Abstract
Shikimate pathway is an essential synthetic pathway for microbes and parasites but absent in mammals. The end product, chorismate, serves as a precursor for the synthesis of aromatic amino acids, including tyrosine and phenylalanine. The importance of shikimate pathway makes it a potential antimicrobial target for new drug discovery. Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway, which catalyzes the oxidation of shikimate to 3-dehydroshikimate. The structure of shikimate dehydrogenase from Helicobacter pylori had been determined in previous studies and several potential inhibitors had been identified, including NSC408168. Here, we focused on the study of inhibitor NSC408168 toward our target enzyme by performing site-directed mutagenesis. Five HpSDH mutant structures (Y210A, Y210S, Q237A, Q237N, and Q237K) had been solved and they showed subtle differences compared with the wild type structure. The isothermal titration calorimetry analysis suggested that the HpSDH mutants had weaker binding affinities with NSC408168 if compared to the wild type. In addition, a structure of SDH from a clinical isolated H. pylori strain v2356, which retained 93% sequence identity with wild type HpSDH, had been solved in this study and it showed higher structure flexibility and enzyme activity compared to the wild type structure. Surprisingly, NSC408168 showed better inhibition toward HpSDH-v2356 with the IC50 value of 0.82 μM. Together, these results may provide clues for the inhibition mechanism of NSC408168 toward HpSDH and serve as a template for designing more effective inhibitors in the future. On the other hand, 3-dehydroquinate synthase (DHQS) involved in the second step of shikimate pathway, which catalyzes the conversion of 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) to 3-dehydroquinate, by using NAD+ as a cofactor. In this study, we had solved the complex structures of DHQS from Mycobacterium tuberculosis with the cofactor (NAD+) and substrate (DAHP), at a resolution of 2.47 Å and 1.80 Å, respectively. This is also the first DAHP-complexed structure of DHQS being discovered among different species. The two structures show an open form and close form transition. The high resolution of MtDHQS structures provide us an insight in the catalytic mechanism and potential inhibitors can be identified for antimicrobial treatment.