Distinct properties of the five UDP-D-glucose/UDP-D-galactose 4-epimerase isoforms of Arabidopsis thaliana

Plant genomes contain genetically encoded isoforms of most nucleotide sugar interconversion enzymes. Here we show that Arabidopsis thaliana has five genes encoding functional UDP-D-glucose/ UDP-(D)-galactose 4-epimerase (named UGE1 to UGE5). All A. thaliana UDP-(D)-glucose 4-epimerase isoforms are dimeric in solution, maximally active in vitro at 30-40 degrees C, and show good activity between pH 7 and pH 9. In vitro, UGE1, -3, and -5 act independently of externally added NAD(+), whereas cofactor addition stimulates the activity of UGE2 and is particularly important for UGE4 activity. UGE1 and UGE3 are most efficiently inhibited by UDP. The five isoforms display k(cat)(UDP-Gal) values between 23 and 128 s(-1) and K-m(UDP-Gal) values between 0.1 and 0.3mM. This results in enzymatic efficiencies ranging between 97 and 890 mM(-1)s(-1) for UGE4 = UGE1 < UGE3 < UGE5 < UGE2. The K-m(UDP-Glc) values, derived from the Haldane relationship, were 0.76mM for UGE1, 0.56mM for UGE4, and between 0.13 and 0.23 mM for UGE2, -3, and -5. The expression of UGE isoforms is ubiquitous and displays developmental and cell type-dependent variations. UGE1 and -3 expression patterns globally resemble enzymes involved in carbohydrate catabolism, and UGE2, -4, and -5 expression is more related to carbohydrate biosynthesis. UGE1, -2, and -4 are present in the cytoplasm, whereas UGE4 is additionally enriched close to Golgi stacks. All UGE genes tested complement the UGE4(rhd1) phenotype, confer increased galactose tolerance in planta, and complement the galactose metabolization deficiency in the Saccharomyces cerevisiae gal10 mutant. We suggest that plant UGE isoforms function in different metabolic situations and that enzymatic properties, gene expression pattern, and subcellular localization contribute to the differentiation of isoform function.