• BMB Reports
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• 제40권5호
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• pp.697-707
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• 2007

Cytochrome $cbb_3$ oxidase is a member of the heme-copper oxidase superfamily that catalyses the reduction of molecular oxygen to the water and conserves the liberated energy in the form of a proton gradient. Comparison of the amino acid sequences of subunit I from different classes of heme-copper oxidases showed that transmembrane helix VIII and the loop between transmembrane helices IX and X contain five highly conserved polar residues; Ser333, Ser340, Thr350, Asn390 and Thr394. To determine the relationship between these conserved amino acids and the activity and assembly of the $cbb_3$ oxidase in Rhodobacter capsulatus, each of these five conserved amino acids was substituted for alanine by site-directed mutagenesis. The effects of these mutations on catalytic activity were determined using a NADI plate assay and by measurements of the rate of oxygen consumption. The consequence of these mutations for the structural integrity of the $cbb_3$ oxidase was determined by SDS-PAGE analysis of chromatophore membranes followed by TMBZ staining. The results indicate that the Asn390Ala mutation led to a complete loss of enzyme activity and that the Ser333Ala mutation decreased the activity significantly. The remaining mutants cause a partial loss of catalytic activity. All of the mutant enzymes, except Asn390Ala, were apparently correctly assembled and stable in the membrane of the R. capsulatus.

• International Journal of Industrial Entomology and Biomaterials
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• 제19권1호
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• pp.199-204
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• 2009

The newly cloned Bacillus thuringiensis cry1-5 gene showed high activity to both Plutella xylostella and Spodoptera exigua, while cry1Ac only showed high activity against P. xylostella but low to S. exigua. Through the alignment of amino acid sequences between Cry1Ac and Cry1-5, we found 12 different residues in domain I (6 residues) and domain II (6 residues). In this study, the modified cry1Ac gene, which is constructed according to a crop-preferring codon usage, was used as a template to construct mutant B. thuringiensis cry1Ac genes based on cry1-5 gene through multi site-directed mutagenesis. Total 63 various mutant cry genes were obtained at 12 positions randomly. Among them, ten mutant cry genes, whose domain I was totally converted and domain II was randomly, were selected to express in baculovirus expression system as a polyhedrin fusion form. The recombinant proteins were 95 kDa in size and were stably activated as 65 kDa by trypsin. The expressed mutant Cry proteins were applied to bioassays against P. xylostella and S. exigua. All mutants showed high insecticidal activity both to P. xylostella and S. exigua similar to cry1-5. These results suggest that these mutant cry genes might be expected of desirable cry genes for introduction to transgenic crops.

• Journal of Microbiology and Biotechnology
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• 제21권11호
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• pp.1116-1122
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• 2011

In this study, site-directed mutagenesis was performed on the ${\beta}$-agarase AgaA gene from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutant enzymes, S63K, C253I, and S63K-C253I, were 126% (1,757.78 U/mg), 2.4% (33.47 U/mg), and 0.57% (8.01 U/mg), respectively, relative to the wild-type ${\beta}$-agarase AgaA (1,392.61 U/mg) at $40^{\circ}C$. The stability of the mutant S63K enzyme was 125% of the wild-type up to $45^{\circ}C$, where agar is in a sol state. The mutant S63K enzyme produced 166%, 257%, and 220% more neoagarohexaose, and 230%, 427%, and 350% more neoagarotetraose than the wild-type in sol, gel, and nonmelted powder agar, respectively, at $45^{\circ}C$ over 24 h. The mutant S63K enzyme produced 50% more neoagarooligosaccharides from agar than the wild-type ${\beta}$-agarase AgaA from agarose under the same conditions. Thus, mutant S63K ${\beta}$-agarase AgaA may be useful for the production of functional neoagarooligosaccharides.

• BMB Reports
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• 제32권4호
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• pp.358-362
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• 1999

dTDP-D-glucose 4,6-dehydratase as an oxidoreductase catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, which is essential for the formation of 6-deoxysugars. dTDP-D-glucose 4,6-dehydratase shows remarkable sterochemical convergence in which displacement of the C-6 hydroxyl group by a C-4 hydrogen proceeds intramolecularly with inversion of configuration. The reaction mechanism is known to be oxidation, dehydration, and reduction by bases mediating proton transfer and $NAD^+$ cofactor. In this study, the bases in the active site domain are proposed to be His-79 and His-300 from a comparison of the peptides of the dehydratase and UDP-D-glucose epimerase. His-79 and His-300 were mutated to prepare the mutants H79L (mutation of histidine to leucine at the 79th amino acid) and H300A (mutation of histidine to alanine at the 300th amino acid) by site-directed mutagenesis. The H79L protein was inactive, showing that His-79 participates in the reaction mechanism.

