• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to previous page

Molecular characterization of all the six enzymes and their corresponding genes involved in de novo pyrimidine biosynthesis in trypanosomatids

Research Project

Project/Area Number 12470061
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field 寄生虫学(含医用動物学)
Research InstitutionJuntendo University

Principal Investigator

AOKI Takashi  Juntendo University School of Medicine, Department of Molecular and Cellular Parasitology, Professor, 医学部, 教授 (20053283)

Co-Investigator(Kenkyū-buntansha) NARA Takeshi  Juntendo University School of Medicine, Department of Molecular and Cellular Parasitology, Assistant Professor, 医学部, 講師 (40276473)
嶋田 淳子  順天堂大学, 医学部, 講師 (20211964)
高宮 信三郎  順天堂大学, 医学部, 助教授 (90138206)
Project Period (FY) 2000 – 2002
Project Status Completed (Fiscal Year 2002)
Budget Amount *help
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2002: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 2001: ¥3,900,000 (Direct Cost: ¥3,900,000)
Fiscal Year 2000: ¥6,700,000 (Direct Cost: ¥6,700,000)
KeywordsPyrimidine nucleotide biosynthesis / Pyrimidine-biosynthetic (pyr) gene cluster / Trypanosoma / Over-expression of pyr gene products / pyr genes knockout by homologous recombination / Enzyme assays for recombinant pyr sene products / Evolution of a metabolic pathway / 代謝経路の進化 / ピリミジンヌクレオチド / 新生合成経路 / pyr遺伝子 / 遺伝子共発現 / 過剰発現 / RNAi / pyr遺伝子群 / 遺伝子クラスター / リーシュマニア / 多機能タンパク質
Research Abstract

Recently, we reported the presence of the pyrimidine-biosynthetic (pyr) gene cluster as a polycistronic transcription unit, that contained pyrl, pyr3, pyr6/5, pyr2, and pyr4 encoding all the six enzymes of the pathway, in Trypanosoma cruzi. Leishmania mexicana also had a homologous pyr gene cluster. The six Leishmania enzymes resembled the corresponding T. cruzi counterparts; the first 3 enzymes (CPS II, ACT, DHO) may have resulted from early eukaryotic ancestors, the fourth (DHOD) and sixth (OMPDC) enzymes may have been acquired by horizontal gene transfers, and the covalently linked sixth/fifth (OPRT) enzymes possess a C-terminal SKL motif, an essential targeting signal into the glycosome (a peculiar organelle in trypanosomatids).
The previously established recombinant DHOD was expressed in E. coli, affinity-purified, and the enzyme activity was measured as the amount of orotate production spectrophotometrically. Crude methanol-extracts from brown algae, Fucus evanescent and Pelvetia … More babingtonii, showed 50 and 70% reduction in the DHOD activity, respectively, at the concentration of 50 micro g/ml. Kinetic analysis exhibited that the mode of action of these extracts on the parasite enzyme was non-competitive with respect to the substrate, dihydroorotate. To estimate the effects of these two extracts on the infection rate, the percentage of host cells infected by T. cruzi, EtOH- reconstituted samples were added to the in vitro infection system, T. cruzi-HeLa cell cultures. Both samples, at the concentration of 10 micro g/ml, markedly lowered the rate of infection to approximately 40% of the control. These results imply that F. evanescens and P. babingtonii contain substances inhibitory to the T. cruzi DHOD activity and to the infection rate of host cells. Further screening of other marine algae and purification of the effective compound(s) may facilitate the discovery of a new, anti-trypanosomal lead compound
Further, we examined effect of the trypanosomal CPS II overproduction in the protozoan using a shattle vector pTEX harboring pyrl; the transformed T. cruzi grew faster in the infected HeLa cells and a greater number of trypomastigotes appeared in the culture medium. The results suggest that a higher level of expression of CPS II. The rate-limiting enzyme of the pyrimidine-biosynthetic pathway, may have resulted in a higher level of pathogenicity, namely, faster growth. Further characterization of the T. cruzi OPRT and OMPDC is of great interest, particularly with respect to the comparison with the Plasmodium enzymes (because of the P. falciparum genome project completed) Less

Report

(4 results)
  • 2002 Annual Research Report   Final Research Report Summary
  • 2001 Annual Research Report
  • 2000 Annual Research Report
  • Research Products

    (28 results)

All Other

All Publications (28 results)

