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

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

CTP:CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, EC 2.7.7.38) 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.

Original languageEnglish
Pages (from-to)313-320
Number of pages8
JournalBioorganic and Medicinal Chemistry
Volume3
Issue number3
DOIs
Publication statusPublished - 1995
Externally publishedYes

Fingerprint

Escherichia coli
Cytidine Monophosphate
Enzymes
Genes
Cytidine Triphosphate
Cytidine
Tromethamine
Magnesium Chloride
Acids
Polymerase chain reaction
Hydrogen Bonding
Hydrogen bonds
Phosphates
Oxygen
Decomposition
Polymerase Chain Reaction
cytidine-5'-monophosphate-3-deoxy-manno-octulosonic acid
3-deoxy-manno-octulosonate cytidyltransferase
DNA
Substrates

ASJC Scopus subject areas

  • Biochemistry
  • Clinical Biochemistry
  • Molecular Biology
  • Molecular Medicine
  • Organic Chemistry
  • Drug Discovery
  • Pharmaceutical Science

Cite this

CMP-KDO synthetase : Overproduction and application to the synthesis of CMP-KDO and analogs. / Sugai, Takeshi; Lin, Chun Hung; Shen, Gwo Jenn; Wong, Chi Huey.

In: Bioorganic and Medicinal Chemistry, Vol. 3, No. 3, 1995, p. 313-320.

Research output: Contribution to journalArticle

Sugai, Takeshi ; Lin, Chun Hung ; Shen, Gwo Jenn ; Wong, Chi Huey. / CMP-KDO synthetase : Overproduction and application to the synthesis of CMP-KDO and analogs. In: Bioorganic and Medicinal Chemistry. 1995 ; Vol. 3, No. 3. pp. 313-320.
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