An arginyl residue in rice UDP-arabinopyranose mutase is required for catalytic activity and autoglycosylation |
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Authors: | Tomoyuki Konishi Kazumi Funane Teruko Konishi Tadashi Ishii |
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Affiliation: | a Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan b National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan |
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Abstract: | Plants use UDP-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing complex carbohydrates. UDP-Araf itself is formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). However, the mechanism by which this enzyme catalyzes the interconversion of UDP-Arap and UDP-Araf has not been determined. To gain insight into this reaction, functionally recombinant rUAMs were reacted with UDP-Glc or UDP-Araf. The glycosylated recombinant UAMs were fragmented with trypsin, and the glycopeptides formed were then identified and sequenced by LC-MS/MS. The results of these experiments, together with site-directed mutagenesis studies, suggest that in functional UAMs an arginyl residue is reversibly glycosylated with a single glycosyl residue, and that this residue is required for mutase activity. We also provide evidence that a DXD motif is required for catalytic activity. |
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Keywords: | Araf, arabinofuranose Arap, arabinopyranose FAD, flavin adenine dinucleotide GST, glutathion S-transferase HPLC, high-performance liquid chromatography LC/MS, liquid chromatography-mass spectrometry MALDI-TOFMS, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry PCR, polymerase chain reaction RGP, reversibly glycosylated polypeptides rUAMs, recombinant UAMs SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis UAM, UDP-arabinopyranose mutase UDP-Araf, UDP- smallcaps" >l-arabinofuranose UDP-Arap, UDP- smallcaps" >l-arabinopyranose UDP-Glc, UDP-glucose, UDP-Xyl, UDP-xylose UDP, uridine diphosphate |
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