FUNCTIONAL EXPRESSION OF URIDINE 5'-DIPHOSPHO-GLUCOSE 4-EPIMERASE (EC-5.1.3.2) FROM ARABIDOPSIS-THALIANA IN SACCHAROMYCES-CEREVISIAE AND ESCHERICHIA-COLI

It is our goal to investigate the biosynthesis of galactose-containing compounds in higher plants, Searching a database of expressed sequenc e tags, a cDNA from Arabidopsis thaliana (clone 108G20T7) with sequenc e similarity to UDP-glucose epimerase was identified and further analy zed. The 1356-bp-long cDNA included an open reading frame predicted to encode a 351 amino acid protein of 39 kDa. The presumed protein seque nce showed a high degree of similarity to UDP-glucose epimerase sequen ces from bacteria, rat, and yeast. Complementation of the Saccharomyce s cerevisiae gal10 mutant and expression of an active enzyme in Escher ichia coli demonstrated that the cDNA encoded a functional UDP-glucose epimerase. The recombinant enzyme was purified to homogeneity. It sho wed a broad pH optimum of 7.0 to 9.5 and a K-m of 0.11 mM. The UDP-glu cose epimerase activity was not dependent on the addition of the cofac tor NAD(+) and was only moderately inhibited by high salt concentra ti ons. Tissue-specific Northern analysis showed that the gene is express ed in all tissues of A. thaliana with highest expression levels in the stems and roots. Eased on Southern analysis, there seems to be a sing le gene encoding UDP-glucose epimerase in A. thaliana. The cDNA analyz ed during this study is the first known to encode a sugar-nucleotide m odifying enzyme from higher plants. Its availability provides the mean s to investigate the role of UDP-glucose epimerase for the biosynthesi s of UDP-galactose as precursor of galactolipids and cell wall polysac charides. (C) 1996 Academic Press, Inc.