• 대한화학회지
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• 제64권4호
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• pp.225-230
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• 2020

• 한국생명과학회:학술대회논문집
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• 한국생명과학회 2007년도 제48회 학술심포지움 및 추계국제학술대회
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• pp.107.1-107.1
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• 2007

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• Bulletin of the Korean Chemical Society
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• 제28권12호
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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.

• Journal of Photoscience
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• 제2권1호
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• pp.27-29
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• 1995

Site directed mutagenesis has been introduced to determine active site(s) and molecular structure of E. coli RecA protein. Recombinant DNAs were constructed by point mutation of Tyr-264 to Phe which assumed active site for binding and hydrolysis of ATP. RecA proteins were purified from recombinants containing wild type and mutant genes and analyzed for ATPase activity assay. Result suggests that Tyr-264 is involved in ATP binding rather than ATP hydrolysis.

• Journal of Microbiology and Biotechnology
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• 제17권8호
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• pp.1249-1253
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• 2007

A hyperthermostable endoglucanase from Pyrococcus horikoshii with the capability of hydrolyzing crystalline cellulose was analyzed. A protein engineering study was carried out to obtain a reduced-size mutant. Five amino acid residues at both the N- and C-terminus were found to be removable without any loss of activity or thermal stability. Site-directed mutagenesis was also performed on R102, N200, E201, H297, Y299, E342, and W377, residues possibly involved in the active center or in the recognition and binding of a cellulose substrate. The activity of the resulting mutants was considerably decreased, confirming that the mutated residues were all important for activity. A reduced-size enzyme, as active as the wild-type endoglucanase, was successfully obtained, plus the residues critical for its activity and specificity were confirmed. Consequently, an engineered enzyme with a reduced size was obtained, and the amino acids essential for activity were confirmed by site-directed mutagenesis and comparison with a known three-dimensional structure.

• 한국가축번식학회지
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• 제25권4호
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• pp.381-388
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• 2001

본 연구는 돼지에서 성장호르몬과 결합되는 부위에 변이를 유도하여 결합력이 향상된 성장호르몬 결합단백질을 획득하기 위하여 수행되었다. 돼지의 지방으로부터 얻은 성장호르몬 수용체 RNA 내 성장호르몬 결합단백질 부분을 756 bp의 cDNA로 전향하고 클로닝한 후 site-directed mutagenesis 방법을 이용하여 26과 122번째 아미노산을 변이시켰다. 26번째 아미노산은 성장 호르몬과의 결합에 관련이 있다고 알려져 있는 돼지 성장호르몬 수용체 외막에 존재하는 다섯 군데의 N-linked glycosylation 부위와 가까이 위치한 부분이고, 122번째 아미노산은 소에서의 결합부위로 알려져 있다. 이렇게 변이를 유도한 성장호르몬 결합 단백질을 pET-32(c) 발현벡터에 삽입시키고 과발현시켰고 이를 정제하여 30 kDa의 변이를 유도한 성장호르몬 결합 단백질을 얻었다. 이러한 방법으로 성장호르몬 결합 단백질을 성장기에 있는 세포나 동물에 주입한다면 보다 향상된 성장을 볼 수 있을 것으로 사료된다.

• BMB Reports
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• 제29권1호
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• pp.17-21
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• 1996

Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers $Mn^{2+}$ to $Mn^{2+}$ as the cofactor. In $Mn^{2+}$ buffer the mutant is as active as the wild type enzyme in $Mn^{2+}$ buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.

• Preventive Nutrition and Food Science
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• 제6권1호
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• pp.79-81
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• 2001

Deoxynucleoside kinases exist as heterodimeric pairs specific for deoxyadenosine/deoxyguanosine kinase (dAK/dGK) and deoxyadenosine/deoxycytidine kinase (dAK/dCK). The aspartic acid-84 in dGK was mutated to alanine, asparagine and glutamic acid by site-directed mutagenesis. The mutation resulted in a drastic decease in dGK activity compared to the unmodified cloned enzyme while it increased production of dAK activity. The mutated dak/dgk genes, which synthesize tandem deoxyadenosine/deoxyguanosine kinase, were inserted back to the Lactobacillus acidophilus and Lactococcus lactis by electroporation to determine the effect of site-directed mutation of he enzymes on the microbial growth. However, no significant change was observed in cell growth and lactic acid production between wild type and mutant lactic acid bacteria.

• Bulletin of the Korean Chemical Society
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• 제28권5호
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• pp.772-776
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• 2007

In order to study the role of residue in the active site of glutathione S-transferase (GST), Arg13 residue in human GST P1-1 was replaced with alanine, lysine and leucine by site-directed mutagenesis to obtain mutants R13A, R13K and R13L. These three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. Mutation of Arg13 into Ala caused a substantial reduction of the specific activity by 10-fold. Km GSH, Km DCNB and Km EPNP values of R13A were approximately 2-3 fold larger than those of the wild type. Mutation of Arg13 into Ala also significantly affected I50 values of S-methyl-GSH that compete with GSH and ethacrynic acid, an electrophilic substrate-like compound. These results appeared that the substitution of Arg13 with Ala resulted in significant structural change of the active site. Mutation of Arg13 into Leu reduced the catalytic activity by approximately 2-fold, whereas substitution by Lys scarcely affected the activity, indicating the significance of a positively charged residue at position 13. Therefore, arginine 13 participates in catalytic activity as mainly involved in the construction of the proper electrostatic field and conformation of the active site in human GST P1-1.

• 대한의생명과학회지
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• 제14권2호
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• pp.119-122
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• 2008

The baculovirus/insect cell expression system is of great value in the study of structure-function relationships in mammalian glucose-transport proteins by site-directed mutagenesis and for the large-scale production of these proteins for mechanistic and biochemical studies. In order to exploit this, the effects of substitution at the highly conserved residue glutamine 282 of the human erythrocyte-type glucose transporter have been examined by in vitro site-directed mutagenesis. The modified human transport protein has been expressed in Spodoptera frugiperda 21 cells by using the recombinant baculovirus AcNPV-GTL. To assess the functional integrity of the expressed transporter, measurements of the transport inhibitor cytochalasin B binding were performed, involving the membranes prepared from 4 days post infection with no virus, with wild-type virus or AcNPV-GTL virus. Data obtained showed that there was little or no D-glucose-inhibitable binding in cells infected with the wild type or no virus. Only the recombinant virus infected cells exhibited specific binding, which is inhibitable by D- but not by L-glucose. However, there was a notable reduction in the affinity for the potent inhibitor cytochalasin B when binding measurements of AcNPV-GTL were compared with those of AcNPV-GT, which has no substitution. It is thus suggested that although the modified and unmodified human transporters differed slightly in their affinity for cytochalasin B, the glutamine substitution did not interfere the heterologous expression of the human transporter in the insect cells.