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.

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National Institute of General Medical Sciences (NIGMS)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Virginia
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