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 kcat of 7.7 s)1 and Km of 0.148 mm
toward shikimate, kcat of 7.1 s)1 and Km of 0.182 mm toward NADP, kcat
of 5.2 s)1 and Km of 2.9 mm 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)-4H-chromen-7-yl 3-chlorobenzoate (2), butyl 2-{[3-
(2-naphthyloxy)-4-oxo-2-(trifluoromethyl)-4H-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 IC50
values of 15.4, 3.9, 13.4, 2.9, and 3.5 lm, 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 lg?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.