Real-time NMR monitoring of intermediates and labile products of the bifunctional enzyme UDP-apiose/UDP-xylose synthase

The conversion of UDP-alpha-D-glucuronic acid to UDP-alpha-D-Xylose and UDP-alpha-D-apiose by a bifunctional potato enzyme UDP-apiose/UDP-xylose synthase was studied using real-time nuclear magnetic resonance (NMR) spectroscopy. UDP-alpha-D-glucuronic acid is converted via the intermediate uridine 5'-beta-L-threo-pentapyranosyl-4 ''-ulose diphosphate to UDP-alpha-D-apiose and simultaneously to UDP-alpha-D-xylose. The UDP-alpha-D-apiose that is formed is unstable and is converted to alpha-D-apio-furanosyl-1,2-cyclic phosphate and LIMP. High-resolution real-time NMR spectroscopy is a powerful tool for the direct and quantitative characterization of previously undetected transient and labile components formed during a complex enzyme-catalyzed reaction. (C) 2009 Elsevier Ltd. All rights reserved.