Tài liệu Báo cáo khoa học: Biochemical characterization and inhibitor discovery of shikimate

Biochemical characterization and inhibitor discovery

of shikimate dehydrogenase from Helicobacter pylori

Cong Han1

, Lirui Wang1

, Kunqian Yu1

, Lili Chen1

, Lihong Hu1

, Kaixian Chen1

, Hualiang Jiang1,2

and Xu Shen1,2

1 Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of

Sciences, Shanghai, China

2 School of Pharmacy, East China University of Science and Technology, Shanghai, China

Helicobacter pylori is a gram-negative, microaerophilic,

motile, and spiral-shaped bacterium that colonizes the

gastric mucosa. Since it was discovered by Marshall

and Warren in 1982 [1], H. pylori has been recognized

as one of the most common human pathogens, prob￾ably infecting about 50% of the world’s human popu￾lation [2]. H. pylori is a major causative factor for

several gastrointestinal illnesses, including gastritis,

Keywords

antibacterial agent; drug target; enzyme

inhibition; Helicobacter pylori; shikimate

dehydrogenase

Correspondence

X. Shen, H. Jiang, and L. Hu, Shanghai

Institute of Materia Medica, Chinese

Academy of Sciences, 555 Zu Chong Zhi

Road, Zhangjiang Hi-Tech Park, Shanghai

201203, China.

Tel ⁄ Fax: +86 21 50806918

E-mail: [email protected],

[email protected],

[email protected]

Database

The sequence reported in this paper has

been submitted to GenBank database under

accession number AY738333

(Received 23 April 2006, revised 11 July

2006, accepted 16 August 2006)

doi:10.1111/j.1742-4658.2006.05469.x

Shikimate dehydrogenase (SDH) is the fourth enzyme involved in the shiki￾mate pathway. It catalyzes the NADPH-dependent reduction of 3-dehy￾droshikimate 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 recom￾binant 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 maes￾aquinone 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 inhibi￾tion pattern, and compound 4 displays competitive inhibition pattern with

respect to shikimate. Compounds 1, 4, and 5 display noncompetitive inhibi￾tion 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 lgÆmL)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.

Abbreviations

AfSDH, Archaeoglobus fulgidus shikimate dehydrogenase; EcSDH, Escherichia coli shikimate dehydrogenase; EPSP synthase, 5-enoylpyruvyl

shikimate phosphate synthase; HpSDH, Helicobacter pylori shikimate dehydrogenase; IPTG, isopropyl thio-b-D-galactoside; MIC, minimal

inhibitory concentration; MtSDH, Mycobacterium tuberculosis shikimate dehydrogenase; SDH, shikimate dehydrogenase.

4682 FEBS Journal 273 (2006) 4682–4692 ª 2006 The Authors Journal compilation ª 2006 FEBS