• 제목/요약/키워드: Nucleoside

검색결과 284건 처리시간 0.023초

Isolation and Characterization of Engineered Nucleoside Deoxyribosyltransferase with Enhanced Activity Toward 2'-Fluoro-2'-Deoxynucleoside

  • Yoo, Yeon-Jin;Choi, Kang-Hyun;Kim, Byoung-Kyun;Choi, Si-Sun;Kim, Eung-Soo
    • Journal of Microbiology and Biotechnology
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    • 제32권8호
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    • pp.1041-1046
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    • 2022
  • Nucleoside deoxyribosyltransferase (NDT) is an enzyme that replaces the purine or pyrimidine base of 2'-deoxyribonucleoside. This enzyme is generally used in the nucleotide salvage pathway in vivo and synthesizes many nucleoside analogs in vitro for various biotechnological purposes. Since NDT is known to exhibit relatively low reactivity toward nucleoside analogs such as 2'-fluoro-2'-deoxynucleoside, it is necessary to develop an enhanced NDT mutant enzyme suitable for nucleoside analogs. In this study, molecular evolution strategy via error-prone PCR was performed with ndt gene derived from Lactobacillus leichmannii as a template to obtain an engineered NDT with higher substrate specificity to 2FDU (2'-fluoro-2'-deoxyuridine). A mutant library of 214 ndt genes with different sequences was obtained and performed for the conversion of 2FDU to 2FDA (2'-fluoro-2'-deoxyadenosine). The E. coli containing a mutant NDT, named NDTL59Q, showed 1.7-fold (at 40℃) and 4.4-fold (at 50℃) higher 2FDU-to-2FDA conversions compared to the NDTWT, respectively. Subsequently, both NDTWT and NDTL59Q enzymes were over-expressed and purified using a His-tag system in E. coli. Characterization and enzyme kinetics revealed that the NDTL59Q mutant enzyme containing a single point mutation of leucine to glutamine at the 59th position exhibited superior thermal stability with enhanced substrate specificity to 2FDU.

An improved synthesis of 2'-fluoro-2'-deoxyarabinofuranosyl pyrimidine nucleosides

  • Chun, Moon-Woo
    • Archives of Pharmacal Research
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    • 제6권1호
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    • pp.79-81
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    • 1983
  • Several potent anti-herpes virus nucleosides, 2'fluoro-2'-deoxyarabinofuranosyl pyrimidine nucleosides, were prepared in good yields by a new condensation method using sodium iodide and a new solvent system, and FMAU could be also prepared directly from thymine by this method.

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부추 잎으로부터 Adenosine의 분리와 유리 아미노산 조성 (Isolation of Adenosine and Free Amino Acid Composition from the Leaves of Allium tuberosum)

  • Park, Jae-Sue;Kim, Jae-Yeun;Lee, Ji-Hyon;Young, Han-Suk;Lee, Tae-Woong
    • 한국식품영양과학회지
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    • 제21권3호
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    • pp.286-290
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    • 1992
  • 부추 잎으로부터 purine nucleoside인 adenosine을 분리하고 그 구조를 기기적인 분석방법에 의하여 동정하였으며 또한 유리 아미노산 관련 화합물들의 조성과 상대함량을 표준품과 동일 조건하에서 아미노산 자동 분석 기기로 비교 검토하였다. 가장 함량이 많은 유리 아미노산들은 ala-nine, glutamic acid, aspartic acid 그리고 valine이었다.

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Interaction Characteristics of Nucleoside Analogues with Human Organic Anion Transporter 1 and 3

  • Choi, Jun-Shik;Cheon, Eun-Pa;Han, Hyo-Kyung
    • Journal of Pharmaceutical Investigation
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    • 제36권4호
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    • pp.283-286
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    • 2006
  • The present study aimed to investigate the interaction of nucleoside analogs with human organic anion transporter 1 and 3(hOAT1 and hOAT3) that play a primary role in the tubular uptake of endogenous and exogenous organic anions in the kidney. The interactions of ddC, ara-C, ara-A and ara-U with hOAT1 and hOAT3 were examined using MDCK cells stably overexpressing hOAT1 or hOAT3. Among the tested drugs, ddC showed the highest affinity to hOAT1 with $IC_{50}$ values of 5.2 mM, while ara-A, ara-C and ara-U weakly inhibited the cellular uptake of $[^3H]-PAH$ in MDCK-hOAT1 cells at 1 mM. In contrast, all the tested drugs did not have any inhibition effect on the cellular uptake of $[^3H]-estrone$ sulfate in MDCK-hOAT3 cells over the drug concentration of 0.01-2 mM, implying that they might not interact with hOAT3. Taken all together, the present study suggests that hOAT1 could weakly interact with nucleoside analogues such as ddC, ara-C, ara-A and ara-U but the interaction with hOAT3 during the urinary excretion of these nucleoside analogues may be negligible in the kidney.

