Member # 1
posted 12. July 2003 08:54
Biochemistry, ASAP Article 10.1021/bi034769a S0006-2960(03)04769-X
Web Release Date: June 24, 2003
Copyright (c) 2003 American Chemical Society
Evolutionary Potential of (beta/alpha)8 Barrels: Functional Promiscuity Produced by Single Substitutions in the Enolase Superfamily
Dawn M. Z. Schmidt, Emily C. Mundorff, Michael Dojka, Ericka Bermudez, Jon E. Ness, Sridhar Govindarajan, Patricia C. Babbitt, Jeremy Minshull, and John A. Gerlt
Abstract: (beta/alpha)8-barrel domains in both the L-Ala-D/L-Glu epimerase from Escherichia coli (AEE) and the muconate lactonizing enzyme II from Pseudomonas sp. P51 (MLE II) that catalyze the o-succinylbenzoate synthase (OSBS) reaction as well as the wild-type reaction. These enzymes are members of the MLE subgroup of the superfamily, share conserved lysines on opposite sides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. A comparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutant of AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neither wild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotroph and have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEE and the E323G mutant of MLE II are each located at the end of the eighth (beta/alpha)8-barrel and alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutions relax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reaction with the assistance of the active site lysines. The generation of functionally promiscuous and mechanistically diverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution of enzymatic activities and also highlights the utility of the (beta/alpha)8-barrel as a scaffold for new function.