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UDP-GlcA 4-epimerase (UGlcAE) catalyzes the epimerization of UDP-α-D-glucuronic acid (UDP-GlcA) to UDP-α-D-galacturonic acid (UDP-GaIA). UDP-GalA is a precursor for the synthesis of numerous cell-surface polysaccharides in bacteria and plants. Using a biochemical screen, a gene encoding AtUGlcAE1 in Arabidopsis (Arabidopsis thaliana) was identified and the recombinant enzyme biochemically characterized. The gene belongs to a small gene family composed of six isoforms. All members of the UGlcAE gene family encode a putative type-II membrane protein and have two domains: a variable N-terminal region approximately 120 amino acids long composed of a predicted cytosolic, transmembrane, and stem domain, followed by a large conserved C-terminal catalytic region approximately 300 amino acids long composed of a highly conserved catalytic domain found in a large protein family of epimerase/dehydratases. The recombinant epimerase has a predicted molecular mass of approximately 43 kD, although size-exclusion chromatography suggests that it may exist as a dimer (approximately 88 kD). AtUGlcAE1 forms UDP-GalA with an equilibrium constant value of approximately 1.9 and has an apparent K^sub m^ value of 720 µM for UDP-GlcA. The enzyme has maximum activity at pH 7.5 and is active between 20°C and 55°C. Arabidopsis AtUGlcAE1 is not inhibited by UDP-Glc, UDP-Gal, or UMP. However, the enzyme is inhibited by UDP-Xyl and UDP-Ara, suggesting that these nucleotide sugars have a role in regulating the synthesis of pectin. The cloning of the AtUGlcAE1 gene will increase our ability to investigate the molecular factors that regulate pectin biosynthesis in plants. The availability of a functional recombinant UDP-GlcA 4-epimerase will be of considerable value for the facile generation of UDPD-GalA in the amounts required for detailed studies of pectin biosynthesis.
GalA is a major sugar residue of plant pectic polysaccharides (Mohnen, 2002) and a minor component of some plant arabinogalactan proteins (Darvill et al., 1980; Yates et al., 1996). GalA is also found in various cell-surface polysaccharides of different Gramnegative bacteria, including human pathogenic bacteria Klebsiella pneumoniae (for review, see Hoist et al., 1998), Shigella spp. (Feng et al., 2004), and Vibrio cholera (Adeyeye et al., 2003); plant pathogenic bacteria Agrobacterium larrymoorei (Molinaro et al., 2003) and Erwinia chrysanthemi spp. (Gray et al., 2000); plant symbiotic rhizobacteria (Forsberg and Carlson, 1998); aerobic bacteria from the deep sea (Raguénès et al., 2003); and...