PMID:7606392
| Citation |
Sugai, T, Lin, CH, Shen, GJ and Wong, CH (1995) CMP-KDO synthetase: overproduction and application to the synthesis of CMP-KDO and analogs. Bioorg. Med. Chem. 3:313-20 |
|---|---|
| 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 (t1/2 = 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. |
| Links | |
| Keywords |
Base Sequence; Cloning, Molecular; Cytidine Monophosphate/analogs & derivatives; Cytidine Monophosphate/biosynthesis; Gene Expression; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Molecular Structure; Nucleotidyltransferases/biosynthesis; Nucleotidyltransferases/genetics; Nucleotidyltransferases/isolation & purification; Nucleotidyltransferases/metabolism; Polymerase Chain Reaction; Substrate Specificity; Sugar Acids/chemistry; Sugar Acids/metabolism |
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