CMP-KDO synthetase: Overproduction and application to the synthesis of CMP-KDO and analogs

Takeshi Sugai, Chun Hung Lin, Gwo Jenn Shen, Chi Huey Wong

研究成果: Article査読

14 被引用数 (Scopus)


CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, EC has been cloned and overexpressed in Escherichia coli. The structure gene was amplified from the total DNA of E coli K-235 through the primer-directed polymerase chain reaction. The gene was then cloned into lambda ZAP vector at the EcoRI and XbaI restriction sites and overexpressed in E coli Sure strain at a level approximately 400 times as much as that produced in the host strain. Application of the enzyme to the synthesis of cytidine 5′-monophospho-3-deoxy-d-manno-2-octulosonic acid (CMP-KDO) and analogs was studied. Of several KDO analogs tested, 5-fluoro-2-keto-3,5-dideoxyoctulosonic acid (5-FKDO) was found to be a good substrate of the enzyme, and the product (CMP-5-FKDO) was prepared and characterized, representing the first stable CMP-KDO analog prepared enzymatically to date. The natural enzyme product, CMP-KDO, was however quite unstable (t 1 2 ≈ 19 min, in 50 mM MgCl2 0.2 M Tris buffer, pH 9.0). A mechanism for the decomposition of CMP-KDO involving the hydrogen bonding interactions between the OH groups of C-5 and C-7 (and/or C-8) and the phosphate oxygens was proposed.

ジャーナルBioorganic and Medicinal Chemistry
出版ステータスPublished - 1995 3

ASJC Scopus subject areas

  • 生化学
  • 分子医療
  • 分子生物学
  • 薬科学
  • 創薬
  • 臨床生化学
  • 有機化学


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