The Structural Proteomics in Toronto Centre has developed high-throughput methods for purification of thousands of unknown recombinant proteins. Using this protein pipeline, we have designed an experimental approach for rapid enzymatic and structural characterization of unknown proteins. This approach involves gene cloning and over-expression in E. coli, purification and crystallization of individual proteins, and use of general enzymatic assays to screen purified proteins for enzymatic activity. The assays have relaxed substrate specificity and are designed to identify the subclass or sub-subclasses of enzymes. Further biochemical characterization of novel enzymes can be facilitated by the application of secondary screens with specific substrates. We have demonstrated the feasibility and merits of this approach for hydrolases, oxidoreductases, and transferases and have discovered enzymatic activity in over 250 proteins.

      Substrate profiling of new enzymes showed that most proteins exhibit significant substrate promiscuity. Enzymatic screening of the haloacid dehalogenase-like hydrolases from E. coli and yeast has identified many new phosphatases and disclosed the relationship between sequence similarity and substrate preference of these enzymes. Biochemical and structural characterization of the polyphosphate kinase family-2 has revealed the presence of two groups of enzymes catalyzing the polyphosphate-dependent generation of ATP from AMP and ADP. Crystal structures of novel enzymes have provided insights into molecular mechanisms of substrate selectivity and catalysis.