  • [Publications] Nara T, Hashimoto T, Aoki T: "Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes"Gene. 257. 209-222 (2000)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara T, Zou C, Mikami Y, Aoki T: "Two-hybrid analysis of the de novo pyrimidine biosynthetic enzymes from Trypanosoma cruzi"Proceedings of the 10th International Congress of Parasitology, Monduzzi Editore, Italy. 269-272 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Takashima E, Inaoka DK, Osanai A, Nara T, Odaka M, Aoki T, Inaka K, Harada S, Kita K: "Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi"Molecular and Biochemical Parasitology. 122. 189-200 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara T, Hashimoto T, Aoki T: "Evolution of de novo pyrimidine biosynthetic pathway in parasitic protists"Tenth International Congress of Parasitology抄録集. (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara T, Zou C, Aoki T: "Protein interaction of the first three enzymes of de novo pyrimidine biosynthetic pathway in Trypanosoma cruzi"Tenth International Congress of Parasitology抄録集. (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] 奈良武司, 青木 孝: "トリパノソーマのピリミジン生合成遺伝子クラスター"蛋白質 核酸 酵素. 47. 13-20 (2002)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara T, Hirayama-Noguchi Y, Gao G, Murai E, Annoura T, Aoki T: "Diversity of aspartate carbamoyltransferase of Trypanosoma cruzi."International Journal for Parasitology. (印刷中). (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara,T, Hashimoto,T, Aoki,T: "Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes"Gene. 257. 209-222 (2000)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara,T, Zou,C, Mikami,Y, Aoki,T: "Two-hybrid analysis of the de novo pyrimidine biosynthetic enzymes from Trypanosoma cruzi"Proceedings of the tenth International Congress of Parasitology. 269-272 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Takashima,E, Inaoka,DK, Osanai,A, Nara,T, Odaka,M, Aoki,T, Inaka,K, Harada,S, Kita,K: "Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi"Molecular and Biochemical Parasitology. 122. 189-200 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara,T, Hashimoto,T, Aoki,T: "Evolution of de novo pyrimidine biosynthetic pathway in parasitic protists"Tenth Internatinal Congress of Parasitology (abstract). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara,T, Zou,C, Aoki,T: "Protein interaction of the first three enzymes of de novo pyrimidine biosynthetic pathway in Trypanosoma cruzi"Tenth International Congress of Parasitology (abstract). (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara,T, Aoki,T: "The pyrimidine-biosynthetic (pyr) gene cluster in trypanosomes"Protein, Nucleic Acid and Enzyme. 47 in Japanese. 13-20 (2002)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara,T, Hirayama-Noguchi,Y, Gao,G, Murai,E, Annoura,T, Aoki,T: "Diversity of aspartate carbamoy ltransferase of Trypanosoma cruzi"International Journal for Parasitology. in press.

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2002 Final Research Report Summary
  • [Publications] Nara T, Zou C, Mikami Y, Aoki T: "Two-hybrid analysis of the de novo pyrimidine biosynthetic enzymes from Trypanosoma cruzi"Proceedings of the 10th International Congress of Parasitology, Monduzzi Editore, Italy. 269-272 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Takashima E, Inaoka DK, Osanai A, Nara T, Odaka M, Aoki T, Inaka K, Harada S, Kita K: "Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi"Molecular and Biochemical Parasitology. 122. 189-200 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nara T, Hashimoto T, Aoki T: "Evolution of de novo pyrimidine biosynthetic pathway in parasitic protists"Tenth International Congress of Parasitology 抄録集. (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] Nara T, Zou C, Aoki T: "Protein interaction of the first three enzymes of de novo pyrimidine biosynthetic pathway in Trypanosoma cruzi"Tenth International Congress of Parasitology 抄録集. (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 案浦健, 奈良武司, 牧内貴志, 青木孝: "トリパノソーマ科原虫におけるピリミジン合成経路第4酵素dihydroorotate dehydrogenaseの起原"第25回日本分子生物学会抄録集. 464 (2002)

    • Related Report
      2002 Annual Research Report
  • [Publications] 奈良武司, 三上由祐子, 青木孝: "Trypanosoma cruziのカルバモイルリン酸合成酵素II(CPS II)遺伝子導入株の解析"第72回日本寄生虫学会大会抄録集. 84 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] 牧内貴志, 奈良武司, 案浦健, 大島泰郎, 青木孝: "植物鞭毛虫euglenaおよびトリパノソーマ科原虫におけるジヒドロオロト酸デヒドロゲナーゼ(DHOD)の分子進化学的解析"第72回日本寄生虫学会大会抄録集. 84 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] 案浦健, 奈良武司, 牧内貴志, 青木孝: "キネトプラスト目原虫におけるピリミジン合成経路第4酵素dihydroorotate dehydrogenaseの分子進化学的解析"第72回日本寄生虫学会大会抄録集. 84 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] 奈良武司, 青木 孝: "トリパノソーマのピリミジン生合成遺伝子クラスター"蛋白質 核酸 酵素. 47・1. 13-20 (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 奈良武司, 青木孝他: "Trypanosoma cruziのピリミジン生合成経路第4酵素dihydroorotate dehydrogenaseに対する海草抽出液の阻害効果"第71回日本寄生虫学会大会記録. (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] 案浦 健, 青木孝他: "Trypanosoma cruziにおけるピリミジン合成経路第4酵素dihydroorotate dehydrogenase遺伝子のノックアウト株の作製と表現型の解析"第71回日本寄生虫学会大会記録. (2002)

    • Related Report
      2001 Annual Research Report
  • [Publications] Nara T,Hashimoto T,Aoki T: "Evolutionary implications of the mosaic pyrimidine-biosynthetic pathway in eukaryotes"Gene. 257(2). 209-222 (2000)

    • Related Report
      2000 Annual Research Report
  • [Publications] 奈良武司,三上由祐子,青木孝: "クルーズトリパノソーマにおけるピリミジン生合成経路初段3酵素のタンパク質相互作用"第70回日本寄生虫学会大会記録. 102-102 (2001)

    • Related Report
      2000 Annual Research Report
  • [Publications] 高島英造,奈良武司 他: "Trypanosoma cruziのコハク酸/フマル酸酸化還元系について"第70回日本寄生虫学会大会記録. 103-103 (2001)

    • Related Report
      2000 Annual Research Report

URL: 

Published: 2000-04-01   Modified: 2016-04-21  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi