Identification and characterization of a strict and a promiscuous N-acetylglucosamine-1-P uridylyltransferase in Arabidopsis.
UDP-GlcNAc is an essential precursor for glycoprotein and glycolipid synthesis. In the present study, a functional nucleotidyltransferase gene from Arabidopsis encoding a 58.3 kDa GlcNAc1pUT-1 (N-acetylglucosamine-1-phosphate uridylyltransferase) was identified. In the forward reaction the enzyme catalyses the formation of UDP-N-acetylglucosamine and PPi from the respective monosaccharide 1-phosphate and UTP. The enzyme can utilize the 4-epimer UDP-GalNAc as a substrate as well. The enzyme requires divalent ions (Mg2+ or Mn2+) for activity and is highly active between pH 6.5 and 8.0, and at 30-37 degrees C. The apparent Km values for the forward reaction were 337 microM (GlcNAc-1-P) and 295 microM (UTP) respectively. Another GlcNAc1pUT-2, which shares 86% amino acid sequence identity with GlcNAc1pUT-1, was found to convert, in addition to GlcNAc-1-P and GalNAc-1-P, Glc-1-P into corresponding UDP-sugars, suggesting that subtle changes in the UT family cause different substrate specificities. A three-dimensional protein structure model using the human AGX1 as template showed a conserved catalytic fold and helped identify key conserved motifs, despite the high sequence divergence. The identification of these strict and promiscuous gene products open a window to identify new roles of amino sugar metabolism in plants and specifically their role as signalling molecules. The ability of GlcNAc1pUT-2 to utilize three different substrates may provide further understanding as to why biological systems have plasticity.
Yang
Ting
T
Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602, U.S.A.
Echols
Merritt
M
Martin
Andy
A
Bar-Peled
Maor
M
D000117:Acetylglucosamine / Q000031:analogs & derivatives* / Q000378:metabolism; D017360:Arabidopsis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008968:Molecular Conformation; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D029681:Arabidopsis Proteins; C026146:N-acetylglucosamine-1-phosphate; D009713:Nucleotidyltransferases; D000117:Acetylglucosamine