Elucidation of 2-keto-3-deoxy-6-phosphogluconate aldolase and 2-deoxyribose-5-phosphate aldolase structure-function relationships via rational mutagenesis and directed evolution

This dissertation describes efforts to understand the relationship between primary amino acid sequence of an enzyme and the activity of the fully folded protein. Understanding how protein structure affects function facilitates the design or redesign of enzymes to carry out synthetically useful reactions. We consider this question in the context of two bacterial aldolases: 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase and 2-deoxyribose-5-phosphate aldolase (DERA). Both rational mutagenesis and directed evolution approaches were used to probe the structural basis of catalysis by these enzymes.; A distantly related T. maritima homologue of the E. coli KDPG aldolase was identified cloned and characterized. Kinetic parameters for the enzyme were evaluated in the retroaldol cleavage direction with the natural substrate KDPG and in the synthetic direction with a range of electrophilic aldehydes. In order to explore the structural basis for differences between the E. coli and T. maritima KDPG aldolases, site-directed mutants of the T. maritima enzyme were created and the activities of these mutants were evaluated. Results of these studies are interpreted in light of a recently solved crystal structure of the T. maritima KDPG aldolase.; In a second set of experiments, the development of a novel bacterial in vivo selection for evolved pyruvate aldolase activity is described. The selection exploits the E. coli PB25 cell line, which lacks pyruvate kinase activity and thus requires exogenous pyruvate when grown on a five carbon sugar as the sole carbon source. Rescue of the auxotroph by retro-aldol cleavage of a 2-keto-4-hydroxybutyrate adduct to produce pyruvate provides the basis for the selection. An initial round of selection against 2-keto-4-hydroxyoctonate (KHO), a non-substrate for wild-type E. coli KDPG aldolase, identified seven mutants that convert this unnatural substrate. Several mutants showed a decrease in Km against the KHO substrate versus the wild-type enzyme.; Finally, the structural basis of aldolase activity was considered in experiments comparing the E. coli KDPG and 2-deoxyribose-5-phosphate aldolases. These enzymes show remarkable similarities with relation to overall structure and positioning of the key catalytic lysine and apparently achieve orthogonal substrate specificities through subtle differences in the placement of catalytic bases. Site-directed mutants were created to interchange the positions of the key basic residues in the two enzymes. Evolutionary studies were also carried out on DERA to further explore the structural basis of substrate specificity in this enzyme. Characterization of the site-directed mutants and results from the directed evolution studies are reported.