Evolutionary Potential of (/)8-Barrels
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- 作者：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
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：July 22, 2003
- 年：2003
- 卷：42
- 期：28
- 页码：8387 - 8393
- 全文大小：281K
- 年卷期：v.42,no.28(July 22, 2003)
- ISSN：1520-4995
文摘
The members of the mechanistically diverse, (
/
)8-barrel fold-containing enolase superfamilyevolved from a common progenitor but catalyze different reactions using a conserved partial reaction.The molecular pathway for natural divergent evolution of function in the superfamily is unknown. Wehave identified single-site mutants of the (/)8-barrel domains in both the L-Ala-D/L-Glu epimerase fromEscherichia 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. Theseenzymes are members of the MLE subgroup of the superfamily, share conserved lysines on oppositesides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. Acomparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutantof AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neitherwild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotrophand have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEEand the E323G mutant of MLE II are each located at the end of the eighth -strand of the (/)8-barreland alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutionsrelax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reactionwith the assistance of the active site lysines. The generation of functionally promiscuous and mechanisticallydiverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution ofenzymatic activities and also highlights the utility of the (/)8-barrel as a scaffold for new function.
/)8-barrel fold-containing enolase superfamilyevolved from a common progenitor but catalyze different reactions using a conserved partial reaction.The molecular pathway for natural divergent evolution of function in the superfamily is unknown. Wehave identified single-site mutants of the (/)8-barrel domains in both the L-Ala-D/L-Glu epimerase fromEscherichia 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. Theseenzymes are members of the MLE subgroup of the superfamily, share conserved lysines on oppositesides of their active sites, but catalyze acid- and base-mediated reactions with different mechanisms. Acomparison of the structures of AEE and the OSBS from E. coli was used to design the D297G mutantof AEE; the E323G mutant of MLE II was isolated from directed evolution experiments. Although neitherwild-type enzyme catalyzes the OSBS reaction, both mutants complement an E. coli OSBS auxotrophand have measurable levels of OSBS activity. The analogous mutations in the D297G mutant of AEEand the E323G mutant of MLE II are each located at the end of the eighth -strand of the (/)8-barreland alter the ability of AEE and MLE II to bind the substrate of the OSBS reaction. The substitutionsrelax the substrate specificity, thereby allowing catalysis of the mechanistically diverse OSBS reactionwith the assistance of the active site lysines. The generation of functionally promiscuous and mechanisticallydiverse enzymes via single-amino acid substitutions likely mimics the natural divergent evolution ofenzymatic activities and also highlights the utility of the (/)8-barrel as a scaffold for new function.