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J. Biol. Chem., Vol. 279, Issue 44, 45728-45736, October 29, 2004

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

Toshihisa Kotake, Daisuke Yamaguchi, Hiroshi Ohzono, Sachiko Hojo, Satoshi Kaneko¶, Hide-ki Ishida||, and Yoichi Tsumuraya

From the Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan, ^¶Biological Function Division, National Food Research Institute, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan, and the ^||Noguchi Institute, 1-8-1 Kaga, Itabashi-ku, Tokyo 173-0003, Japan

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 °C in the presence of 2 mM Mg²⁺. 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.

Received for publication, July 30, 2004 , and in revised form, August 23, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB178642.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. Tel.: 81-48-858-3955; Fax: 81-48-858-3384; E-mail: kotake{at}molbiol.saitama-u.ac.jp.

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