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

Abstract

The conversion of UDP-α-d-gdlucuronic acid to UDP-α-d-xylose and UDP-α-d-apiose by a bifunctional potato enzyme UDP-apiose/UDP-xylose synthase was studied using real-time nuclear magnetic resonance (NMR) spectroscopy. UDP-α-d-glucuronic acid is converted via the intermediate uridine 5′-β-l-threo-pentapyranosyl-4″-ulose diphosphate to UDP-α-d-apiose and simultaneously to UDP-α-d-xylose. The UDP-α-d-apiose that is formed is unstable and is converted to α-d-apio-furanosyl-1,2-cyclic phosphate and UMP. 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.

Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution

“…7 and supplemental Table S1) confirmed that UDP-Api is the first product formed. SpUAS produces UDP-Api and UDP-Xyl in a ratio of ϳ1.7: 1.0, which is similar to potato UAS (30). Our studies with SpUAS also confirm that some of the UDP-Api is converted to the apiofuranosyl-1,2-cyclic phosphate during the in vitro reaction (Fig.…”

“…Real time NMR-based assays provide the opportunity to detect transient intermediates (30). Our real time 1 H NMR data using recombinant SpUAS confirm that UDP-4-keto-xylose is an intermediate formed during the conversion of UDP-GlcA to UDP-Api (Fig.…”

“…Continuous 1 H NMR spectroscopic monitoring of reactions (180-l volume) was carried out at 37°C in a mixture of D 2 O/H 2 O (9:1, v/v) containing 0.83 mM 2,2-dimethyl-2-silapentane-5-sulfonate (internal reference), 50 mM Tris-HCl, pH 7.9, 1 mM UDP-GlcA, 1 mM NAD ϩ , and purified recombinant enzyme (30 g). One-dimensional 1 H NMR spectra with the water resonance signal presaturated were collected 5 min after addition of enzyme to optimize spectrometer settings, and then spectra were continuously averaged every 2 min for up to 9 h. All chemical shifts are referenced to 2,2-dimethyl-2-silapentane-5-sulfonate at 0.00 ppm (30).…”

UUAT1 Is a Golgi-Localized UDP-Uronic Acid Transporter That Modulates the Polysaccharide Composition of Arabidopsis Seed Mucilage

“…Substrate interconversion is required for polysaccharide biosynthesis, and in Arabidopsis leaves, polysaccharides containing sugars derived from UDP-GlcA account for nearly 50% of the cell wall biomass (Zablackis et al, 1995). The cytosol contains some of the enzymes for the interconversion of UDP-GlcA, such as soluble UDP-xylose synthase (UXS) (Harper and Bar-Peled, 2002;Pattathil et al, 2005;Kuang et al, 2016), UDP-apiose/UDP-xylose synthase (Guyett et al, 2009;Mølhøj et al, 2003), and UDP-arabinose mutase Rautengarten et al, 2011). There are also Golgi-localized interconverting enzymes.…”

UDP-Glucuronic Acid Decarboxylases of Bacteroides fragilis and Their Prevalence in Bacteria

“…The UDP-xylose synthase (Uxs) family enzymes found in animals produce UDPxylose by decarboxylation of UDP-GlcA using NAD (NAD ϩ ). Similarly, the enzymes of the UDP-apiose/UDP-xylose synthase (Uaxs) family found in plants convert UDP-GlcA to UDP-apiose and UDP-xylose (21).…”