Title

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

Date of Completion

January 2004

Keywords

Biology, Molecular|Biology, Botany|Biology, Plant Physiology

Degree

Ph.D.

Abstract

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. ^