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1
Expression, crystallization, and preliminary X-ray crystallographic analysis of peptide deformylase from Acinetobacter baumanii

Thien-Hoang Ho,Kyoungho Jung,Inho Lee,Kim-Hung Huynh,Diem-Quynh Nguyen,Hyunjae Park,Sang Hee Lee,Lin-Woo Kang 한국구조생물학회 2017 Biodesign Vol.5 No.1
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The emergences of multi-drug resistant bacteria such as Acinetobacter baumanni have emphasized the necessity of new antibiotics. Peptidyl deformylase (PDF) catalyzes the removal of the formyl group from the N-terminal formylated methionine residue present in all nascent polypeptides in bacteria. In this study, the PDF gene from Acinetobacter baumannii K0420859 was cloned and its protein was overexpressed in E. coli, purified, and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal diffracted to 2.4 Å resolution. The crystal belonged to the trigonal space group P3 2 with unit cell parameters of a = b= 39.4 Å and c = 187.9 Å. Two protomers were presented in the asymmetric unit with a corresponding V M of 2.10 Å 3 Da -1 and a solvent content of 41.5%.
2
Crystallographic snapshots of active site metal shift in E. coli fructose 1,6-bisphosphate aldolase

( Huyen-thi Tran ),( Seon-hwa Lee ),( Thien-hoang Ho ),( Seung-hye Hong ),( Kim-hung Huynh ),( Yeh-jin Ahn ),( Deok-kun Oh ),( Lin-woo Kang ) 생화학분자생물학회(구 한국생화학분자생물학회) 2016 BMB Reports Vol.49 No.12
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Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between 1.8 A and 2.0 A. Native EcFBA structures showed two separate sites of Zn1 (interior position) and Zn2 (active site surface position) for Zn2+ ion. Citrate and TRIS bound EcFBA structures showed Zn2+ position exclusively at Zn2. Crystallo-graphic snapshots of EcFBA structures with and without ligand binding proposed the rationale of metal shift at the active site, which might be a hidden mechanism to keep the trace metal cofactor Zn2+ within EcFBA without losing it. [BMB Reports 2016; 49(12): 681-686]
3
Inhibitory effect of α-terpinyl acetate on cytochrome P450 2B6 enzymatic activity

Lee, Yejin,Park, Hyoung-Goo,Kim, Vitchan,Cho, Myung-A.,Kim, Harim,Ho, Thien-Hoang,Cho, Kyoung Sang,Lee, Im-Soon,Kim, Donghak Elsevier Pub. Co 2018 Chemico-biological interactions Vol.289 No.-
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Abstract Human cytochrome P450 2B6 is an important hepatic enzyme for the metabolism of xenobiotics and clinical drugs. Recently, more attention has been paid to P450 2B6 because of the increasing number of drugs it metabolizes. It has been known to interact with terpenes, the major constituents of the essential oils used for various medicinal purposes. In this study, the effect of monoterpenes on P450 2B6 catalytic activity was investigated. Recombinant P450 2B6 was expressed in Escherichia coli and purified using Ni-affinity chromatography. The purified P450 2B6 enzyme displayed bupropion hydroxylation activity in gas-mass spectrometry (GC-MS) analysis with a k cat of 0.5 min−1 and a K m of 47 μM. Many terpenes displayed the type I binding spectra to purified P450 2B6 enzyme and α-terpinyl acetate showed strong binding affinity with a K d value of 5.4 μM. In GC-MS analysis, P450 2B6 converted α-terpinyl acetate to a putative oxidative product. The bupropion hydroxylation activity of P450 2B6 was inhibited by α-terpinyl acetate and its IC50 value was 10.4 μM α-Terpinyl acetate was determined to be a competitive inhibitor of P450 2B6 with a K i value of 7.6 μM. The molecular docking model of the binding site of the P450 2B6 complex with α-terpinyl acetate was constructed. It showed the tight binding of α-terpinyl acetate in the active site of P450 2B6, which suggests that it could be a competitive substrate for P450 2B6. Highlights <UL> <LI> Recombinant P450 2B6 was expressed and purified. </LI> <LI> Purified P450 2B6 displayed the bupropion hydroxylation activity. </LI> <LI> Terpenes displayed the typical type I binding spectra to P450 2B6. </LI> <LI> α-Terpinyl acetate showed strong binding affinity to P450 2B6. </LI> <LI> α-Terpinyl acetate is a competitive inhibitor of P450 2B6 to bupropion. </LI> </UL> Graphical abstract [DISPLAY OMISSION]