31P and 13C NMR studies of oxygen transfer during catalysis by 3-deoxy-D-manno-octulosonate cytidylyltransferase from Escherichia coli.
[18O]3-Deoxy-D-manno-octulosonate (KDO), labeled at the anomeric oxygen, was prepared by exchange with [18O]H2O and used to follow the route of oxygen transfer during cytidine 5'-monophosphate-3-deoxy-D-manno-octulosonate (CMP-KDO) formation catalyzed by 3-deoxy-D-manno-octulosonate cytidylyl-transferase (CMP-KDO synthetase). The 31P-NMR signal of the phosphoryl group of CMP-KDO (-5.85 ppm), which appeared as a single resonance when CMP-KDO formation took place with unenriched KDO, appeared as two peaks when CMP-KDO formation took place in the presence of a mixture of [16O]-and [18O]KDO. These results demonstrate the retention of 18O during CMP-KDO formation. Confirmation that the labeled oxygen in CMP-KDO was retained in the "bridge" position between CMP and KDO came from 13C-NMR studies of CMP-KDO formed in the presence of 90% [2-13C, 18O] KDO. The prominent C-2 KDO resonance in CMP-KDO, which is normally a doublet at 101.4 ppm (Kohlbrenner, W.E., and Fesik, S.W. (1985) J. Biol. Chem. 260, 14695-14700), appeared as four peaks when a mixture of [2-13C,16O]- and [2-13C, 18O]KDO was used, confirming the direct bonding of 18O to the C-2 of KDO in CMP-KDO. These results are consistent with a nucleophilic displacement mechanism for CMP-KDO formation.
D002247:Carbon Isotopes; D055598:Chemical Phenomena; D002621:Chemistry; D004926:Escherichia coli / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes; D010758:Phosphorus
D002247:Carbon Isotopes; D010103:Oxygen Isotopes; D010758:Phosphorus; D009713:Nucleotidyltransferases; C028984:3-deoxy-manno-octulosonate cytidyltransferase; D010100:Oxygen