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. 2008 Jul;29(7):870–876. doi: 10.1111/j.1745-7254.2008.00808.x

Natural product juglone targets three key enzymes from Helicobacter pylori: inhibition assay with crystal structure characterization

Yun-hua Kong 1, Liang Zhang 1, Zheng-yi Yang 1, Cong Han 1, Li-hong Hu 1,✉, Hua-liang Jiang 1, Xu Shen 1,✉
PMCID: PMC7091819  PMID: 18565285

Abstract

Aim:

To investigate the inhibition features of the natural product juglone (5-hydroxy-1,4-naphthoquinone) against the three key enzymes from Helicobacter pylori (cystathionine γ-synthase [HpCGS], malonyl-CoA:acyl carrier protein transacylase [HpFabD], and β-hydroxyacyl-ACP dehydratase [HpFabZ]).

Methods:

An enzyme inhibition assay against HpCGS was carried out by using a continuous coupled spectrophotometric assay approach. The inhibition assay of HpFabD was performed based on the α-ketoglutarate dehydrogenase-coupled system, while the inhibition assay for HpFabZ was monitored by detecting the decrease in absorbance at 260 nm with crotonoyl-CoA conversion to β-hydroxybutyryl-CoA. The juglone/FabZ complex crystal was obtained by soaking juglone into the HpFabZ crystal, and the X-ray crystal structure of the complex was analyzed by molecular replacement approach.

Results:

Juglone was shown to potently inhibit HpCGS, HpFabD, and HpFabZ with the half maximal inhibitory concentration IC50 values of 7.0±0.7, 20±1, and 30±4 μmol/L, respectively. An inhibition-type study indicated that juglone was a non-competitive inhibitor of HpCGS against O-succinyl-L-homoserine (Ki=αKi=24 μmol/L), an uncompetitive inhibitor of HpFabD against malonyl-CoA (αKi=7.4 μmol/L), and a competitive inhibitor of HpFabZ against crotonoyl-CoA (Ki=6.8 μmol/L). Moreover, the crystal structure of the HpFabZ/juglone complex further revealed the essential binding pattern of juglone against HpFabZ at the atomic level.

Conclusion:

HpCGS, HpFabD, and HpFabZ are potential targets of juglone.

Keywords: cystathionine γ-synthase, malonyl-CoA:acyl carrier protein transacylase, β-hydroxyacyl-ACP dehydratase, inhibitor type, complex structure

Glossary

IC50

the half maximal inhibitory concentration

Ki

the dissociation constant for inhibitor binding

CoA

coenzyme A

ACP

acyl carrier protein transacylase

PDB

Protein Data Bank

HO-HxoDH

D-2-Hydroxyisocaproate dehydrogenase

Footnotes

This work was supported by the National Natural Science Foundation of China (No 30525024, 20721003, and 90713046).

Contributor Information

Li-hong Hu, FAX: 86-21-5080-6918, Email: simmhulh@mail.shcnc.ac.cn.

Xu Shen, FAX: 86-21-5080-6918, Email: xshen@mail.shcnc.ac.cn.

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