• BMB Reports
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• v.49 no.6
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• pp.349-354
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• 2016

The archaeon Sulfolobus solfataricus P1 carboxylesterase is a thermostable enzyme with a molecular mass of 33.5 kDa belonging to the mammalian hormone-sensitive lipase (HSL) family. In our previous study, we purified the enzyme and suggested the expected amino acids related to its catalysis by chemical modification and a sequence homology search. For further validating these amino acids in this study, we modified them using site-directed mutagenesis and examined the activity of the mutant enzymes using spectrophotometric analysis and then estimated by homology modeling and fluorescence analysis. As a result, it was identified that Ser151, Asp244, and His274 consist of a catalytic triad, and Gly80, Gly81, and Ala152 compose an oxyanion hole of the enzyme. In addition, it was also determined that the cysteine residues are located near the active site or at the positions inducing any conformational changes of the enzyme by their replacement with serine residues.

• Journal of Integrative Natural Science
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• v.5 no.3
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• pp.168-174
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• 2012

Gal (1-3) receptors are members of GPCR superfamily with seven transmembrane helices. The neuropeptide galanin mediates its effects through the receptor subtypes Gal1, Gal2, and Gal3 and has been implicated in anxiety and depression related behaviors. Galanin receptors are considered to be important targets for the development of novel antidepressant drugs. Owing to the importance of these receptors, a short communication about the sequential and structural studies about the functional Galanin (1-3) receptors has been reported. Structural studies have been hampered due to the lack of X-ray crystal structures. However with the availability of templates with close homologs comparative modeling could be encouraging. Sequence analysis was done for each receptors and homology modeling of each receptors were done with recently reported templates. Comparative analyses were done between these receptors to identify the relationships between them sequentially. Phylogram was generated between these receptors to identify the close homologue between this receptor and found that Gal2 and Gal3 receptors are closer. Our results could be useful for further structure based drug design targeting Gal1, Gal2 and Gal3 receptors.

• 한국당과학회:학술대회논문집
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• 2006.11a
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• pp.31-31
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• 2006

• BMB Reports
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• v.37 no.3
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• pp.304-313
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• 2004

Three-dimensional (3D) models for the 65-kDa activated Cry4A and Cry4B $\delta$-endotoxins from Bacillus thuringiensis subsp. israelensis that are specifically toxic to mosquito-larvae were constructed by homology modeling, based on atomic coordinates of the Cry1Aa and Cry3Aa crystal structures. They were structurally similar to the known structures, both derived 3D models displayed a three-domain organization: the N-terminal domain (I) is a seven-helix bundle, while the middle and C-terminal domains are primarily comprise of anti-parallel $\beta$-sheets. Circular dichroism spectroscopy confirmed the secondary structural contents of the two homology-based Cry4 structures. A structural analysis of both Cry4 models revealed the following: (a) Residues Arg-235 and Arg-203 are located in the interhelical 5/6 loop within the domain I of Cry4A and Cry4B, respectively. Both are solvent exposed. This suggests that they are susceptible to tryptic cleavage. (b) The unique disulphide bond, together with a proline-rich region within the long loop connecting ${\alpha}4$ and ${\alpha}5$ of Cry4A, were identified. This implies their functional significance for membrane insertion. (c) Significant structural differences between both models were found within domain II that may reflect their different activity spectra. Structural insights from this molecular modeling study would therefore increase our understanding of the mechanic aspects of these two closely related mosquito-larvicidal proteins.

• Journal of Life Science
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• v.25 no.2
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• pp.121-129
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• 2015

Alginates are found in marine brown seaweeds and in extracellular biofilms secreted by some bacteria. Previously, we reported an oligoalginate lyase from Sphingomonas sp. MJ-3 (MJ3-Oal) that had an exolytic activity and protein sequence homology with endolytic polymannuronate (polyM) lyase in the N-terminal region. In this study, the MJ3-Oal was tested for both exolytic and endolytic activity by homology modeling using the crystal structure of Alg17c from Saccharophagus degradans 2-40T. The tyrosine residue at the $426^{th}$ position, which possibly formed a hydrogen bond with the substrate, was mutated to phenylalanine. The FPLC profiles showed that MJ3-Oal degraded alginate quickly to monomers as a final product through the oligmers, whereas the Tyr426Phe mutant showed only exolytic alginate lyase activity. $^1H$-NMR spectra also showed that MJ3-Oal degraded the endoglycosidic bond of polyM and polyMG (polymannuronate-guluronate) blocks. These results indicate that oligoalginate lyase from Sphingomonas sp. MJ-3 probably catalyzes the degradation of both exo- and endo-glycosidic bonds of alginate.

• BMB Reports
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• v.45 no.8
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• pp.476-481
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• 2012

Flavodoxin (Fld) has been demonstrated to bind to ferredoxin-NADP$^+$ reductase A (FprA) in Pseudomonas putida. Two residues ($Phe^{256}$, $Lys^{259}$) of FprA are likely to be important for interacting with Fld based on homology modeling. Site-directed mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-$Ala^{259}$ and FprA-$Asp^{259}$ proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-$Ala^{259}$ and FprA-$Asp^{259}$ were examined by isothermal titration calorimetry. We demonstrated, for the first time, that $Phe^{256}$ and $Lys^{259}$ are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex.

