Biochemical characterization and inhibitor discovery of shikimate dehydrogenase from Helicobacter pylori.
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 mm toward shikimate, k(cat) of 7.1 s(-1) and K(m) of 0.182 mm toward NADP, k(cat) of 5.2 s(-1) and K(m) 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 degrees 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 IC(50) values of 15.4, 3.9, 13.4, 2.9, and 3.5 microm, 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 microg.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.
Han
Cong
C
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, China.
Wang
Lirui
L
Yu
Kunqian
K
Chen
Lili
L
Hu
Lihong
L
Chen
Kaixian
K
Jiang
Hualiang
H
Shen
Xu
X
D000429:Alcohol Oxidoreductases / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000737:chemistry / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D015870:Gene Expression; D016480:Helicobacter pylori / Q000737:chemistry / Q000187:drug effects / Q000201:enzymology*; D013058:Mass Spectrometry; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C022641:Shikimate dehydrogenase