• BMB Reports
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• 제41권5호
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• pp.369-375
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• 2008

Polygonatum cyrtonema Lectin (PCL), which is classified as a monocot mannose-binding lectin, has received great regards for its uniquely biological activities and potentially medical applications in cancer cells. This paper was initially aimed to study apoptosis of PCL on Hela cells. Thus, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method was carried out. Through observation of cell morphologic changes and Lactate dehydrogenase (LDH) activity-based cytotoxicity assays, PCL induced HeLa cell apoptosis in a dose-dependent manner. To further gain structural basis, multiple alignments, homology modeling and docking experiments were performed to analyze the correlation between its biological activities and mannose-binding sites. Eventually, considering docking data, chemical modification properties on the three mannose-binding sites were analyzed by a series of biological experiments (e.g., hemagglutinating and mitogenic activity assays, fluorescence and Circular Dichrosim (CD) spectroscopy) to profoundly identify the role of some key amino acids in the structure-function relationship of PCL.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2002년도 생물공학의 동향 (X)
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• pp.457-460
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• 2002

In an effort to isolate novel strains expressing a thermostable esterase that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened various soils and composts from broad ecological niches in which the activity was expected to be found. Three hundreds of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily at high growth and incubation temperature and further compared the ability to ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain JYl44 was isolated as a novel strain that produced a (R)-stereospecific esterase with high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme indeed stables at a broad range of temperature, upto 65 $^{\circ}C$, and pH ranging from 6.0 to 10.0. The optimal temperature and pH for enzymatic conversion were 50 $^{\circ}C$ and 9.0, respectively. Based on the observations that resulted a poor cell growth, and enzyme expression in wild type strain, we further attempted the gene cloning into a general host Escherichia coli and determined its primary structure, concomitantly resulting a high level expression of the enzyme. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa, and its primary structure showed a relative high homology (45-52 %) to the esterases from Streptomyces and Bacillus strains. The recombinant whole cell enzyme could efficiently convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with optical purity of 99 % and yield of 49 %.

• Journal of Microbiology and Biotechnology
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• 제26권12호
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• pp.2019-2029
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• 2016

Zinc finger proteins are among the most extensively applied metalloproteins in the field of biotechnology owing to their unique structural and functional aspects as transcriptional and translational regulators. The classical zinc fingers are the largest family of zinc proteins and they provide critical roles in physiological systems from prokaryotes to eukaryotes. Two cysteine and two histidine residues ($Cys_2His_2$) coordinate to the zinc ion for the structural functions to generate a ${\beta}{\beta}{\alpha}$ fold, and this secondary structure supports specific interactions with their binding partners, including DNA, RNA, lipids, proteins, and small molecules. In this account, the structural similarity and differences of well-known $Cys_2His_2$-type zinc fingers such as zinc interaction factor 268 (ZIF268), transcription factor IIIA (TFIIIA), GAGA, and Ros will be explained. These proteins perform their specific roles in species from archaea to eukaryotes and they show significant structural similarity; however, their aligned amino acids present low sequence homology. These zinc finger proteins have different numbers of domains for their structural roles to maintain biological progress through transcriptional regulations from exogenous stresses. The superimposed structures of these finger domains provide interesting details when these fingers are applied to specific gene binding and editing. The structural information in this study will aid in the selection of unique types of zinc finger applications in vivo and in vitro approaches, because biophysical backgrounds including complex structures and binding affinities aid in the protein design area.

• Journal of Plant Biotechnology
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• 제29권2호
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• pp.79-84
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• 2002

Suppression subtractive hybridization (SSH)를 통해 상처에 의해 발현이 유도되는 cDNA들을 분리하였고, 그 중 하나인 gmwi33은 $\beta$-amyrin synthase 유전자들과 높은 유사성을 보였다. gmwi33의 전장 cDNA인 GmAMS1은 2416 bp 길이에 739개 아미노산으로 구성된 긴 open reading frame(ORF)를 포함하고 있었다. GmAMS1 단백질은 감초의 $\beta$-amyrin synthase인 GgbAS와 89%, 완두의 OSCPSY와 86%의 유사성을 보였다. 암조건 하에 5일간 기른 콩나물에서, GmAMS1는 빛을 쪼여주었을 때 가장 강하게 발현되었고 methyl jasmonate 처리와 저온처리 시에도 발현이 유도된 반면, UV-B나 elicitor를 처리하였을 때는 발현이 유도되지 않았다. 이러한 GmAMS1의 발현양상은 사포닌의 활성산소 제거기능과 밀접한 연관이 있을 것으로 추측된다.

