- 學位論文
Biochemical characterization and drug discovery of shikimate dehydrogenase from Helicobacter pylori
研究胃幽門螺旋菌shikimate dehydrogenase 生物化學特性與抑制劑之開發
摘要
The shikimate biosynthetic pathway is essential to microorganisms and parasites but absent from mammals. Shikimate dehydrogenase (SDH), the fourth enzyme in this pathway, catalyzes the NADPH-dependent reduction of 3-dehydroshikimate to shikimate. Therefore, SDH is considered as an attractive target for the discovery of antimicrobial agent. In this work, we have expressed and purified SDH from Helicobacter pylori (HpSDH) and Mycobacterium tuberculosis (MtSDH). Enzymatic analysis of HpSDH showed the Km values as 130 μM toward shikimate, and 186 μM toward NADP, respectively. Using pharmacophore hotspot model, six potent inhibitors (compounds A-F) with good IC50 values (1.4−6.9 μM) were identified (Figure 16). Compound C had a noncompetitive inhibition pattern, whereas compound A, B, D, E and F displayed uncompetitive inhibition pattern with respect to shikimate. Compound C displayed noncompetitive inhibition mode, and compounds A, B, D, E and F showed uncompetitive inhibition modes with respect to NADP. In addition, a combination of site-directed mutagenesis in the shikimate binding pocket (S16A, T65A, K69A, D105A, Y210A and Q237A) and isothermal titration calorimetry (ITC) studies was used to demonstrate the importance of these conserved residues. These results provide useful information for the development of novel antibiotics to treat H. pylori-associated infection.