We propose to study the catalytic mechanism of a novel nuclease from the important bacterial pathogen Serratia marcescens by determining its three dimensional structure using x-ray diffraction techniques. Its properties include activity against single and double stranded DNA, as well as RNA, without any apparent base preference. The Serratia nuclease is not homologous with Staph nuclease, the only other DNA/RNA nuclease for which a structure exists, nor is it homologous with other nucleases that have been solved by x-ray diffraction such as the RNA nuclease, Ribonuclease A, or the DNA endonuclease EcoRI. The structure of this enzyme should, therefore, provide valuable information about a new class of nuclease. At present we have succeeded in obtaining large, high quality nuclease crystals in Space Group P212121 that diffract to beyond 2A. Low resolution native data sets have been recorded and a search is under way for heavy atom derivatives. Our overall goal will be to solve the structure of the wild type enzyme and use the sequence to interpret the electron density. Next, we hope to study the structure, in the context of known kinetic research on this enzyme, in order to identify its active site and potentially important catalytic residues. Finally, we hope to further probe any putative mechanism of nuclease action through structure determination of enzyme-inhibitor complexes and structural studies of important mutant enzymes. All of the structural work will be interpreted in light of current bioorganic studies on the enzyme.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
First Independent Research Support & Transition (FIRST) Awards (R29)
Project #
5R29GM046866-04
Application #
2184351
Study Section
Molecular and Cellular Biophysics Study Section (BBCA)
Project Start
1992-02-01
Project End
1997-01-31
Budget Start
1995-02-01
Budget End
1996-01-31
Support Year
4
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Houston
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77204