Chemical Modification of Nucleic Acids toward Functional Nucleic Acid Systems

  • Venkatesan, Natarajan;Seo, Young-Jun;Bang, Eun-Kyoung;Park, Sun-Min;Lee, Yoon-Suk;Kim, Byeang-Hyean
    • Bulletin of the Korean Chemical Society
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    • 제27권5호
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    • pp.613-630
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    • 2006
  • Nucleic acids are virtually omnipresent; they exist in every living being. These macromolecules constitute the most important genetic storage material: the genes. Genes are conserved throughout the evolution of all living beings; they are transmitted from the parents to their offspring. Many interdisciplinary research groups are interested in modifying nucleic acids for use in a wider variety of applications. These modified oligonucleotides are used in many diverse fields, including diagnostics, detection, and therapeutics. In this account, we summarize our research efforts related to modified nucleic acid systems. First, we discuss our syntheses of modified oligonucleotides containing fluorescent tags for use as molecular probes (molecular beacons) to detect single-nucleotide polymorphisim (SNP) in nucleic acids and to distinguish between the B and Z forms of DNA. We also describe our research efforts into oligonucleotides functionalized with steroid derivatives to enhance their cell permeability, and the synthesis of several calix[4]arene-oligonucleotide conjugates possessing the ability to form defined triplexes. In addition, we have performed systematic studies to have an understanding about the functional groups necessary for a given nucleoside to behave as an organo or hydrogelator. The aggregation properties of a number of nucleoside-based phospholipids have been examined in different solvents; some of these derivatives are potential candidates for use as nucleoside-based liposomes. Finally, we also describe our research efforts toward the preparation of isoxazole- and isoxazoline-containing nucleoside derivatives and the determination of their antiviral activities.

Saccharomyces cerevisiae에서 얻은 Purine Nucleoside Phosphorylase의 반응 속도론적 분석 (Kinetic Analysis of Purine Nucleoside Phosphorylase in Saccharomyces cerevisiae)

  • Choi, Hye-Seon
    • 미생물학회지
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    • 제31권2호
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    • pp.148-156
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    • 1993
  • Kinetic parameters of purine nucleoside phosphorylase (PNP) from Saccharomyces cerevisiae were measured. The Michaelis constants determined for substrates of the enzyme were $ 2.0 * 10^{-4}$ M for inosine, $2.0 *10^{-3}$ M for deoxyinosine, $ 2.0 * 10^{-5}$ M for guanosine and $2.0 10 ^{-5}$ M for deoxyguanosine. According to the ratio of relative $K_{cat}$Km, substrate specificity of each nucleoside was in the order of guanosine or deoxyguanosine, inosine and deoxyinosine. Cosubstrate, phosphate, revealed downward curvature in Lineweaver-Burk plot at high concentrations, indicating a negative cooperativity between subunits. The inhibition constants for purine analogs were measured to be $ 6 * 10^{-4}$ M for formycin B as the competitive inhibitor of inosine, $ 9 * 10^{-6}$ M for guanine as the competitive inhibitor of guanosine, $2 * 10^{-4}$ M for hypoxanthine as the non competitive inhibitor of guanosine and $4.5 * 10 ^{-4}$ M for 6-mercaptopurine as the non competitive inhibitor of guanosine. Alternative substrates, guanosine, deoxyguanosine and adenosine were found to act as competitive inhibitors with Ki values o $f^ 2.0 * 10 {-5}$ M, $2.6 * 10^{-5}$ M and $8.5 * 10 ^{-4}$ M, respectively, when inosine was the variable substrate. Guanosine and deoxyguanosine were also observed as competitive inhibitors with the Ki values of $1.8 * 10^{-5}$ M and $ 3.0 * 10^{-5}$ M, respectively, when deoxyinesine was the variable substrate. The results of alternative substrate sstudies suggested that a single enzyme acted on different nucleosides, inosine, deoxyinosine, adenosine, guanosine and deoxyguanosine.e.

