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 i dentified as useful tools for the organic chemist. However, their utility i s limited in part by their narrow substrate utilization. Site-directed muta genesis of various enzymes to alter their specificity has been performed fo r many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffo ldings. This approach allows random mutation of the gene acid then selects for fitness to purpose those proteins with the desired activity. To date su ch approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic resid ue been altered while activity is retained.