The biosynthesis of UDP-sugars in plants: Cloning and characterization of nucleotide sugar interconversion enzymes in Arabidopsis thaliana

The biosynthesis of plant cell wall polymers is dependent on the availability of activated sugar precursors, which are synthesized by a series of enzymatic reactions that are commonly referred to as nucleotide sugar interconversion pathways. Bioinformatics approaches led to the identification of candidate genes for UDP-L-rhamnose synthases (RHMs), UDP-D-apiose/UDP-D-xylose synthases ( AXSs), UDP-D-glucose 4-epimerases (UGEs), UDP-D-glucuronic acid 4-epimerases (GAEs) and UDP-D-xylose 4-epimerases (UXEs). An evaluation of the Arabidopsis genome sequence suggests that these enzymes are encoded by small gene families. Although a number of these enzymes are predicted to be soluble proteins, some contain an N-terminal hydrophobic extension containing a single transmembrane domain. This suggests that these enzymes co-localize with glycosyltransferases within the Golgi. ^ The RHM1 gene was shown to encode for a soluble UDP- L-rhamnose synthase, which is bipartite in structure. Expression of the N-terminal and C-terminal domains of RHM1 showed that the N-terminal domain functions as a UDP-D-glucose-4,6-dehydratase, and the C-terminal domain functions as a UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase-4-reductase in vitro. ^ The biosynthesis of UDP-D-apiose involves the decarboxylation of UDP-D-glucuronate and a complex ring re-arrangement mechanism, which results in the formation of UDP-D-apiose and UDP- D-xylose. The AXS1 genes of Arabidopsis and Nicotiana benthamiana were shown to encode soluble NAD +-dependent UDP-D-apiose/UDP-D-xylose synthases. The recombinant AtAXS1 protein has a K m of ∼7.6 μM for UDP-D-glucuronate with a kcat of 0.27 min−1. Analysis of cell wall material from NbAXS1 silenced plants showed ∼50% reduction in the contents of 2-O-methyl-sugars suggesting that the AXS1 activity is required for the synthesis of the rhamnogalacturonan II side chains, which are involved in borate-mediated cross-linking of the RG-II polymer chains. ^ The GAE1 and UXE1 genes encode for proteins with a transmembrane domain that catalyze the 4-epimerization of UDP-D-glucuronate and UDP-D-xylose, respectively. Expression analysis suggests that GAE1 is expressed ubiquitously, whereas the UXE isoforms showed some differences in their expression patterns. The UGE isoforms encode for soluble proteins catalyzing the 4-epimerization of both UDP-D-galactose and UDP-D-xylose. Kinetic analysis suggests that some of the UGE isoforms are involved in generating cytoplasmic pools of UDP- L-arabinose. However, this remains to be characterized in further detail using genetic approaches.