| To combat the increased resistance of pathogenic bacteria, there needs to be incessant expansion of our standard antibacterial agents. 3-Deoxy- D-arabino-heptulosonate-7-phosphate synthase (DAHPS), the first enzyme in the shikimate pathway, which catalyzes the formation of DAHP from phospho enolpyruvate (PEP) and D-erythrose 4-phosphate, proves to be an interesting target for the design of antimicrobial agents since the shikimate pathway is absent in mammals.; DAHPSs from microorganisms vary in length of primary amino acid sequence, as well as their modes of regulation or lack of regulation. These differences have a profound effect on how inhibitors function; therefore, there is an urgent need to understand these differences for the rational design of inhibitors.; This thesis examines several different DAHPSs, including an archaeal DAHPS from Aeropyrum pernix (DAHPSAp), to illustrate that DAHPSS evolved from an ancient (beta/alpha) 8 barrel scaffold to more sophisticated (beta/alpha)8 barrels containing domains and insertions for feedback regulation. DAHPS Ap and the 260 residue Thermoanaerobacter tengcongensis DAHPS (DAHPSTt-short) are both unregulated DAHPS. The crystal structure of DAHPSAp shows only the (beta/alpha)8 barrel, which catalyzes the formation of DAHP. The 338 residue Thermoanaerobacter tengcongensis DAHPS (DAHPSTt-long) and the previously characterized Thermotoga maritima DAHPS (DAHPSTm) each have a regulatory domain at the N-terminus and are regulated by L-Phe and L-Tyr. Furthermore, the implications of domain truncation, appendage, and/or swapping are examined through creation and characterization of several truncated and fusion proteins from DAHPSAp, DAHPSTm, DAHPSTt-long, and DAHPSTt-short. Finally, a model mimicking the feedback inhibition of DAHPTm by L-Tyr is created from preliminary crystallographic data and characterization results. |
