title: X-ray diffraction analysis and in vitro characterization of the UAM2 protein from Oryza sativa.
abstract: The role of seemingly non-enzymatic proteins in complexes interconverting UDP-arabinopyranose and UDP-arabinofuranose (UDP-arabinosemutases; UAMs) in the plant cytosol remains unknown. To shed light on their function, crystallographic and functional studies of the seemingly non-enzymatic UAM2 protein from Oryza sativa (OsUAM2) were undertaken. Here, X-ray diffraction data are reported, as well as analysis of the oligomeric state in the crystal and in solution. OsUAM2 crystallizes readily but forms highly radiation-sensitive crystals with limited diffraction power, requiring careful low-dose vector data acquisition. Using size-exclusion chromatography, it is shown that the protein is monomeric in solution. Finally, limited proteolysis was employed to demonstrate DTT-enhanced proteolytic digestion, indicating the existence of at least one intramolecular disulfide bridge or, alternatively, a requirement for a structural metal ion.
authors.affiliation: DTU Bioengineering, Technical University of Denmark, Elektrovej, Building 375, 2800 Lyngby, Denmark.
authors.forename: Ditte Hededam
authors.initials: DH
authors.lastname: Welner
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Alex Yi Lin
authors.initials: AY
authors.lastname: Tsai
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Andy M
authors.initials: AM
authors.lastname: DeGiovanni
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Henrik Vibe
authors.initials: HV
authors.lastname: Scheller
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608, USA.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D003460:Crystallization; D018360:Crystallography, X-Ray; D004229:Dithiothreitol / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D019751:Intramolecular Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012275:Oryza / Q000737:chemistry* / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020860:Subtilisin / Q000737:chemistry; D014539:Uridine Diphosphate Sugars / Q000737:chemistry* / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D014539:Uridine Diphosphate Sugars; C040817:uridine diphosphate arabinose; D020860:Subtilisin; D019751:Intramolecular Transferases; D004229:Dithiothreitol
keywords: UDP-arabinopyranose mutase; limited proteolysis; reversibly glycosylated polypeptide; vector data collection