• 대한수의학회지
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• 제41권2호
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• pp.189-195
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• 2001

Single base substitution and deletion mutation have been introducted into the verotoxin 2 (VT2)A subunit gene from O157:H7 isolates to reduce cytotoxicity of VT2 and the cytotoxicity between wild type toxin and mutant toxoid were compared. A M13-derived recombinant plasmid pEP19RF containing a 940bp EcoRI-PstI fragment of VT2A gene was constructed for oligonucleotide-directed mutagenesis. The duoble mutant pDOEX was constructed by point and deletion mutation of two different highly conserved regions of VT2A encoding active site cleft of enzymatic domain. The key residue, Glu 167(GAA) and the pentamer(WGRIS) consisting of the enzymatic domain were replaced by ASP(GAC) and completely deleted in nucleotide sequence analysis of mutant, respectively. In the comparision of vero cell cytotoxicity between wide type toxin and toxoid from mutant, the wild type toxin expressed cytotoxicity in dilution of $10^{-6}$, but the toxid from mutant did not show cytotoxicity to vero cells.

• 한국임상수의학회지
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• 제22권4호
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• pp.386-391
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• 2005

This study was done to produce a mutated protein inactivated cytotoxicity of Shigatoxin 2e (Stx2e) of E.coli O139 isolates by deletional mutagenesis of Stx2e A subunit gene encoding active-site cleft of enzymatic domain in ST2e holotoxin. Cytotoxicity of the toxoid expressed from the mutant Stx2e gene was compared with wild type Stx2e for development of vaccine candidate. A recombinant plasmid pED18 containing Stx2e gene ot E.coli O139 isolates was used to generate mutation plasmid. Deletion mutagenesis was conducted for Stx2e A subunit gene encoding enzymatically active domain by polymerase chain reaction (PCR) using ot designed primer to induce deletional mutation. DNA sequence analysis was confirmed that the pentamer (Typ 202- Ser 206) that lies within the proposed active-site cleft in the second region was completely deleted. A DNA fragment of 1.1 kb that encode the new mutant Stx2eA gene was inserted into plasmid pRSET vector digested with EcoRV-Hind III and named pEDSET The PEDSET was transformed in E. coli for expression of mutant protein and the protein was confirmed by SDS-PACE and Western-blotting. The protein expressed by the mutant was tested to confirm the reduction of cytotoxic activities on Vero cell using microcytotoxicity assay compared with wild type Stx2e, the cytotoxicity of deletional mutant protein was at least reduced by 3,000-fold on Vero cell.

• BMB Reports
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• 제51권12호
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• pp.609-610
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• 2018

Glycosylation is one form of protein modification and plays a key role in protein stability, function, signaling regulation and even cancer. NleB and SseK are bacterial effector proteins and possess glycosyltransferase activity, even though they have different substrate preferences. NleB/SseKs transfer the GlcNAc sugar to an arginine residue of host proteins, leading to reduced $NF-{\kappa}B-dependent$ responses. By combining X-ray crystallography, NMR, molecular dynamics, enzyme kinetic assays and in vivo experiments, we demonstrated that a conserved HEN (His-Glu-Asn) motif in the active site plays a key role in enzyme catalysis and virulence. The lid-domain regulates the opening and closing of the active site and the HLH domain determines the substrate specificity. Our findings provide evidence for the enzymatic mechanism by which arginine can be glycosylated by SseK/NleB enzymes.

• Journal of Microbiology and Biotechnology
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• 제30권7호
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• pp.1104-1107
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• 2020

The N-terminal domain of the Pseudomonas sp. FB15 phytase increases low-temperature activity and catalytic efficiency. In this study, the 3D structure of the N-terminal domain was predicted and substitutions for the amino acid residues of the region assumed to be the active site were made. The activity of mutants, in which alanine (A) was substituted for the original residue, was investigated at various temperatures and pH values. Significant differences in enzymatic activity were observed only in mutant E263A, suggesting that the amino acid residue at position 263 of the N-terminal domain is important in enzyme activity.

• BMB Reports
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• 제32권6호
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• pp.599-604
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• 1999

A highly conserved amino acid, glutamic acid (Glu), present at position 152 in the catalytic domain of the human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein has been known to be critical for enzymatic function since substitution of Glu 152 with other residues results in a complete loss of enzymatic activities. In order to better understand the role of Glu 152 as a conserved residue in enzymatic action, intragenic second site mutations have been introduced around residue 152 of a mutant IN (E152A), and their biochemical properties were analyzed in terms of enzymatic activities. Disintegration activities were found to be significantly restored in several second site mutant INs, while integration activities were only recovered weakly. However, endonucleolytic activities were not discovered in all the mutant INs. These findings indicate that the second site mutations can partially restore that catalytic structure of the active site disturbed by the E152A mutation and lead to the regaining of integration and disintegration activities. In addition, it is also suggested that endonucleolytic activity requires a more accurate structure of the catalytic site than that for the integration and disintegration activities.

