To explore the functional potential of a contemporary bacterial proteome, our laboratory recently challenged Escherichia coli K-12 for growth at the expense of several metabolically inert compounds. High-level overexpression of specific genes affords an expansion of the metabolic capabilities of this bacterium by virtue of latent catalytic activities embedded within the genome. This report reveals the experimental strategies that led to the identification of these promiscuous catalysts and describes the preliminary kinetic and mechanistic characterization of these activities. The implications of these findings for the natural and directed evolution of enzymes with useful applications will be discussed.