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더덕에서 Nucleoside Diphosphate Kinase 1 분리 및 분석 (Isolation and Characterization of Nucleoside Diphosphate Kinase 1 of Codonopsis lanceolata)

  • 김종학;양덕춘
    • 한국자원식물학회지
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    • 제16권3호
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    • pp.257-263
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    • 2003
  • 더덕의 재배는 수익성이 높고 재배면적도 증가하지만 수요를 만족시킬 만큼 공급이 따르지 못하고 있다. 또한 재 배 상의 어려운점은 병충해, 기계수확에 의한 대규모 재배를 더욱더 곤란하게 하고 있는 실정이다. 이러한 문제점 및 환경적 스트레스에 저항하여 자랄 수 있는 식물체를 얻기 위해 더덕의 cDNA를 분석하여 스트레스 관한 유전자 Nucleoside diphosphates kinase 1(NDK 1)을 분리하여 분석하여 148개의 아미노산 서열과 다른 식물체들의 NDK 1과 높은 유사성을 가진다는 것을 알았고, 더덕의 각 조직에서 나타나는 ClNDK1의 발현을 알아보기 위해 캘러스, 잎, 줄기, 뿌리 조직의 전체 RNA를 추출하여 cDNA를 합성하고 PCR을 수행하였다 RT­PCR 분석 결과, ClNDK1은 조직 특이성 없이 캘러스, 잎, 줄기, 뿌리 조직에 대해서 모두 발현이 되었으며, 발현량, 역시 큰 차이 없이 모든 조직에서 동일하게 발현되었다. NDKs 는 환경 스트레스에 저항성을 가진다고 알려져 있지만 NDK 1 대한 연구는 아직까지 부족한 상태이다. 우리는 더덕에서 분리한 ClNDK1의 스트레스 저항성에 대해서 지속적으로 연구를 수행 할 것이다.

Bradykinin Receptor의 발현에 미치는 녹농균유래 Nucleoside Diphosphate Kinase 및 Flagellin의 효과 (Upregulaton of Bradykinin Receptor Mediated by Nucleoside Diphosphate Kinase and Flagellin from Pseudomonas aeruginosa)

  • 김용재;신희성;;하운환
    • 미생물학회지
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    • 제50권4호
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    • pp.281-284
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    • 2014
  • 병원성 균주인 녹농균(Pseudomonas aeruginosa) 감염에 대응하여 나타나는 면역반응은 인체의 항상성 유지에 중요하다. 선행 연구에서 녹농균의 감염에 대응하여 bradykinin receptor (BR)의 발현이 증가됨을 보고하였지만, 발현유도에 관여하는 녹농균 유래인자에 대해서는 보고한 바가 없었다. 이번 연구에서는 녹농균에 의한 BR의 발현은 Type III secretion system (T3SS)이 관여하지만, 기존에 알려진 T3SS인자가 아닌 nucleoside diphosphate kinase (Ndk)에 의한 것으로 조사되었다. 하지만 pDNNDK를 이용한 transfection 실험 결과, Ndk 만으로는 BR의 발현이 유도되지 않았으며, Ndk와 함께 flagella가 필요함을 발견하였다. 이러한 결과는 기존에 보고된 주요 pathogen-associated molecular patterns (PAMPs)인 flagella와 더불어 감염대응에 관여하는 Ndk를 발굴한 의미가 있으며, 녹농균에 의한 질병기전을 이해하는데 도움을 줄 수 있다.

Facile Synthesis of 2',5'-Dideoxy-, 2',3'-Dideoxy- and 3'-Deoxy-1, N6-ethenoadenosine Nucleosides

  • Chae, Whi-Gun
    • Biotechnology and Bioprocess Engineering:BBE
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    • 제4권1호
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    • pp.17-20
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    • 1999
  • Facile synthetic methods of 2',5'-dideoxy-, 2',3'-dideoxy- and 3'-deoxy-1, N6-ethenoadenosine nucleosides by either an enzymatic dideoxyribosyl transfer reaction or a simple chemical reaction were proposed. The synthesis products were isolated and purified by preparative HPLC and their structures were confirmed by 1H NMR (500 MHz) and FAB-MS including high resolution mass measurement. These modified nucleoside analogs have not been reported yet. Therefore, these modified nucleoside analogs are of potential value to be studied further for biological activity such as anticancer or antiviral.

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