A facile enzymatic synthesis of uridine diphospho-[14C]galacturonic acid.
Galacturonic acid (GalA) is a major component of plant cell-wall-derived pectins. It can be also found in the cell-surface polysaccharides of different microorganisms, including several symbiotic and pathogenic bacteria. Uridine diphosphogalacturonic acid (UDP-GalA) is a likely donor for GalA during the biosynthesis of these polysaccharides. A highly efficient, yet simple, method is presented for generating and purifying UDP-[14C]GalA. Commercially available UDP-[14C]-galactose was quantitatively oxidized (>95% conversion) to UDP-[14C]GalA in the presence of high levels of galactose oxidase and catalase, at prolonged incubation times. Following this one-step enzymatic oxidation, UDP-[14C]GalA was purified using a polyethyleneimine cellulose column with a single-step 1 M NaCl elution. The authenticity of the purified UDP-[14C]GalA was verified by its relative mobility on thin-layer chromatograms, analysis of its chemical hydrolysis products, and 1H NMR spectroscopy. Our yield of >90% is much higher than by previously described methods. The method may serve as a prototype for the preparation of other radiolabeled uronic acids and their nucleotide derivatives.
Basu
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Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Dotson
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Raetz
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D002250:Carbon Radioisotopes; D002374:Catalase / Q000737:chemistry*; D005692:Galactose Oxidase / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D014539:Uridine Diphosphate Sugars / Q000138:chemical synthesis*
D002250:Carbon Radioisotopes; D014539:Uridine Diphosphate Sugars; C050306:UDP-galacturonic acid; D005692:Galactose Oxidase; D002374:Catalase