title: Probing a CMP-Kdn synthetase by 1H, 31P, and STD NMR spectroscopy.
abstract: CMP-Kdn synthetase catalyses the reaction of sialic acids (Sia) and cytidine-5'-triphosphate (CTP) to the corresponding activated sugar nucleotide CMP-Sia and pyrophosphate PP(i). STD NMR experiments of a recombinant nucleotide cytidine-5'-monophosphate-3-deoxy-d-glycero-d-galacto-nonulosonic acid synthetase (CMP-Kdn synthetase) were performed to map the binding epitope of the substrate CTP and the product CMP-Neu5Ac. The STD NMR analysis clearly shows that the anomeric proton of the ribose moiety of both investigated compounds is in close proximity to the protein surface and is likely to play a key role in the binding process. The relative rates of the enzyme reaction, derived from (1)H NMR signal integrals, show that Kdn is activated at a rate 2.5 and 3.1 faster than Neu5Ac and Neu5Gc, respectively. Furthermore, proton-decoupled (31)P NMR spectroscopy was successfully used to follow the enzyme reaction and clearly confirmed the appearance of CMP-Sia and the inorganic pyrophosphate by-product.
authors.affiliation: Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 60 Gold Coast Mail Centre, Queensland 9726, Australia.
authors.forename: Thomas
authors.initials: T
authors.lastname: Haselhorst
authors.forename: Anja K
authors.initials: AK
authors.lastname: Münster-Kühnel
authors.forename: Anita
authors.initials: A
authors.lastname: Stolz
authors.forename: Melanie
authors.initials: M
authors.lastname: Oschlies
authors.forename: Joe
authors.initials: J
authors.lastname: Tiralongo
authors.forename: Ken
authors.initials: K
authors.lastname: Kitajima
authors.forename: Rita
authors.initials: R
authors.lastname: Gerardy-Schahn
authors.forename: Mark
authors.initials: M
authors.lastname: von Itzstein
mesh_terms: D002384:Catalysis; D003570:Cytidine Triphosphate / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000378:metabolism*; D010759:Phosphorus Isotopes; D014316:Tritium
chemical_list: D010759:Phosphorus Isotopes; D014316:Tritium; D003570:Cytidine Triphosphate; C079494:CTP-CMP-3-deoxynonulosonate cytidyltransferase; D009713:Nucleotidyltransferases; D019158:N-Acetylneuraminic Acid