UDP-sugar pyrophosphorylase with broad substrate specificity toward various monosaccharide 1-phosphates from pea sprouts.
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.
Kotake
Toshihisa
T
Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan. kotake@molbiol.saitama-u.ac.jp
Yamaguchi
Daisuke
D
Ohzono
Hiroshi
H
Hojo
Sachiko
S
Kaneko
Satoshi
S
Ishida
Hide-Ki
HK
Tsumuraya
Yoichi
Y
D000595:Amino Acid Sequence; D005695:Galactosephosphates / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D006600:Hexosephosphates / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000378:metabolism*; D018532:Pisum sativum / Q000201:enzymology*; D013379:Substrate Specificity; D013403:Sugar Phosphates / Q000378:metabolism; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D005695:Galactosephosphates; D005958:Glucosephosphates; D006600:Hexosephosphates; D013403:Sugar Phosphates; D014539:Uridine Diphosphate Sugars; C104973:arabinose-1-phosphate; C029973:galactose-1-phosphate; C031590:glucose-1-phosphate; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase