Abstract
Nucleotide sugars are building blocks for carbohydrate polymers in plant cell walls. They are synthesized from sugar-1-phosphates or epimerized as nucleotide sugars. The main precursor for primary cell walls is UDP-glucuronic acid, which can be synthesized via two independent pathways. One starts with the ring cleavage of myo-inositol into glucuronic acid, which requires a glucuronokinase and a pyrophosphorylase for activation into UDP-glucuronate. Here we report on the purification of glucuronokinase from Lilium pollen. A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization. Glucuronokinase is a novel member of the GHMP-kinase superfamily having an unique substrate specificity for d-glucuronic acid with a Km of 0.7 mm. It requires ATP as phosphate donor (Km 0.56 mm). In Arabidopsis, the gene is expressed in all plant tissues with a preference for pollen. Genes for glucuronokinase are present in (all) plants, some algae, and a few bacteria as well as in some lower animals.
Highlights
A 40-kDa protein was purified combining six chromatographic steps and peptides were de novo sequenced. This allowed the cloning of the gene from Arabidopsis thaliana and the expression of the recombinant protein in Escherichia coli for biochemical characterization
In many plants a supplementary “salvage” pathway exists, which enables cells to convert free sugars into NDP sugars via phosphorylated intermediates allocated by the successive work of sugar kinases and nucleotide sugar pyrophosphorylases
These salvage pathways presumably play a primary role in the recycling of sugars derived from the conversion of macromolecules like polysaccharides, proteoglycans, and galactolipids, which are substituted with sugar components
Summary
Materials—Bulbs of L. longiflorum were purchased from BulbsOnaWire (Alkmaar, The Netherlands) and plants were grown outside in the botanical garden. The desalted sample was applied on a 4.0 ϫ 1.0-cm inner diameter (MoBiTec GmbH, Goettingen, Germany) blue dextran-agarose (Sigma) column equilibrated with the same HEPES buffer and proteins were eluted with a linear gradient of 0 –100 mM KCl (total volume, 42 ml). Proteins were eluted with a linear gradient of 0 – 450 mM NaCl (total volume, 25 ml) and positive fractions were combined and dialyzed against a 50 mM Tris-Cl, pH 8.0, buffer using an Amicon centrifugation device. The concentrated proteins (final volume, 1 ml) were loaded onto a 0.5 ϫ 5.0-cm inner diameter Toyopearl DEAE-5 PW column (Tosho Bioscience GmbH, Stuttgart, Germany) equilibrated with 50 mM Tris-Cl buffer and eluted with a linear gradient of 0 –550 mM NaCl (total volume, 20 ml). Measurement was performed in 96-well microplates (Greiner BioOne, Kremsmunster, Austria) at a wavelength of 340 nm for 30 min
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