• Journal of Photoscience
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• v.2 no.1
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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 the Korean Chemical Society
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• v.64 no.4
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• pp.225-230
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• 2020

• Bulletin of the Korean Chemical Society
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• v.31 no.12
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• pp.3515-3516
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• 2010

• Proceedings of the Korean Society of Life Science Conference
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• 2007.10a
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• pp.107.1-107.1
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• 2007

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• Journal of Microbiology and Biotechnology
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• v.17 no.8
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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.

• 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.

• Journal of Microbiology and Biotechnology
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• v.19 no.3
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• pp.217-228
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• 2009

Mobile genetic segments, or transposons, are also referred to as jumping genes as they can shift from one position in the genome to another, thus inducing a chromosomal mutation. According to the target site-specificity of the transposon during a transposition event, the result is either the insertion of a gene of interest at a specific chromosomal site, or the creation of knockout mutants. The former situation includes the integration of conjugative transposons via site-specific recombination, several transposons preferring a target site of a conserved AT-rich sequence, and Tn7 being site-specifically inserted at attTn7, the downstream of the essential glmS gene. The latter situation is exploited for random mutagenesis in many prokaryotes, including IS (insertion sequence) elements, mariner, Mu, Tn3 derivatives (Tn4430 and Tn917), Tn5, modified Tn7, Tn10, Tn552, and Ty1, enabling a variety of genetic manipulations. Randomly inserted transposons have been previously employed for a variety of applications such as genetic footprinting, gene transcriptional and translational fusion, signature-tagged mutagenesis (STM), DNA or cDNA sequencing, transposon site hybridization (TraSH), and scanning linker mutagenesis (SLM). Therefore, transposon-mediated genetic engineering is a valuable discipline for the study of bacterial physiology and pathogenesis in living hosts.

• Bulletin of the Korean Chemical Society
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• v.28 no.5
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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.

• BMB Reports
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• v.29 no.1
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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.

• Biomedical Science Letters
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• v.14 no.2
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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.