project to predict the structures of their substrates and, therefore, their functions. 3) Sequence divergent members of unknown function will be structurally characterized to facilitate prediction of the identities of the substrates by the computational project. The proteins will be screened for activities using libraries of potential substrates, thereby providing additional constraints to the computational project.