SUMMARY (from the funded grant application) Metabolic enzymes in cells rarely function in isolation, Often their activities are coordinated by physical or covalent association with each other and cellular structures, A consequence of these associations is that metabolic intermediates do not equilibrate with the cellular milieu and are instead channeled between enzyme active sites, Despite the widespread recognition that protein-protein interactions are ubiquitous, the molecular mechanisms of substrate channeling remain obscure, and the impact of channeling at the cellular and organismal levels is largely unknown, We seek to narrow these knowledge gaps by exploring substrate channeling within and between the enzymes of praline catabolism, The proposed experiments will explore long-distance, allosteric communication between the active sites of the bifunctional enzyme PutA using kinetic crystallography, assess the contributions of substrate channeling to bacterial fitness and pathogenesis, and determine the first structure of a novel bifunctional enzyme that moonlights as a transcriptional repressor using cryo-EM.