Evolution of DAHP synthase: From archaea to eubacteria.

Abstract

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<italic> enol</italic>pyruvate (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 <italic>Aeropyrum pernix</italic> (DAHPS<super>Ap</super>), to illustrate that DAHPS_S evolved from an ancient (beta/alpha)₈ barrel scaffold to more sophisticated (beta/alpha)₈ barrels containing domains and insertions for feedback regulation. DAHPS<super> Ap</super> and the 260 residue <italic>Thermoanaerobacter tengcongensis</italic> DAHPS (DAHPS<super>Tt-short</super>) are both unregulated DAHPS. The crystal structure of DAHPS<super>Ap</super> shows only the (beta/alpha)₈ barrel, which catalyzes the formation of DAHP. The 338 residue <italic> Thermoanaerobacter tengcongensis</italic> DAHPS (DAHPS<super>Tt-long</super>) and the previously characterized <italic>Thermotoga maritima</italic> DAHPS (DAHPS<super>Tm</super>) 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 DAHPS<super>Ap</super>, DAHPS<super>Tm</super>, DAHPS<super>Tt-long</super>, and DAHPS<super>Tt-short</super>. Finally, a model mimicking the feedback inhibition of DAHP<super>Tm</super> by L-Tyr is created from preliminary crystallographic data and characterization results.