• Journal of Microbiology and Biotechnology
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• v.23 no.10
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• pp.1395-1402
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• 2013

Reductases convert some achiral ketone compounds into chiral alcohols, which are important materials for the synthesis of chiral drugs. The Saccharomyces cerevisiae reductase YOR120W converts ethyl-4-chloro-3-oxobutanoate (ECOB) enantioselectively into (R)-ethyl-4-chloro-3-hydroxybutanoate ((R)-ECHB), an intermediate of a pharmaceutical. As YOR120W requires NADPH as a cofactor for the reduction reaction, a cofactor recycling system using Bacillus subtilis glucose dehydrogenase was employed. Using this coupling reaction system, 100 mM ECOB was converted to (R)-ECHB. A homology modeling and site-directed mutagenesis experiment were performed to determine the NADPH-binding site of YOR120W. Four residues (Q29, K264, N267, and R270) were suggested by homology and docking modeling to interact directly with 2'-phosphate of NADPH. Among them, two positively charged residues (K264 and R270) were experimentally demonstrated to be necessary for NADPH 2'-phosphate binding. A mutant enzyme (Q29E) showed an enhanced enantiomeric excess value compared with that of the wild-type enzyme.

• Bulletin of the Korean Chemical Society
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• v.35 no.8
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• pp.2317-2325
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• 2014

P-glycoprotein (P-gp) is a member of the ATP-Binding Cassette transporter superfamily and mediates transmembrane efflux of many drugs. Since it is involved in multi-drug resistance activity in various cancer cells, the development of P-gp inhibitor is one of the major concerns in anticancer therapy. Human P-gp protein has at least two "functional" drug binding sites that are called "H" site and "R" site, hence it has multi-binding-specificities. Though the amino acid residues that constitute in drug binding pockets have been proposed by previous experimental evidences, the shapes and the binding poses are not revealed clearly yet. In this study, human P-gp structure was built by homology modeling with available crystal structure of mouse P-gp as a template and docking simulations were performed with inhibitors such as verapamil, hoechst33342, and rhodamine123 to construct the interaction between human P-gp and its inhibitors. The docking simulations were performed 500 times for each inhibitor, and then the interaction frequency of the amino acids at the binding poses was analyzed. With the analysis results, we proposed highly contributing residues that constitute binding pockets of the human P-gp for the inhibitors. Using the highly contributing residues, we proposed the locations and the shapes of verapamil binding site and "R" site, and suggested the possible position of "H" site.

• Journal of Microbiology and Biotechnology
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• v.18 no.1
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• pp.48-54
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• 2008

A putative ${\omega}$-aminotransferase gene, cc3143 (aptA), from Caulobacter crescentus was screened by bioinformatical tools and overexpressed in E. coli, and the substrate specificity of the ${\omega}$-aminotransferase was investigated. AptA showed high activity for short-chain ${\beta}$-amino acids. It showed the highest activity for 3-amino-n-butyric acid. It showed higher activity toward aromatic amines than aliphatic amines. The 3D model of the ${\omega}$-aminotransferase was constructed by homology modeling using a dialkylglycine decarboxylase (PDB ID: 1DGE) as a template. Then, the ${\omega}$-aminotransferase was rationally redesigned to increase the activity for 3-amino-3-phenylpropionic acid. The mutants N285A and V227G increased the relative activity for 3-amino-3-phenylpropionic acid to 3-amino-n-butyric acid by 11-fold and 3-fold, respectively, over that of wild type.

• Proceeding of EDISON Challenge
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• 2017.03a
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• pp.46-53
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• 2017

알츠하이머병은 치매를 유발하는 가장 주된 원인 질환으로 환자들은 인지장애를 겪게 된다. 현재 치료약으로 사용되는 약으로는 acetylcholinesterase 저해재가 있지만 이 약들의 효과는 미비하다. 그래서 인지기능에 영향을 미친다고 알려진 신경전달물질인 GABA, Glutamate, acetylcholine의 수치를 조절 할 수 있는 $5-HT_6$ receptor antagonist가 현재 개발되고 있다. 현재 여러 antagonist들이 임상실험 되었고, 인지 능력향상에 효과를 보이고 있다. 그러나, $5-HT_6$ receptor의 구조가 밝혀지지 않아 아직 원자적 수준의 결합 분석이 이루어지지 않았으므로 이 부분에 대한 연구가 필요하다. 따라서 본 연구에서는 Homology modeling을 통해 receptor의 구조를 예측하고, 현재 임상실험 중인 antagonist들 중 7개를 docking을 통해 단백질과 리간드의 결합을 예측하였다. Edison에서 Galaxy TBM과 Galaxy Refine을 사용하여 Homology modeling 한 결과 GPCR의 전형적인 모델에 특징적으로 긴 cterminal을 가졌다는 것을 확인 할 수 있었다. 생성된 구조를 가지고 Edison의 Dock 프로그램으로 7개의 antagonist가 어떠한 결합을 하는지 분석하였다. 그 결과, binding pose에 공통적으로 Trp102, Asp106, Val107, Pro177, Phe188, Val189, Ala192, Phe284, Phe285, Asn288, Thr306, Tyr310이 관여하는 것을 docking을 통해 알 수 있었다. 특히, Phe285는 7개의 antagonist 중에 4개와의 interaction을 하고 있는 것을 관찰하였다. 이 연구를 통하여 $5-HT_6$에 효과적으로 결합하여 치료효과를 낼 수 있는 신약을 개발할 수 있다.