• The Plant Pathology Journal
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• 제23권4호
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• pp.272-280
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• 2007

Isolates of Cucumber mosaic virus (CMV) collected in Korea, were compared with their pathological features in tobacco and zucchini squash. Full-length cDNA clone of RNA3 was generated by using long-distance RT-PCR. Transcript RNA3 from the cDNA clone was inoculated onto host plants with transcripts RNA1 and RNA2 of Fny strain, generating RNA3-pseudorecombinant CMV. Timing and severity of systemic symptom was not significantly different among the pseudorecombinant CMVs in tobacco, compared with strains Fny-CMV and Pf-CMV. However, the pseudorecombinant CMVs induced two different systemic symptoms (mosaic vs. chlorotic spot) in zucchini squash. Based on symptom induction, the pseudorecombinant CMVs were categorized into two classes. The severity and timing of symptoms were correlated with viral RNA accumulations in systemic leaves of zucchini squash, suggesting that different kinetics of virus movement associated with CMV proteins are crucial for systemic infection and symptom development in zucchini squash. The analysis of movement proteins (MP) of CMV strains showed high sequence homology, but the differences of several amino acids were found in the C-terminal region between Class-I-CMV and Class-II-CMV. The analysis of coat proteins (CP) showed that the CMV isolates tested belonged to CMV subgroup I and the viruses shared overall 87-99% sequence identity in their genomes. Phylogenetic analysis of MP and CP suggested that biological properties of Korean CMV isolates have relationships associated with host species.

• Journal of Microbiology and Biotechnology
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• 제20권12호
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• pp.1689-1695
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• 2010

Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Here, we report the identification, cloning, and characterization of panK-sp from Streptomyces peucetius ATCC 27952. The gene encoded a protein of 332 amino acids with a calculated molecular mass of 36.8 kDa and high homology with PanK from S. avermitilis and S. coelicolor A3(2). To elucidate the putative function of PanK-sp, it was cloned into pET32a(+) to construct pPKSP32, and the PanK-sp was then expressed in E. coli BL21(DE3) as a His-tag fusion protein and purified by immobilized metal affinity chromatography. The enzyme assay of PanK-sp was carried out as a coupling assay. The gradual decrease in NADH concentration with time clearly indicated the phosphorylating activity of PanK-sp. Furthermore, the ca. 1.4-fold increase of DXR and the ca. 1.5-fold increase of actinorhodin by in vivo overexpression of panK-sp, constructed in pIBR25 under the control of a strong $ermE^*$ promoter, established its positive role in secondary metabolite production from S. peucetius and S. coelicolor, respectively.

• Journal of Microbiology and Biotechnology
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• 제30권11호
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• pp.1750-1759
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• 2020

The characterization of cytochrome P450 CYP125A13 from Streptomyces peucetius was conducted using cholesterol as the sole substrate. The in vitro enzymatic assay utilizing putidaredoxin and putidaredoxin reductase from Pseudomonas putida revealed that CYP125A13 bound cholesterol and hydroxylated it. The calculated K_D value, catalytic conversion rates, and Km value were 56.92 ± 11.28 μM, 1.95 nmol min^-1 nmol^-1, and 11.3 ± 2.8 μM, respectively. Gas chromatography-mass spectrometry (GC-MS) analysis showed that carbon 27 of the cholesterol side-chain was hydroxylated, characterizing CYP125A13 as steroid C27-hydroxylase. The homology modeling and docking results also revealed the binding of cholesterol to the active site, facilitated by the hydrophobic amino acids and position of the C27-methyl group near heme. This orientation was favorable for the hydroxylation of the C27-methyl group, supporting the in vitro analysis. This was the first reported case of the hydroxylation of cholesterol at the C-27 position by Streptomyces P450. This study also established the catalytic function of CYP125A13 and provides a solid basis for further studies related to the catabolic potential of Streptomyces species.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2018년도 춘계학술발표회
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• pp.17-17
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• 2018

