Cloning of Glucuronokinase from Arabidopsis thaliana, the last missing enzyme of the myo-inositol oxygenase pathway to nucleotide sugars.
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 K(m) of 0.7 mm. It requires ATP as phosphate donor (K(m) 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.
Pieslinger
Anja Maria
AM
Department of Cell Biology, Plant Physiology, University of Salzburg, Hellbrunnerstrasse 34, 5020 Salzburg, Austria.
Hoepflinger
Marion Christine
MC
Tenhaken
Raimund
R
D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D003001:Cloning, Molecular; D050562:Inositol Oxygenase / Q000737:chemistry / Q000235:genetics*; D007700:Kinetics; D027762:Lilium / Q000201:enzymology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000737:chemistry; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics*; D010802:Phylogeny; D011108:Polymers / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid
D009711:Nucleotides; D011108:Polymers; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D050562:Inositol Oxygenase; D017853:Phosphotransferases (Alcohol Group Acceptor); C020591:glucuronokinase