Abstract
UDP-sugars, activated forms of monosaccharides, are synthesized through de novo and salvage pathways and serve as substrates for the synthesis of polysaccharides, glycolipids, and glycoproteins in higher plants. A UDP-sugar pyrophosphorylase, designated PsUSP, was purified about 1,200-fold from pea (Pisum sativum L.) sprouts by conventional chromatography. The apparent molecular mass of the purified PsUSP was 67,000 Da. The enzyme catalyzed the formation of UDP-Glc, UDP-Gal, UDP-glucuronic acid, UDP-l-arabinose, and UDP-xylose from respective monosaccharide 1-phosphates in the presence of UTP as a co-substrate, indicating that the enzyme has broad substrate specificity toward monosaccharide 1-phosphates. Maximum activity of the enzyme occurred at pH 6.5-7.5, and at 45 degrees C in the presence of 2 mm Mg(2+). The apparent K(m) values for Glc 1-phosphate and l-arabinose 1-phosphate were 0.34 and 0.96 mm, respectively. PsUSP cDNA was cloned by reverse transcriptase-PCR. PsUSP appears to encode a protein with a molecular mass of 66,040 Da (600 amino acids) and possesses a uridine-binding site, which has also been found in a human UDP-N-acetylhexosamine pyrophosphorylase. Phylogenetic analysis revealed that PsUSP can be categorized in a group together with homologues from Arabidopsis and rice, which is distinct from the UDP-Glc and UDP-N-acetylhexosamine pyrophosphorylase groups. Recombinant PsUSP expressed in Escherichia coli catalyzed the formation of UDP-sugars from monosaccharide 1-phosphates and UTP with efficiency similar to that of the native enzyme. These results indicate that the enzyme is a novel type of UDP-sugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.
Highlights
It is highly probable that the relative amounts and the architecture of cell wall polysaccharides and of the sugar moieties of glycolipids and glycoproteins in higher plants are regulated by the level of nucleotide sugars available, as well as by the levels of glycosyltransferases that incorporate monosaccharide
Further evidence came from the case of the mur1 mutant of Arabidopsis with dwarf phenotype, which shows a decrease in L-fucose (L-Fuc) content in rhamnogalacturonan II because of a defect in the conversion of GDP-Man to GDP-L-Fuc in the de novo pathway [3]
These observations indicate that the network of the synthesis of nucleotide sugars is regulated through concerted actions of various enzymes involved in both de novo and the salvage pathways
Summary
L-Ara, L-arabinose; PsUSP, P. sativum UDP-sugar pyrophosphorylase; L-Fuc, L-fucose; GlcA, D-glucuronic acid; Xyl, xylose; UDP-HexNAc, UDP-N-acetylhexosamine; HPLC, high performance liquid chromatography; BSA, bovine serum albumin; MOPS, 3-morpholinopropanesulfonic acid; RACE, rapid amplification of cDNA ends. UDP-Xyl is produced by xylokinase and UDP-Xyl pyrophosphorylase (UTP: ␣-D-xylose-1-phosphate uridylyltransferase, EC 2.7.7.11) The latter enzyme has been detected in a crude enzyme preparation from mung bean seedlings [7], but it has been neither purified nor cloned so far. We discuss the characteristics of the enzyme based on the comparison of amino acid sequences with other pyrophosphorylases as well as its possible function in vivo
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