Sugai T, Lin C H, Shen G J, Wong C H
Department of Chemistry, Scripps Research Institute, La Jolla, CA 92037, USA.
Bioorg Med Chem. 1995 Mar;3(3):313-20. doi: 10.1016/0968-0896(95)00023-a.
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.
CMP-3-脱氧甘露糖辛酮酸胞苷酰转移酶（CMP-KDO合成酶，EC 2.7.7.38）已被克隆并在大肠杆菌中过表达。通过引物定向聚合酶链反应从大肠杆菌K-235的总DNA中扩增结构基因。然后将该基因克隆到λZAP载体的EcoRI和XbaI限制位点，并在大肠杆菌Sure菌株中过表达，其表达水平约为宿主菌株的400倍。研究了该酶在胞苷5'-单磷酸-3-脱氧-D-甘露糖-2-辛酮酸（CMP-KDO）及其类似物合成中的应用。在测试的几种KDO类似物中，发现5-氟-2-酮-3,5-二脱氧辛酮酸（5-FKDO）是该酶的良好底物，并制备和表征了产物（CMP-
