Divergent Evolution of Enzymatic Activity: Functional Assignment and in Vitro Evolution in the MLE Subgroup of the Enolase Superfamily
Schmidt, Dawn Marie Zelley
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https://hdl.handle.net/2142/84800
Description
Title
Divergent Evolution of Enzymatic Activity: Functional Assignment and in Vitro Evolution in the MLE Subgroup of the Enolase Superfamily
Author(s)
Schmidt, Dawn Marie Zelley
Issue Date
2003
Doctoral Committee Chair(s)
Gerlt, John A.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Chemistry, Biochemistry
Language
eng
Abstract
The mechanistically-diverse members of the enolase enzyme superfamily provide an opportunity to understand generalized strategies for the divergent evolution of new enzymatic function, particularly within the context of enzymes containing the ubiquitous (beta/alpha)8-barrel fold. We have used the paradigm of the enolase superfamily and genomic context to functionally assign unknown proteins as L-Ala-D/L-Glu epimerases in several bacterial organisms, and studied those proteins from Escherichia coli and Bacillus subtilis in detail. The discovery of the L-Ala-D/L-Glu epimerases expands the repertoire of reactions catalyzed by the enolase superfamily, and highlights the usefulness of enzyme family relationships in assignment of function in newly sequenced genomes. In an effort to probe our understanding of the structure-function relationships in the enolase superfamily, in vitro evolution experiments were performed with the intent of converting the activity of one superfamily member to that of another enolase superfamily member. By rational design, a single amino acid change in the L-Ala-D/L-Glu epimerase (AEE) from E. coli was sufficient to evolve o-succinylbenzoate synthase (OSBS) activity in that enzyme. Subsequent directed evolution of the promiscuous AEE/OSBS single mutant sought to improve the level of OSBS activity. Directed evolution experiments also accomplished the evolution of OSBS activity by a single amino acid change in the muconate-lactonizing enzyme II (MLE II) from Pseudomonas sp . P54. The single amino acid changes in the AEE and the MLE II mutants are homologous in position, located in the active site at the end of the eighth beta-strand of the (beta/alpha)8-barrel. Substrate design was used to identify possible substrates for a third activity for the MLE II mutant, 1,1-proton transfer. The location of the mutation conferring the evolved OSBS activity upon AEE and MLE II at the end of the eighth beta-strand of the (beta/alpha) 8-barrel highlights the inherent functional plasticity of the barrel; the active site residues located at the C-terminal ends of the beta-strands of the barrel can be varied independently of one another and the surrounding structure of the barrel. This in vitro evolution of enzymatic activity likely mimics the process by which Nature evolves new activities, by proceeding through promiscuous intermediates.
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