• 대한화장품학회:학술대회논문집
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• 대한화장품학회 2003년도 IFSCC Conference Proceeding Book I
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• pp.630-644
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• 2003

A human Cu, Zn-superoxide dismutase (Cu, Zn-SOD) was fused with a Tat PTD of HIV-1 to produce a novel anti-aging ingredient, Tat-SOD for cosmeceuticals. Test of stability and evaluation of transduction efficacy and enzymatic activity suggest Tat-SOD is an effective active ingredient for anti-aging treatment.

• 산업기술연구
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• 제26권B호
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• pp.163-171
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• 2006

Human immunodeficiency virus type 1 (HIV-1) integrase plays a critical role in the life cycle of the HIV virus. An ability to accurately map its electrostatic potential, and then use this information to predict the manner in which DNA will bind to the active site of the catalytic domain could provide a foundation for inhibitory design. Attempts to discern the crystal structure of HIV-1 integrase have proven problematic, especially in the region of enzymatic activity, that being those residues involved in the catalysis of the integration of viral DNA into the host cell. However, there is a structural correlation in to the region of interest with avian sarcoma virus (ASV), so a homology model utilizing this similarity was constructed to approximate the behavior/structure of the undetermined portions of the HIV-1 integrase crystal. After this model was constructed and its energy minimized, electrostatic calculations were carried out on the substance, so that an electrostatic potential map was constructed. Using this information, it was determined that DNA binding was oriented so as to exploit the regions of positive potential nearby the active site, as well as the positive potential of the magnesium cofactors.

• Molecules and Cells
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• 제42권1호
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• pp.79-86
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• 2019

Endolysins are bacteriophage-derived enzymes that hydrolyze the peptidoglycan of host bacteria. Endolysins are considered to be promising tools for the control of pathogenic bacteria. LysB4 is an endolysin produced by Bacillus cereus-infecting bacteriophage B4, and consists of an N-terminal enzymatic active domain (EAD) and a C-terminal cell wall binding domain (CBD). LysB4 was discovered for the first time as an L-alanoyl-D-glutamate endopeptidase with the ability to breakdown the peptidoglycan among B. cereus-infecting phages. To understand the activity of LysB4 at the molecular level, this study determined the X-ray crystal structure of the LysB4 EAD, using the full-length LysB4 endolysin. The LysB4 EAD has an active site that is typical of LAS-type enzymes, where $Zn^{2+}$ is tetrahedrally coordinated by three amino acid residues and one water molecule. Mutational studies identified essential residues that are involved in lytic activity. Based on the structural and biochemical information about LysB4, we suggest a ligand-docking model and a putative endopeptidase mechanism for the LysB4 EAD. These suggestions add insight into the molecular mechanism of the endolysin LysB4 in B. cereus-infecting phages.

• BMB Reports
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• 제43권4호
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• pp.233-244
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• 2010

Parkinson's disease (PD) is the second most common neurodegenerative disease, and 5-10% of the PD cases are genetically inherited as familial PD (FPD). LRRK2 (leucine-rich repeat kinase 2) was first reported in 2004 as a gene corresponding to PARK8, an autosomal gene whose dominant mutations cause familial PD. LRRK2 contains both active kinase and GTPase domains as well as protein-protein interaction motifs such as LRR (leucine-rich repeat) and WD40. Most pathogenic LRRK2 mutations are located in either the GTPase or kinase domain, implying important roles for the enzymatic activities in PD pathogenic mechanisms. In comparison to other PD causative genes such as parkin and PINK1, LRRK2 exhibits two important features. One is that LRRK2's mutations (especially the G2019S mutation) were observed in sporadic as well as familial PD patients. Another is that, among the various PD-causing genes, pathological characteristics observed in patients carrying LRRK2 mutations are the most similar to patients with sporadic PD. Because of these two observations, LRRK2 has been intensively investigated for its pathogenic mechanism (s) and as a target gene for PD therapeutics. In this review, the general biochemical and molecular features of LRRK2, the recent results of LRRK2 studies and LRRK2's therapeutic potential as a PD target gene will be discussed.

• BMB Reports
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• 제42권6호
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• pp.350-355
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• 2009

Although previous studies on hexokinase (HK) II indicate both the N- and C-terminal halves are catalytically active, we show in this study the N-terminal half is significantly more catalytic than the C-terminal half in addition to having a significantly higher $K_m$ for ATP and Glu. Furthermore, truncated forms of intact HK II lacking its first N-terminal 18 amino acids ($\Delta$18) and a truncated N-terminal half lacking its first 18 amino acids ($\Delta$18N) have higher catalytic activity than other mutants tested. Similar results were obtained by PET-scan analysis using $^{18}F-FDG$. Our results collectively suggest that each domain of HK II possesses enzyme activity, unlike HK I, with the N-terminal half showing higher enzyme activity than the C-terminal half.