The long term objectives of the proposed research are twofold: to understand the structural basis of catalytic activity and regulatory specificity in feedback-regulated enzymes and to elucidate the evolutionary pathways and molecular mechanisms by which specific branch point enzymes have acquired end product regulation. Continued studies of two critical enzyme systems of aromatic biosynthesis in bacteria are proposed. One system consists of the three isozymes of 3-deoxy-D-arabino-heptulosonate-7- phosphate synthase (DAHPS) from Escherichia coli which catalyze the first step of the common aromatic biosynthetic pathway. Although the isozymes are highly homologous, each is specifically regulated by negative feedback inhibition by one of the three aromatic amino acids. The second system is the multifunctional anthranilate synthase-phosphoribosyl transferase (AS- PRT) complex from Salmonella typhimurium which catalyzes the first two steps of the tryptophan biosynthetic pathway. The AS-PRT complex is a heterotetrameric (TrpE2TrpD2) enzyme whose multiple activities are feedback regulated by tryptophan, mediated by its binding to a regulatory site on the TrpE subunit. Regions and residues of the three DAHPS polypeptides that are essential for catalytic and regulatory activity will be identified by site-directed mutagenesis. Specific target sites in the three isozymes will be based on the recent results of random mutagenesis of the DAHPS(Trp) isozyme. In addition, a strategy for the non-selective mutational scanning of the entire DAHPS(Phe) polypeptide by doped oligonucleotide-directed mutagenesis will be initiated. Mutant enzymes will be tested for structural, catalytic and regulatory deficiencies by rapid screening methods. Analysis of the AS-PRT complex will continue to concentrate on structure/function studies of the TrpE subunit. Molecular genetic approaches will be used to define further the structure of the catalytic and regulatory domains of the TrpE subunit. Mutational strategies will be used to characterize in more detail the structure of the active site and feedback site in TrpE, to define the catalytic and regulatory mechanisms of the AS reaction and to probe the conformational and assembly properties of the complex. Spectroscopic and crystallographic studies of the DAHPS and AS-PRT enzymes will be undertaken in collaboration with others.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM035889-07
Application #
3289274
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1986-09-01
Project End
1996-06-30
Budget Start
1993-07-01
Budget End
1994-06-30
Support Year
7
Fiscal Year
1993
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
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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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