Preparation of UDP-galacturonic acid using UDP-sugar pyrophosphorylase.
UDP-galacturonic acid, the activated form of galacturonic acid (GalUA), is synthesized both de novo and by salvage pathways. The UDP-GalUA pyrophosphorylase gene involved in the salvage pathway has not been identified. Here we show that UDP-sugar pyrophosphorylase from Pisum sativum with a broad specificity has UDP-GalUA pyrophosphorylase activity. The enzyme catalyzed the formation of UDP-GalUA and pyrophosphate from GalUA 1-phosphate and UTP with an equilibrium constant value of 0.24. The recombinant UDP-sugar pyrophosphorylase had optimal pH of 6.0, and the apparent K(m) values for GalUA 1-phosphate, UTP, UDP-GalUA, and pyrophosphate were 2.27, 1.15, 0.70, and 1.26 mM, respectively. In the presence of inorganic pyrophosphatase, the recombinant enzyme produced UDP-GalUA in an 84% yield (based on the GalUA 1-phosphate substrate) on a preparative scale. Thus, this UDP-sugar pyrophosphorylase is useful for the highly efficient production of UDP-GalUA for studies on pectin biosynthesis.
Ohashi
Takao
T
Department of Chemistry, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan.
Cramer
Nicolai
N
Ishimizu
Takeshi
T
Hase
Sumihiro
S
D002384:Catalysis; D011756:Diphosphates / Q000138:chemical synthesis / Q000737:chemistry; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods / Q000592:standards; D009713:Nucleotidyltransferases / Q000737:chemistry*; D018532:Pisum sativum / Q000201:enzymology; D011994:Recombinant Proteins / Q000737:chemistry; D012015:Reference Standards; D013997:Time Factors; D014539:Uridine Diphosphate Sugars / Q000138:chemical synthesis / Q000737:chemistry*
D011756:Diphosphates; D011994:Recombinant Proteins; D014539:Uridine Diphosphate Sugars; C050306:UDP-galacturonic acid; D009713:Nucleotidyltransferases; C023226:UDPacetylglucosamine pyrophosphorylase