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Random and rational evolution of tautomerase superfamily members : analysis and implications

P[Kappa]a is not responsible for the improved activity. Hence, stabilization of an enediolate intermediate may be important for catalysis. In the second part of this work, the Chloroflexus aurantiacus J-10-fl heterohexameric 4-OT tautomerase was employed in random and rational directed evolution studies to introduce a CaaD activity. Genetic selection and a high throughput screening assay were used to identify mutants. Genetic selection was unsuccessful due to plasmid instability in the host strain. A small mutant library in the screening assay precluded the identification of any mutants with CaaD activity. Finally, rational design using structure-function relationships was investigated and a single mutant was discovered for hh4-OT that incorporated CaaD activity into the enzyme, the [alpha]L9R hh4-OT, this mutant has been characterized kinetically and the evolutionary implications for the tautomerase superfamily are described. / text

Identiferoai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/23942
Date10 April 2014
CreatorsDarty, Joseph Edward
Source SetsUniversity of Texas
LanguageEnglish
Detected LanguageEnglish
TypeThesis
Formatelectronic
RightsCopyright is held by the author. Presentation of this material on the Libraries' web site by University Libraries, The University of Texas at Austin was made possible under a limited license grant from the author who has retained all copyrights in the works.

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