Functional Expression of Uridine 5′-Diphospho-Glucose 4-Epimerase (EC 5.1.3.2) fromArabidopsis thalianainSaccharomyces cerevisiaeandEscherichia coli

Abstract

It is our goal to investigate the biosynthesis of galactose-containing compounds in higher plants. Searching a database of expressed sequence tags, a cDNA fromArabidopsis thaliana(clone 108G20T7) with sequence similarity to UDP-glucose epimerase was identified and further analyzed. The 1356-bp-long cDNA included an open reading frame predicted to encode a 351 amino acid protein of 39 kDa. The presumed protein sequence showed a high degree of similarity to UDP-glucose epimerase sequences from bacteria, rat, and yeast. Complementation of theSaccharomyces cerevisiae gal10mutant and expression of an active enzyme inEscherichia colidemonstrated that the cDNA encoded a functional UDP-glucose epimerase. The recombinant enzyme was purified to homogeneity. It showed a broad pH optimum of 7.0 to 9.5 and aKmof 0.11 mM. The UDP-glucose epimerase activity was not dependent on the addition of the cofactor NAD+and was only moderately inhibited by high salt concentrations. Tissue-specific Northern analysis showed that the gene is expressed in all tissues ofA. thalianawith highest expression levels in the stems and roots. Based on Southern analysis, there seems to be a single gene encoding UDP-glucose epimerase inA. thaliana.The cDNA analyzed during this study is the first known to encode a sugar-nucleotide modifying enzyme from higher plants. Its availability provides the means to investigate the role of UDP-glucose epimerase for the biosynthesis of UDP-galactose as precursor of galactolipids and cell wall polysaccharides.