The objectives of the proposed research are to define the relationship between molecular structure and catalytic function in complex enzymes and to elucidate the evolutionary pathways and mechanisms that have led to the attainment of end product regulation by specific branch point enzymes in metabolism. Parallel studies of two enzymes of aromatic amino acid biosynthesis will be undertaken. These are the aroH DAHP (deoxyarobinoheptulosonate-phosphate) synthase of Escherichia coli, which is one of the three isoenzymes that catalyze the first specific step of the common aromatic amino acid pathway, and the trpE subunit of the anthranilate synthase of Salmonella typhimurium, which catalyzes the first specific step of the tryptophan pathway. Although catalytically distinct, both enzymes are specifically regulated by the same feedback inhibitor, L-tryptophan. Molecular genetic, biochemical and immunological methodologies will be employed to analyze the aroH and trpE genes and polypeptides. The catalytic and regulatory domains of the proteins will be identified and mapped by mutagenesis and DNA sequence analysis of cloned aroH and trpE genes, and, in the case of aroH, by the exchange of domains between it and the tyrosine-sensitive (aroF) and the phenylalanine-sensitive (aroG) DAHP synthase isozymes. These studies should reveal whether the tryptophan-binding domains of the trpE and aroH polypeptides are homologous in sequence or structure, whether they might be of common evolutionary origin, and whether domain recruitment has served as a mechanism for the evolution of feedback-inhibitable enzymes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035889-05
Application #
3289273
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-09-01
Project End
1992-06-30
Budget Start
1990-09-01
Budget End
1992-06-30
Support Year
5
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Arts and Sciences
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Wagner, T; Kretsinger, R H; Bauerle, R et al. (2000) 3-Deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli. Model of binding of phosphoenolpyruvate and D-arabinose-5-phosphate. J Mol Biol 301:233-8
Wagner, T; Shumilin, I A; Bauerle, R et al. (2000) Structure of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: comparison of the Mn(2+)*2-phosphoglycolate and the Pb(2+)*2-phosphoenolpyruvate complexes and implications for catalysis. J Mol Biol 301:389-99
Tolbert, W D; Chatterji, S; Bauerle, R et al. (1999) Crystallization and preliminary crystallographic studies of the anthranilate synthase partial complex from Salmonella typhimurium. Acta Crystallogr D Biol Crystallogr 55:305-6
Shumilin, I A; Kretsinger, R H; Bauerle, R H (1999) Crystal structure of phenylalanine-regulated 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. Structure 7:865-75
Park, O K; Bauerle, R (1999) Metal-catalyzed oxidation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: inactivation and destabilization by oxidation of active-site cysteines. J Bacteriol 181:1636-42
Akowski, J P; Bauerle, R (1997) Steady-state kinetics and inhibitor binding of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tryptophan sensitive) from Escherichia coli. Biochemistry 36:15817-22
Shumilin, I A; Kretsinger, R H; Bauerle, R (1996) Purification, crystallization, and preliminary crystallographic analysis of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. Proteins 24:404-6
Tolbert, W D; Moll, J R; Bauerle, R et al. (1996) Crystallization and preliminary crystallographic studies of 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli. Proteins 24:407-8
Morollo, A A; Bauerle, R (1993) Characterization of composite aminodeoxyisochorismate synthase and aminodeoxyisochorismate lyase activities of anthranilate synthase. Proc Natl Acad Sci U S A 90:9983-7
Stephens, C M; Bauerle, R (1992) Essential cysteines in 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli. Analysis by chemical modification and site-directed mutagenesis of the phenylalanine-sensitive isozyme. J Biol Chem 267:5762-7

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