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Biochemical characterization and inhibitor discovery of shikimate dehydrogenase from Helicobacter pylori
Author(s) -
Han Cong,
Wang Lirui,
Yu Kunqian,
Chen Lili,
Hu Lihong,
Chen Kaixian,
Jiang Hualiang,
Shen Xu
Publication year - 2006
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2006.05469.x
Subject(s) - shikimate pathway , stereochemistry , chemistry , biochemistry , enzyme , enzyme kinetics , nad+ kinase , sulfanyl , aromatic amino acids , active site
Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shikimate pathway. It catalyzes the NADPH‐dependent reduction of 3‐dehydroshikimate to shikimate, and has been developed as a promising target for the discovery of antimicrobial agent. In this report, we identified a new aroE gene encoding SDH from Helicobacter pylori strain SS1. The recombinant H. pylori shikimate dehydrogenase (HpSDH) was cloned, expressed, and purified in Escherichia coli system. The enzymatic characterization of HpSDH demonstrates its activity with k cat of 7.7 s −1 and K m of 0.148 m m toward shikimate, k cat of 7.1 s −1 and K m of 0.182 m m toward NADP, k cat of 5.2 s −1 and K m of 2.9 m m toward NAD. The optimum pH of the enzyme activity is between 8.0 and 9.0, and the optimum temperature is around 60 °C. Using high throughput screening against our laboratory chemical library, five compounds, curcumin ( 1 ), 3‐(2‐naphthyloxy)‐4‐oxo‐2‐(trifluoromethyl)‐4 H ‐chromen‐7‐yl 3‐chlorobenzoate ( 2 ), butyl 2‐{[3‐(2‐naphthyloxy)‐4‐oxo‐2‐(trifluoromethyl)‐4 H ‐chromen‐7‐yl]oxy}propanoate ( 3 ), 2‐({2‐[(2‐{[2‐(2,3‐dimethylanilino)‐2‐oxoethyl]sulfanyl}‐1,3‐benzothiazol‐6‐yl)amino]‐2‐oxoethyl}sulfanyl)‐ N ‐(2‐naphthyl)acetamide ( 4 ), and maesaquinone diacetate ( 5 ) were discovered as HpSDH inhibitors with IC 50 values of 15.4, 3.9, 13.4, 2.9, and 3.5 µ m , respectively. Further investigation indicates that compounds 1 , 2 , 3 , and 5 demonstrate noncompetitive inhibition pattern, and compound 4 displays competitive inhibition pattern with respect to shikimate. Compounds 1 , 4 , and 5 display noncompetitive inhibition mode, and compounds 2 and 3 show competitive inhibition mode with respect to NADP. Antibacterial assays demonstrate that compounds 1 , 2 , and 5 can inhibit the growth of H. pylori with MIC of 16, 16, and 32 µg·mL −1 , respectively. This current work is expected to favor better understanding the features of SDH and provide useful information for the development of novel antibiotics to treat H. pylori ‐associated infection.