The major goal of this proposal is to use evolutionary protein design to explore the molecular mechanism(s) supporting H2O2-mediated oxygenation in heme-enzymes. Cytochrome P450 and chioroperoxidase (CPO) are both heme containing oxidoreductases that stereospecifically catalyze the H2O2-mediated oxygenation of organic substrates. These proteins, however, differ significantly in their catalytic efficiencies. It is not known why CPO catalyzes this reaction more efficiently than P450s and unclear whether a P450 scaffold is compatible with CPO-like activity. To better understand the molecular determinants that allow heme-enzymes to function efficiently, I plan to: 1) engineer P450 variants with CPO-like catalytic efficiency through directed evolution; 2) analyze the mechanism(s) by which P450 variants catalyze oxygenation; and 3) determine the structure of interesting mutants. Through comparison of the activities and structures of wild type and evolved P450 enzymes, I will determine whether P450s adapt a catalytic cycle and active site structure similar to CPO, or if they adapt properties distinct from known heme-enzymes. In addition to providing insight into heme-enzyme activity adaptation, these studies could also aid in the development of single-enzyme monooxygenases with sufficient activity to make them practical biocatalysts for medical and pharmaceutical applications.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32GM064949-02
Application #
6622302
Study Section
Physical Biochemistry Study Section (PB)
Program Officer
Marino, Pamela
Project Start
2002-03-01
Project End
Budget Start
2003-03-01
Budget End
2004-02-29
Support Year
2
Fiscal Year
2003
Total Cost
$46,420
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Engineering
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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Silberg, Jonathan J; Tapley, Tim L; Hoff, Kevin G et al. (2004) Regulation of the HscA ATPase reaction cycle by the co-chaperone HscB and the iron-sulfur cluster assembly protein IscU. J Biol Chem 279:53924-31
Endelman, Jeffrey B; Silberg, Jonathan J; Wang, Zhen-Gang et al. (2004) Site-directed protein recombination as a shortest-path problem. Protein Eng Des Sel 17:589-94
Meyer, Michelle M; Silberg, Jonathan J; Voigt, Christopher A et al. (2003) Library analysis of SCHEMA-guided protein recombination. Protein Sci 12:1686-93