The long range goal of this project is to understand the process of evolution of new enzymatic activities. The system which will be used to study this process consists of the three enzymes of the 4-chlorobenzoate degrading pathway found in adapted soil bacteria. The proposed work is divided into three parts. These parts and their specific aims are as follows: Part I: Evolution of catalysis in 4-chlorobenzoyl-CoA dehalogenase. 1. Determine the energy profile for catalysis. 2. Measure the contributions of active site residues to the free energy of ground states and transition states in catalysis. 3. Test interchange of dehalogenase, crotonase and delta-3-cis, delta-2-trans enoyl-CoA isomerase activities within the dehalogenase scaffold. 4. Test in vivo evolution of 4-fluorobenzoyl-CoA activity and 2-chlorobenzoyl-CoA activity from the 4-chlorobenzoyl-CoA progenitor and in vitro evolution by random mutagenesis and by rational design. Part II: Mechanism and structure of 4-chlorobenzoate:CoA ligase and 4-hydroxybenzoyl-CoA thioesterase. 5. Complete the x-ray structural determination of the crystalline thioesterase. 6. Measure the time course for a single turnover on the thioesterase and test for the formation of a covalent enzyme intermediate. 7. Determine the active site residues essential to substrate binding and catalysis by site directed mutagenesis. Part III: Mechanism and structure of 4-chlorobenzoate:CoA ligase. 8. Crystallize and determine the x-ray structure of the ligase from Pseudomonas and/or Alcaligenes. 9. Locate the CoA binding site by site-directed mutagenesis of conserved residues. 10. Complete the determination of the kinetic mechanism of ligase catalysis.
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