Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli

Background: Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene acid then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained.