• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.491-494
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• 2009

Within the secondary metabolite class of flavonoids, which consist of more than 10,000 known structures, flavones define one of the largest subgroups. The diverse function of flavones in plants as well as their various roles in the interaction with other organisms offers many potential applications including in human nutrition and pharmacology. We used two genes, flavone synthase (PFNS-l) that converts naringenin into apigenin and flavone 7-O-methyltransferase (POMT-7) that converts apigenin into 7-O-methyl apigenin, to synthesize 7-O-methyl apigenenin from naringenin. The PFNS-l gene was subcloned into the E. coli expression vector pGEX and POMT-7 was subcloned into the pRSF vector. Since both constructs contain different replication origins and selection markers, they were cotransformed into E. coli. Using E. coli transformants harboring both PFNS-l and POMT-7, naringenin could be converted into 7-O-methyl apigenin, genkwanin.

• Biomedical Science Letters
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• v.13 no.2
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• pp.135-140
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• 2007

The possible mechanism of decreased catechol-O-methyltransferase (COMT) activity in cholestatic rat liver was studied. Hepatic and serum COMT activities were determined from the experimental rats with choledocho-caval shunt (CCS). The Michaelis-Menten constants in this hepatic enzyme was also measured. The activities of cytosolic, mitochondrial and mircosomal COMT as well as their $V_{max}$ values were found to be decreased significantly in CCS plus taurocholic acid (TCA) injected group than in the control group, such as CCS alone groups. However, their $K_m$ values in the experimental groups did not vary. Seru4m COMT activity increased slightly in the CCS plus TCA injerted group than in the control group. The above results suggest that TCA represses biosynthesis of the COMT in the liver, The elevated activity of the serum COMT is believed to be caused by the increment of membrane permeability of hepatocytes upon TCA mediated liver cell necrosis.

• Bulletin of the Korean Chemical Society
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• v.28 no.12
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• pp.2248-2252
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• 2007

SOMT-9 is an O-methyltransferase that utilizes quercetin to produce 3'-methoxy quercetin. In order to determine which amino acids of SOMT-9 are important for this reaction and its regioselectivity, molecular docking experiments followed by site directed mutagenesis were performed. Molecular modeling and molecular docking experiments identified several amino acid residues involved in metal binding, AdoMet binding, and substrate binding. Site-directed mutagenesis showed that Asp188 is critical for metal binding and that Lys165 assists other metal binding residues in maintaining quercetin in the proper position during the reaction. In addition, Tyr207 was shown to play an important role in the determination of the regioselectivity and Met60 was shown to be involved in formation of the hydrophobic pocket necessary for substrate binding. The molecular modeling and docking experiments discussed in this study could be applicable to future research including prediction of substrate binding and regioselectivity of an enzyme.

• Bulletin of the Korean Chemical Society
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• v.35 no.1
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• pp.62-68
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• 2014

Coptis chinensis 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (HOMT), an essential enzyme in the berberine biosynthetic pathway, catalyzes the methylation of 3'-hydroxy-N-methylcoclaurine (HMC) producing reticuline. A 3D model of HOMT was constructed by homology modeling and further subjected to docking with its ligands and molecular dynamics simulations. The 3D structure of HOMT revealed unique structural features which permitted the methylation of HMC. The methylation of HMC was proposed to proceed by deprotonation of the 4'-hydroxyl group via His257 and Asp258 of HOMT, followed by a nucleophilic attack on the SAM-methyl group resulting in reticuline. HOMT showed high substrate specificity for methylation of HMC. The study evidenced that Gly117, Thr312 and Asp258 in HOMT might be the key residues for orienting substrate for specific catalysis.

• Journal of Life Science
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• v.18 no.4
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• pp.580-584
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• 2008

$O^6-methylguanine-DNA$ methyltransferase (MGMT) is a DNA repair protein that protects cells against the carcinogenic effects of alkylating agents. The loss of MGMT expression was commonly known due to hypermethylation of CpG islands in its promoter region. We evaluated the expression of MGMT by immunohistochemistry in order to examine the relationship between loss of MGMT expression and clinicopathological characteristics in 74 Korean patients with non-small cell lung cancers. Loss of MGMT was detected in 25 (33.8%) of 74 cases. The loss of MGMT expression was frequently seen in the adenocarcinoma than in the squamous cell carcinoma (p=0.021). However, there was no significant differences between loss of MGMT expression and other clinicopathological characteristics, including age, gender, smoking status, tumor size, tumor T stage, and lymph node metastasis (p>0.05). In conclusion, loss of MGMT expression was related with the histologic type of lung cancer. Further methylation study of MGMT promoter is needed to evaluate the relationships with immunohistochemical expression of MGMT and to clarify the role of MGMT in lung cancer.

• Biomolecules & Therapeutics
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• v.3 no.2
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• pp.122-131
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• 1995

Five human cancer cell lines (HeLa S3, Hep 3B, KATO III, Hs 683, HeLa MR) and one human normal cell line (WI-38) were examined cell viability, northern blot analysis, western blot analysis, and in situ hybridization for the expression $O_{6}$ -methylguanine-DNAmethyltransferase (MGMT), which can repair $O_{6}$ -methylguanine produced in DNA by alkylating agents. In cell viability test, the lethal sensitivities of each strain against anti-tumor drug N,N-bis(2-chloroethyl)- N-nitrosourea (BCNU) were counted, and both BCNU treated and untreated cell extracts were examined for their MGMT inducibility by RNA dot blot analysis. Cell lines did not show MGMT induction by BCNU pretreatment. Tlle MGMT activity was assayed by measuring the $^3$H radioactivity transferred from the substrate DNA containing [methyl-$^3$H)-O$_{6}$ -methylguanine to acceptor molecules in the cell extracts. Extracts from the majority of tumor strains and normal cells contained substantial MGMT activity of varying degree, while the known Mer$^{[-10]}$ cell (lacked or severely depleted in MGMT activity) Hela MR, and Hs 683 (proved to be Mer$^{[-10]}$ ) were much more sensitive to BCNU than the rest of tumor strains, as measured by cell viability test. Overall results above, KATO III showed the highest expression level of MGMT among the strains examined. Furthermore, with all the tumor and normal strains tested, a good correlation was observed between MGMT expression and cellular resistance to BCNU. The varying levels of expression of MGMT in human cancer cells found in this study should provide a molecular basis for MGMT expression among tumor strains from different tissue origin, the information of antitumor agents selection for chemotherapy of cancers.

• Journal of Plant Biotechnology
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• v.3 no.3
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• pp.131-134
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• 2001

In $\lambda$-Zap II vector system, a cDNA library was constructed for the developing secondary xylem mRNA from hybrid poplar, Populus nigra x maximowiczii. A cDNA clone of 1.5 kb in size, pOMTB1.4 encoding a lignin-bispecific O-methyltransferase was screened by plaque hybridization using a probe of 540 bp cDNA amplified by polymerase chain reaction from the cDNA library and identified by nucleotide sequencing. Its nucleotide sequence contains one open reading frame of 366 amino acids. The deduced amino acid sequence in comparison with that of Populus tremuloides showed the differences of 9 amino acids and revealed 85-99% homology among alfalfa, poplar and aspen.

• 한국생물공학회:학술대회논문집
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• 2005.04a
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• pp.518-518
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• 2005

• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.987-987
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• 2005

• Bulletin of the Korean Chemical Society
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• v.29 no.7
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• pp.1311-1314
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• 2008

Bacillus halodurans O-methyltransferase (BhOMT) is an S-adenosylmethionine dependent methyltransferase. In our previous study, three dimensional structure of the BhOMT has been determined by comparative homology modeling and automated docking study showed that two hydroxyl groups at 3'- and 4'-position in Bring and structural rigidity of C-ring resulting from the double bond characters between C2 and C3 of flavonoid, were key factors for interaction with BhOMT. In the present study, BhOMT was cloned and expressed. Binding assay was performed on purified BhOMT using fluorescence experiments and binding affinity of luteolin, quercetin, fisetin, and myricetin were measured in the range of $10^7$. Fluorescence quenching experiments indicated that divalent cation plays a critical role on the metal-mediated electrostatic interactions between flavonoid and substrate binding site of BhOMT. Fluorescence study confirmed successfully the data obtained from the docking study and these results imply that hydroxyl group at 7-position of luteolin, quercetin, fisetin, and myricetin forms a stable hydrogen bonding with K211 and carboxyl oxygen of C-ring forms a stable hydrogen bonding with R170. Hydroxyl group at 3'-and 4'-position in the B-ring also has strong $Ca^{2+}$ mediated electrostatic interactions with BhOMT.