Panax ginseng Meyer is a perennial medicinal plant, the roots of which has been used in the traditional formulations in Oriental countries. To understand its floral transition, we isolated Flowering Locus T (FT) from ginseng, the bioinformatics analysis of PgFT has revealed a considerable homology to the higher plants, with the essential amino acids for FT function are conserved. The phylogenetic analysis has shown that the PgFT is belonged to the shrub classes of plants and closest kin to Jatropha curcas FT. The expression profiling from juvenile (2-year-old) were abundant in leaves as well as in root and was concentrated in the secondary leaflet and stem bottom in adult (4-year-old) ginseng plant tissues, moreover PgFT transcript displayed photoperiod dependent oscillation. The ectopic expression of PgFT in Arabidopsis thaliana, exhibit precocious flowering and several floral pathway integrators were up-regulated, interestingly their root length was increased in the transgenic seedlings. Therefore, we could conclude that PgFT encodes a florigen that acts as a key regulator in the flowering pathway in ginseng and hypothesize that, it might involve in the underground organ development as well. We believe our finding could provoke future studies on the physiology and development in P. ginseng.

• 생명과학회지
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• 제17권6호통권86호
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• pp.772-777
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• 2007

식물의 루이신 생합성에 관여하는 3-isopropylmalate isomerase (IPMI) (EC 4.2.1.33) 효소의 소단편을 암호화는 Leucine D유전자를 벼로부터 분리하고 OsLeuD유전자로 명명하였다. OsLeuD유전자는 257개의 아미노산을 암호화하고 있으며 cyanobacteria의 IPMI 단백질과는 약58% 그리고 green sulfur bacteria들의 IPMI 단백질과는 약48%의 상동성을 갖고 있었다. 벼의 OsLeuD 유전자는 japonica벼 (Oryza sativa L.)의 2번 염색체의 26.45 Mb의 위치로서 109.3 cM 거리에 좌위하고 있었다. OsLeuD유전자는 잎과 성숙하는 종자에 많이 발현이 되었으므로 대사가 급증하는 발생단계 에 발현이 조절되는 것으로 여겨진다. OsLeuD유전자와 단백질은 균류와 yeast 보다 광합성 박테리아의 유전자와 높은 동질성을 보이는 것으로 보아 OsLeuD유전자는 식물의 엽록체 유전자 genome 에서 기원하여 핵 genome으로 이동 진화된 유전자로 추측된다.

• Journal of Ginseng Research
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• 제19권2호
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• pp.127-133
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• 1995

The complete open reading frame (ORF) of o-subunit of the $F_1$ ATP synthase (atPA) in Korean ginseng mitochondria was identified by the sequence similarity with atPA genes in other plant mitochondria. The sequence alignment showed that the common translation initiation codon, ATG, in plant genes was replaced with GTG valid codon in Korean ginseng. The atPA gene from GTG to TGA termination codon was 1524 nucleotides long, and the sequence homology of nucleotides and deduced amino acids revealed high values of 92~97%. A deletion event of 6 nucleotides was observed at the 1468th nucleotide from the GTG in Korean ginseng, in contrast to that at the 1450th in other plants such as pea, common bean, soybean, sugar beet, and radish. An unidentified open reading frame (on7) was observed upstream of atmA ORF. No other ATG as an initiation codon was detected in the region between off and atmA ORF in Korean ginseng, although a pyrimidine cluster "TTTTCTTTT" was located in this region as in Oenothera and maize genes. It could be supposed that GTG codon in atpA gene of Korean ginseng mitochondria would act as an initiation codon as in microbial genes.ial genes.