UDP-sugar Pyrophosphorylase with Broad Substrate Specificity Toward Various Monosaccharide 1-Phosphates from Pea Sprouts

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 UDPsugar 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, UDPGal, UDP-glucuronic acid, UDP-L-arabinose, and UDPxylose 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°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 UDPsugar pyrophosphorylase, which catalyzes the formation of various UDP-sugars at the end of salvage pathways in higher plants.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 units from respective nucleotide sugars into the polymers. Activated nucleotide sugars that serve as glycosyl donors for these polymers in higher plants are generated through de novo pathways, in which various UDP-and GDP-sugars are produced through sequential interconversions from UDP-Glc and GDP-Man as the starting substrates (1). In the salvage pathways, alternative routes to synthesize nucleotide sugars, free monosaccharides released during the degradation of polysaccharides and glycoconjugates are first phosphorylated by the action of monosaccharide kinases, then converted into nucleotide sugars by the action of pyrophosphorylases in the presence of the respective nucleotide triphosphates as co-substrates. Pyrophosphorylases principally catalyze both the following forward (synthesis of NDP-sugars) and reverse (pyrophospholysis) reactions (1): monosaccharides 1-phosphates ϩ NTP % NDP-sugars ϩ PP i , where NTP and NDP are nucleoside triphosphates and diphosphates, respectively. Var...

KONJAC1 and 2 Are Key Factors for GDP-Mannose Generation and Affect l-Ascorbic Acid and Glucomannan Biosynthesis in Arabidopsis

“…Generation of GDP-Man relies only on GMPP activity, whereas UDPGlc, the starting substrate in the de novo pathway of UDP-sugars, can be formed by different enzymes: UDP-Glc pyrophosphorylase, USP, or sucrose synthase (Kotake et al, 2004). Out of three GMPP isoforms in Arabidopsis, VTC1 is the main isoform based on the expression level and its mutant phenotypes (Conklin et al, 1999; (A) Monosaccharide composition of the kjc cell wall.…”

“…The homogenate was centrifuged at 8000g for 5 min to remove cell debris (Kotake et al, 2004) and then used as a crude enzyme fraction. The activity of GMPP was determined by monitoring the formation of GDP-Man.…”

“…UDP-Glc pyrophosphorylases generate UDP-Glc, the starting substrate of UDP-sugar metabolism, which is used for the synthesis of cellulose and b-1,3-glucan and for the b-1,4-glucan backbone of xyloglucan (Hayashi et al, 1987;Gordon and Maclachlan, 1989;Cocuron et al, 2007;Park et al, 2010). A plant-specific enzyme, UDP-sugar pyrophosphorylase (USP), converts various monosaccharide 1-Ps to their respective UDP-sugars in the salvage pathway and is required for pollen development (Kotake et al, 2004(Kotake et al, , 2007Schnurr et al, 2006). ADP-Glc pyrophosphorylases provide ADP-Glc for starch synthesis and can be divided into two clades, small (S) subunit and large (L) subunit clades.…”

Generation of nucleotide sugars for biomass formation in plants

“…UDP-sugar pyrophosphorylase (USP, EC 2.7.7.64) is a novel type of nucleotide sugar pyrophosphorylase with broad substrate specificity toward monosaccharide 1-Ps (Kotake et al 2004;Litterer et al 2006;Kotake et al 2007). USP catalyzes the formation of UDP-Glc, UDP-Gal, UDPGlcA, UDP-GalA, UDP-L-Ara, and UDP-Xyl from respective monosaccharide 1-Ps in the presence of UTP in the salvage pathway (Kotake et al 2004;Ohashi et al 2006). One can attribute the activity of UDP-Xyl pyrophosphorylase (EC 2.7.7.11) detected in mung bean seedlings (Ginsburg et al 1956) to the broad substrate specificity of USP toward monosaccharide 1-Ps.…”

Identification of Galacturonic Acid-1-phosphate Kinase, a New Member of the GHMP Kinase Superfamily in Plants, and Comparison with Galactose-1-phosphate Kinase

“…The subsequent pyrophosphorylation of UDP-sugars could be carried out by UDP-sugar pyrophosphorylases (20).…”