Novel method for enzymatic synthesis of CMP-NeuAc.
A novel method for synthesizing CMP-NeuAc was established. We first confirmed that the putative neuA gene of Haemophilus influenzae, identified by its whole genome sequence project, indeed encodes CMP-NeuAc synthetase (EC 2.7.7.43). The enzyme requires CTP as a cytidylyl donor for cytidylylation of NeuAc. The enzyme was coupled with an enzymatic CTP-generating system from CMP and inorganic polyphosphate as a sole phospho-donor driven by the combination of polyphosphate kinase and CMP kinase, where phosphorylation of CMP is done by the combined activity expressed by both enzymes, and subsequent phosphorylation of CDP by polyphosphate kinase itself occurred efficiently. When CMP-NeuAc synthetase of H. influenzae, polyphosphate kinase, and CMP kinase were added to the reaction mixture containing equimolar concentrations (15 mM) of CMP and NeuAc, and polyphosphate (150 mM in terms of phosphate), CMP-NeuAc was synthesized up to 10 mM in 67% yield.
Ishige
K
K
Biochemicals Division, YAMASA Corporation, Choshi, Chiba, Japan. ishige-k@yamasa.com
Hamamoto
T
T
Shiba
T
T
Noguchi
T
T
D003568:Cytidine Monophosphate / Q000031:analogs & derivatives* / Q000138:chemical synthesis*; D003570:Cytidine Triphosphate / Q000138:chemical synthesis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006193:Haemophilus influenzae / Q000201:enzymology / Q000235:genetics; D000216:N-Acylneuraminate Cytidylyltransferase / Q000138:chemical synthesis*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000737:chemistry / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000138:chemical synthesis; D012794:Sialic Acids / Q000138:chemical synthesis*
D011994:Recombinant Proteins; D012794:Sialic Acids; D003570:Cytidine Triphosphate; D017856:Phosphotransferases (Phosphate Group Acceptor); C022424:polyphosphate kinase; D000216:N-Acylneuraminate Cytidylyltransferase; D003568:Cytidine Monophosphate