Toward Transition State Analysis of DAHP Synthase

Abstract

Antibiotic resistance is a major threat to the global health. The overuse and misuse of antibiotics have accelerated the process of antibiotic resistance and the emergence of multiple drug resistant bacteria. Therefore, it is urgent to develop new antibiotics. 3 Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase is a promising target due to its vital role in shikimate pathway in bacteria, and the lack of mammalian counterpart. It catalyzes the aldol-like reaction between erythrose 4-phosphate and phosphoenolpyruvate, the first step in shikimate pathway. Inhibiting DAHPS will disrupt the shikimate pathway, leading to bacteria death. However, no clinical available DAHPS inhibitor makes it important to fully understand DAHPS catalytic mechanism. A tetrahedral intermediate (THI) is formed and broken during the reaction. Two possible mechanisms regarding THI breakdown generate the opposite charge distribution on the transition state (TS). The TS is an ideal target for inhibitor design. Due to the low concentration and short lifetimes, kinetic isotope effects (KIEs) are the only means to determine TS structures in detail. Competitive KIEs measurement involves measurement of the ratio of isotopologues in the same reaction. In this study, we have developed a method for accurate measurement of isotope ratios by quantitative NMR that involves using a less time-consuming pulse program that only flips the magnetization to an ‘Ernst angle’, and processing the NMR peaks with Voigt deconvolution fitting model which considers both the natural line shapes and the contributions from the spectrometer.