Determination of the anomeric specificity of the Escherichia coli CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyltransferase by 13C NMR spectroscopy.

摘要: [99%, 1-13C]- and [90%, 2-13C]3-deoxy-D-manno-octulosonic acid (KDO) were prepared enzymatically used to determine the anomeric specificity of CTP:CMP-3-deoxy-D-manno-octulosonate cytidylyl transferase (CMP-KDO synthetase) by 13C NMR spectroscopy. Addition CMP-KDO synthetase reaction mixtures containing either 1-13C- or 2-13C-labeled KDO resulted in rapid formation which was accompanied a substantial decrease 13C-enriched resonances beta-pyranose form relative other species solution, demonstrating that is preferred substrate. Concomitant with production appearance peaks at 174.3 101.4 ppm when [1-13C]- [2-13C]KDO, respectively, as substrates. The correspondence these enriched carbons confirmed expected 3-bond (3JP,C-1 = 6.9 Hz) 2-bond coupling (2JP,C-2 8.3 between labeled ketosidically linked phosphoryl group. A large (3J 5.7 observed proton-coupled spectra CMP-[1-13C]KDO carbon 1 axial proton 3 KDO. magnitude this constant supports diaxial relationship two groups and, along chemical shift data, indicates retains beta-configuration CMP-KDO.