This renewal proposal requests funds to continue our studies ont he structure-function relationships for the enzyme 5-enolpyruvylshikimate-3- phosphate (EPSP) synthase, while developing further a new method for the structural characterization of enzymatic reactions in general by time- resolved solid-state NMR spectroscopy. The project will be extended to include uridine diphosphate N-acetyl-glucosamine enolpyruvyl transferase (UDP-NAG EPT) and 3-deoxy-D-manno-2-octulosonate-8-phosphate (KD08P) synthase. The research program is designed to determine, using nuclear magnetic resonance (NMR) spectroscopy, the structure of the enzyme-bound intermediates of three enolpyruvyl transfer enzymes; EPSP synthase; UDP- NAG EPT and KD08P synthase. EPSP synthase catalyzes the condensation of shikimate-3-phosphate and phosphoenolpyruvate, and the product, EPSP, is a key intermediate in the biosynthesis of aromatic amino acids. UDP-NAG EPT is a key enzyme in bacterial cell wall biosynthesis, and KD08P synthase is involved in bacterial lipopolysaccharide biosynthesis.
The specific aims of this renewal proposal are to: (1) carry out site-directed mutagenesis studies on specific active site residues of EPSP synthase, (2) carry out time-resolved solid-state REDOR NMR measurements on EPSP synthase, measuring longer distances than was originally proposed in GM43215, and (3) extend this approach to two other enolpyruvyl transferase enzymes, UDP-NAG EPT and KD08P synthase, as time permits. We believe that the consequences of these studies are particularly interesting and exciting, not just for extending our understanding of the structure-function relationship of EPSP synthase and related enolpyruvyl transferase enzymes, but also for developing methodologies that can provide detailed time-resolved structural information on enzymatic reactions in general, which even sophisticated techniques like Laue X-ray diffraction have difficulty obtaining. The long-term goal of our research is to collaborate with a Laue X-ray crystallographer, whose structural information of the protein as whole will be crucial, and generate a """"""""move"""""""" of the molecular details of an enzyme in action. This might enable the rational of antibacterial agents in the future.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM043215-08
Application #
2749862
Study Section
Special Emphasis Panel (ZRG3-BBCA (01))
Project Start
1991-08-01
Project End
1999-07-31
Budget Start
1998-08-01
Budget End
1999-07-31
Support Year
8
Fiscal Year
1998
Total Cost
Indirect Cost
Name
Washington State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164
Kristiansen, Per Eugen; Mitchell, Dan J; Evans, Jeremy N S (2002) Double-quantum dipolar recoupling at high magic-angle spinning rates. J Magn Reson 157:253-66
Kim, Hak Jun; Young, John K; Helms, Gregory L et al. (2002) 1H, 13C, and 15N backbone resonance assignments of the C-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase. J Biomol NMR 24:269-70
Campos-Olivas, Ramon; Aziz, Rehan; Helms, Gregory L et al. (2002) Placement of 19F into the center of GB1: effects on structure and stability. FEBS Lett 517:55-60
Stauffer, M E; Young, J K; Evans, J N (2001) Shikimate-3-phosphate binds to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase. Biochemistry 40:3951-7
Stauffer, M E; Young, J K; Helms, G L et al. (2001) Chemical shift mapping of shikimate-3-phosphate binding to the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase. FEBS Lett 499:182-6
Jordan, P A; Bohle, D S; Ramilo, C A et al. (2001) New insights into the metal center of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase. Biochemistry 40:8387-96
Stauffer, M E; Young, J K; Helms, G L et al. (2001) Sequential assignments of the isolated N-terminal domain of 5-enolpyruvylshikimate-3-phosphate synthase. J Biomol NMR 20:387-8
Krekel, F; Samland, A K; Macheroux, P et al. (2000) Determination of the pKa value of C115 in MurA (UDP-N-acetylglucosamine enolpyruvyltransferase) from Enterobacter cloacae. Biochemistry 39:12671-7
Shuttleworth, W A; Pohl, M E; Helms, G L et al. (1999) Site-directed mutagenesis of putative active site residues of 5-enolpyruvylshikimate-3-phosphate synthase. Biochemistry 38:296-302
Jakeman, D L; Mitchell, D J; Shuttleworth, W A et al. (1998) Effects of sample preparation conditions on biomolecular solid-state NMR lineshapes. J Biomol NMR 12:417-21

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