An arginyl residue in rice UDP-arabinopyranose mutase is required for catalytic activity and autoglycosylation.
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.
Konishi
Tomoyuki
T
Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan.
Ohnishi-Kameyama
Mayumi
M
Funane
Kazumi
K
Miyazaki
Yasumasa
Y
Konishi
Teruko
T
Ishii
Tadashi
T
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics / Q000502:physiology*; D002853:Chromatography, Liquid; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D019751:Intramolecular Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012275:Oryza / Q000201:enzymology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D010940:Plant Proteins; D011994:Recombinant Proteins; D014539:Uridine Diphosphate Sugars; C040817:uridine diphosphate arabinose; D001120:Arginine; D019751:Intramolecular Transferases