Event Information
EXPANDING THE SYNTHETIC UTILITY OF KDPG ALDOLASE
- Abstract:
- An understanding of structure-function relationships of proteins facilitates design or redesign of enzymes to carry out synthetically useful reactions. Efforts to understand the relationship between the primary amino acid sequence of an enzyme and the activity of the fully folded protein have been made in the context of KDPG aldolase. Both rational mutagenesis and directed evolution approaches were used to probe the structural basis of catalysis of this enzyme.
A novel bacterial in vivo selection for evolved pyruvate aldolase activity has been developed. 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. Enzyme catalyzed retro-aldol cleavage of a 2-keto-4-hydroxy-butyrate adduct liberates pyruvate and rescues the host cells; this rescue mechanism is the basis for the selection. An initial round of selection against 2-keto-4-hydroxy-octanoate (KHO), a non-substrate for wild-type E. coli KDPG aldolase, identified 5 mutants that convert this substrate. All five mutants show vastly improved activity over the wild-type enzyme.
Finally, the syntheses of natural and unnatural substrates are described, including a new synthetic route to the 2-keto-4-hydroxy-butyrate moiety. This new methodology provides access to substrates that were previously unavailable.
Ph.D. Defense Examination Seminar
Student Exams Seminar