The growing number of three-dimensional structures available has allowed the identification of protein superfamilies, which are compiled on the basis of their common overall structural fold. Members of enzyme superfamilies catalyze a diverse range of reactions using a large variety of substrates, indicating that many of the amino acid residues that dictate substrate and reaction specificity are likely to be distinct from those residues that define the overall structure of these enzymes. The goal of this proposal is to alter the preferred reaction type of the PLP-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) to that of the aminotransferases within the same superfamily, in order to probe the determinants of substrate and reaction specificity. The novel technology of DNA shuffling will be used to direct the evolution of ACC synthase into an aminotransferase. Comparitive kinetic and spectrophotometric analysis of ACC and the evolved aminotransferase will be performed, and the importance of specific amino acid changes in dictating active site specificity will be investigated by analyzing the effect of restoring the wild-type ACC synthase coding sequence at each position. Collaborative structural analysis of the most interesting evolved enzymes will complement the enzymological studies described in this proposal. The structural basis for active site specificity is a fundamental question that has broad implications for the continued development of efficacious therapeutic agents. The studies described here will help to establish a framework for future work in this area.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM020585-02
Application #
6454940
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Program Officer
Marino, Pamela
Project Start
2001-05-01
Project End
Budget Start
2001-05-01
Budget End
2001-08-24
Support Year
2
Fiscal Year
2001
Total Cost
$13,594
Indirect Cost
Name
University of California Berkeley
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
094878337
City
Berkeley
State
CA
Country
United States
Zip Code
94704