Kotake Toshihisa, Yamaguchi Daisuke, Ohzono Hiroshi, Hojo Sachiko, Kaneko Satoshi, Ishida Hide-Ki, Tsumuraya Yoichi
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan.
J Biol Chem. 2004 Oct 29;279(44):45728-36. doi: 10.1074/jbc.M408716200. Epub 2004 Aug 23.
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.
UDP-糖是单糖的活化形式，通过从头合成途径和补救途径合成，在高等植物中作为多糖、糖脂和糖蛋白合成的底物。一种名为PsUSP的UDP-糖焦磷酸化酶，通过传统色谱法从豌豆（Pisum sativum L.）芽中纯化了约1200倍。纯化后的PsUSP的表观分子量为67,000 Da。该酶在UTP作为共底物存在的情况下，催化从各自的单糖1-磷酸形成UDP-Glc、UDP-Gal、UDP-葡萄糖醛酸、UDP-L-阿拉伯糖和UDP-木糖，表明该酶对单糖1-磷酸具有广泛的底物特异性。该酶的最大活性出现在pH 6.5 - 7.5，以及在2 mM Mg(2+)存在下45℃时。Glc 1-磷酸和L-阿拉伯糖1-磷酸的表观K(m)值分别为0.34和0.96 mM。通过逆转录酶-PCR克隆了PsUSP cDNA。PsUSP似乎编码一种分子量为66,040 Da（600个氨基酸）的蛋白质，并拥有一个尿苷结合位点，该位点也在人UDP-N-乙酰己糖胺焦磷酸化酶中被发现。系统发育分析表明，PsUSP可以与拟南芥和水稻的同源物归为一组，这与UDP-Glc和UDP-N-乙酰己糖胺焦磷酸化酶组不同。在大肠杆菌中表达的重组PsUSP催化从单糖1-磷酸和UTP形成UDP-糖，效率与天然酶相似。这些结果表明，该酶是一种新型的UDP-糖焦磷酸化酶，在高等植物的补救途径末端催化各种UDP-糖的形成。
