This revised renewal proposal outlines our specific objectives for study in the shikimic acid biosynthetic pathway, which is used by microorganisms and plants to produce a diverse array of essential aromatic compounds. Foremost among these objectives is the study of chorismate mutase (CM), which catalyzes the first committed step in phenylalanine and tyrosine biosynthesis. With a detailed knowledge of the structure and function of CM, it should be possible to develop new inhibitors which could provide important leads for new antibiotics. CM inhibitors could join the ranks of such therapeutic agents as trimethoprim and the sulfa antibiotics for antimicrobial chemotherapy. Moreover, mutase inhibitors represent a form of treatment against which no resistance mechanisms have emerged. Specific objectives include the following. (1) Site-specific mutagenesis and crystallographic studies on an engineered monofunctional E. coli chorismate mutase (EcCM) to probe the importance of various side chain residues in forming and stabilizing the E-S complex in BcCM, and in promoting the pericyclic [3,3]-rearrangement (2) Crystallographic studies on bifunctional chorismate mutases. By studying the solid-state structure of the E. coli P-protein (chorismate mutase-prephenate dehydratase), we should learn how prephenate is transferred from the mutase domain to the dehydratase domain on the biosynthetic pathway to phenylalanine. The E. coli T-protein also contains a CM domain that overlaps with a prephenate dehydrogenase activity whose study will provide insight into the key structural elements involved in the biosynthesis of tyrosine. (3) Molecular modelling of chorismate analogs for structure-based drug design on chorismate mutases. Since differences are evident in both the amino acid sequence and the overall secondary structure of EcCM and the monofunctional mutase from Bacillus subtilis (BsCM), it may be possible to achieve the design of selective mutase inhibitors. By comparing and contrasting those differences, clues should emerge for the next generation of enzyme-specific mutase inhibitors. New candidate inhibitors, based on the bicyclic framework of a widely used CM transition state analog, will be designed with help from molecular modelling and active site fitting. (4) Further studies on a putative common intermediate in anthranilate, p- aminobenzoate, and isochorismate synthesis. During the last grant period, we showed that one rearranged allylic alcohol diastereomer of chorismate [anti-3-(1 '-carboxyvinyloxy)-6-hydroxy-cyclohexa- 1 ,4-diene- 1- carboxylic acid] was not incorporated into anthranilate by anthranilate synthase. The corresponding syn-stereoisomer will be synthesized and evaluated, since SN2' displacements are known to occur with either syn or antistereochemistry in nonenzymatic systems.
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