• BMB Reports
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• 제43권6호
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• pp.419-426
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• 2010

Aeromonas hydrophila is a bacterial pathogen that infects a large number of eukaryotes, including humans. The UDP-galactose 4'-epimerase (GalE) catalyzes interconversion of UDP-galactose to UDP-glucose and plays a key role in lipopolysaccharide biosynthesis. This makes it an important virulence determinant, and therefore a potential drug target. Our earlier studies revealed that unlike other GalEs, GalE of A. hydrophila exists as a monomer. This uniqueness necessitated elucidation of its structure and active site. Chemical modification of the 6xHis-rGalE demonstrated the role of histidine residue in catalysis and that it did not constitute the substrate binding pocket. Loss of the 6xHis-rGalE activity and coenzyme fluorescence with thiol modifying reagents established the role of two distinct vicinal thiols in catalysis. Chemical modification studies revealed arginine to be essential for catalysis. Site-directed mutagenesis indicated Tyr149 and Lys153 to be involved in catalysis. Use of glycerol as a cosolvent enhanced the GalE thermostability significantly.

• BMB Reports
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• 제35권3호
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• pp.255-261
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• 2002

GTP cyclohydrolase I (E.C. 3.5.4.16) is a homodecameric protein that catalyzes the conversion of GTP to 7,8-dihydroneopterin triphosphate (H2NTP), the initial step in the biosynthesis of pteridines. It was proposed that the enzyme complex could be composed of a dimer of two pentamers, or a pentamer of tightly associated dimers; then the active site of the enzyme was located at the interface of three monomers (Nar et al. 1995a, b). Using mutant enzymes that were made by site-directed mutagenesis, we showed that a decamer of GTP cyclohydrolase I should be composed of a pentamer of five dimers, and that the active site is located between dimers, as analyzed by a series of size exclusion chromatography and the reconstitution experiment. We also show that the residues Lys 136, Arg139, and Glu152 are of particular importance for the oligomerization of the enzyme complex from five dimers to a decamer.

• Bulletin of the Korean Chemical Society
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• 제33권12호
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• pp.4169-4172
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• 2012

To elucidate the roles of cysteine residues in rice Phi-class GST F3, in this study, all three cysteine residues were replaced with alanine by site-directed mutagenesis in order to obtain mutants C22A, C73A and C77A. Three mutant enzymes were expressed in Escherichia coli and purified to electrophoretic homogeneity by affinity chromatography on immobilized GSH. The substitutions of Cys73 and Cys77 residues in OsGSTF3 with alanine did not affect the glutathione conjugation activities, showing non-essentiality of these residues. On the other hand, the substitution of Cys22 residue with alanine resulted in approximately a 60% loss of specific activity toward ethacrynic acid. Moreover, the ${K_m}^{CDNB}$ value of the mutant C22A was approximately 2.2 fold larger than that of the wild type. From these results, the evolutionally conserved cysteine 22 residue seems to participate rather in the structural stability of the active site in OsGSTF3 by stabilizing the electrophilic substrates-binding site's conformation than in the substrate binding directly.

• Bulletin of the Korean Chemical Society
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• 제32권11호
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• pp.4106-4108
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• 2011

• 미생물과산업
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• 제14권1호
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• pp.22-28
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• 1988

DNA 클로닝과 조작기술의 발전은 어떤 유전자의 특정한 위치에 선택적으로 돌연변이를 도입할 수 있는 site-specific mutagenesis 기술을 창출해 내었다. 이 기술로 DAN 염기의 치환, 결실, 삽입등을 클론된 유전자에 직접 도입할 수가 있게 되어 생체의 유전자 조작이나 유전자의 산물인 단백질의 구조와 기능을 의도적으로 변화시키는 protein engineering에 광범위하게 이용되고 있다. Protein engineering은 주로 단백질의 촉매 및 생리활성의 증가, 효소의 특성및 기질 특이성의 변화, 단백질 구조의 안정화 및 내염성 증가, 분자량의 감소, 효소및 생리활성 단백질의 구조의 안정화및 내열성 증가 등에 활용되고 있으며 산업적 유용성이 큰새로운 단백질의 창조에도 기여할 것으로 기대를 모으고 있다. Site-specific mutagenesis 기술로 현재 가장 널리 이용되는 것이 in vitro상에서 수행하는 oligonucleotide-directed site specific mutagenesis이다. 이 방법은 생화학적으로 합성한 특정한 염기서열을 가진 oligonucleotide들을 일종의 mutagen으로 사용하거나 효소적 DNA 합성을 위한 primer로 사용하여 클론된 DNA의 염기서열을 선택적으로 개조하거나 혹은 다른 조작을 하는 것이다. 여기서는 돌연변이율을 높이는 여러가지 개량된 방법들이 나왔으며 그중의 몇가지를 소개하였다.

• BMB Reports
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• 제33권1호
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• pp.69-74
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• 2000

The Arabidopsis thaliana S-Adenosylmethionine decarboxylase (AdoMetDC) cDNA ($GenBank^{TM}$ U63633) was cloned, then the AdoMetDC protein was expressed and purified. The purified AdoMetDC was inactivated by salicylaldehyde in a pseudo first- order kinetics. The secondorder rate constant for inactivation was 126 $M^{-1}min^{-1}$ with the slope of n=0.73, suggesting that inactivation is the result of the reaction of one lysine residue in the active site of AdoMetDC. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations in conserved $lysine^{81}$ residues. These were chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. Changing $Lys^{81}$ to alanine showed an altered optimal pH. The substrate also provided protection against inactivation by salicylaldehyde. Considering these results, we suggest that the $lysine^{81}$ residue may be involved in catalytic activity.