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

✍ Scribed by Takeshi Sugai; Chun-Hung Lin; Gwo-Jenn Shen; Chi-Huey Wong

• Publisher: Elsevier Science
• Year: 1995
• Tongue: English
• Weight: 625 KB
• Volume: 3
• Category: Article
• ISSN: 0968-0896
• DOI: 10.1016/0968-0896(95)00023-a

✦ Synopsis

CMP-3-deoxy-manno-octulosonate cytidylyltransferase (CMP-KDO synthetase, EC 2.7.7.38) has been cloned and overexpressed in Escherichia coil The structure gene was amplified from the total DNA of E coli K-235 through the primerdirected polymerase chain reaction. The gene was then cloned into lambda ZAP vector at the EeoRI and Xbal 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 eytidine 5'-monophospho-3-deoxy-D-manno-2-oetulosonie acid (CMP-KDO) and analogs was studied. Of several KDO analogs tested, 5-fluoro-2-keto-3,5-dideoxyoetulosonie 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 (tta ~ 19 rain, in 50 mM MgClz 0.2M Trisbuffer, 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.