Evolutionary Potential of (β/α)8-Barrels: In Vitro Enhancement of a “New” Reaction in the Enolase Superfamily†

The repertoire of reactions in the mechanistically diverse enolase superfamily is the result of divergent evolution that conserved enolization of a carboxylate anion substrate but allowed different overall reactions using different substrates. Details of the pathways for the natural evolutionary process are unknown, but the events reasonably involve (1) incremental increases in the level of the “new” reaction that would provide a selective advantage and (2) an accompanying loss of the “old” reaction catalyzed by the progenitor. In an effort to better understand the molecular processes of divergent evolution, the D297G mutant of the l-Ala-d/l-Glu epimerase (AEE) from Escherichia coli was designed so that it could bind the substrate for the o-succinylbenzoate synthase (OSBS) reaction and, as a result, catalyze that reaction [Schmidt, D. M. Z., Mundorff, E. C., Dojka, M., Bermudez, E., Ness, J. E., Govindarajan, S., Babbitt, P. C., Minshull, J., and Gerlt, J. A. (2003) Biochemistry 42, 8387−8393]. The AEE pr...