CMP-KDO synthetase: overproduction and application to the synthesis of CMP-KDO and analogs.
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.
Sugai
T
T
Department of Chemistry, Scripps Research Institute, La Jolla, CA 92037, USA.
Lin
C H
CH
Shen
G J
GJ
Wong
C H
CH
D001483:Base Sequence; D003001:Cloning, Molecular; D003568:Cytidine Monophosphate / Q000031:analogs & derivatives* / Q000096:biosynthesis; D015870:Gene Expression; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009713:Nucleotidyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D013379:Substrate Specificity; D013400:Sugar Acids / Q000737:chemistry / Q000378:metabolism
D013400:Sugar Acids; C094610:cytidine-5'-monophosphate-3-deoxy-manno-octulosonic acid; C002532:2-keto-3-deoxyoctonate; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase; D003568:Cytidine Monophosphate