Abstract

The (beta/alpha)8 (or,TIM) barrel provides an attractive scaffold for the directed evolution of new catalytic activities. I am attempting to evolve chorismate mutase (CM) activity in o-succinylbenzoate synthase (OSBS), which contains a TIM barrel domain. To effect this change in activity, I will make DNA libraries that encode OSBS variants, with specific active site residues changed to a random distribution of standard amino acids. To identify active CMs, these libraries will be subjected to in vivo selections for CM activity. The properties of a laboratory-evolved CM may provide insight into the mechanism of this interesting reaction. To assess the suitability of the TIM barrel fold for this strategy of directed divergent evolution, the libraries will be screened for the in vivo production of soluble protein. To assess the enzymatic versatility of the TIM barrel fold, the libraries will also be subjected to selections for catalysis of other reactions, such as prephenate dehydratase and tyrosine epimerase activities. This research examines the feasibility of using a few strategically placed mutations to create a new catalytic function in a TIM-barrel scaffold. Further, this study aims to develop improved general strategies for obtaining tailor-made catalysts of organic reactions.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM071126-02X1
Application #
7144711
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Fabian, Miles
Project Start
2004-12-01
Project End
2006-05-31
Budget Start
2005-12-01
Budget End
2006-05-31
Support Year
2
Fiscal Year
2006
Total Cost
$7,000
Indirect Cost

  Institution

Name
Swiss Federal Institute of Tech (Eth Zurich)
Department
Type
DUNS #
481907673
City
Zurich
State
Country
Switzerland
Zip Code
8092

  Publications

Beld, Joris; Woycechowsky, Kenneth J; Hilvert, Donald (2008) Catalysis of oxidative protein folding by small-molecule diselenides. Biochemistry 47:6985-7
Beld, Joris; Woycechowsky, Kenneth J; Hilvert, Donald (2007) Selenoglutathione: efficient oxidative protein folding by a diselenide. Biochemistry 46:5382-90
Toscano, Miguel D; Woycechowsky, Kenneth J; Hilvert, Donald (2007) Minimalist active-site redesign: teaching old enzymes new tricks. Angew Chem Int Ed Engl 46:3212-36
Woycechowsky, Kenneth J; Vamvaca, Katherina; Hilvert, Donald (2007) Novel enzymes through design and evolution. Adv Enzymol Relat Areas Mol Biol 75:241-94, xiii
Woycechowsky, Kenneth J; Seebeck, Florian P; Hilvert, Donald (2006) Tunnel plasticity and quaternary structural integrity of a pentameric protein ring. Protein Sci 15:1106-14
Seebeck, Florian P; Woycechowsky, Kenneth J; Zhuang, Wei et al. (2006) A simple tagging system for protein encapsulation. J Am Chem Soc 128:4516-7