title: Purification, characterization and preliminary X-ray crystallographic studies of monodehydroascorbate reductase from Oryza sativa L. japonica.
abstract: Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) is a key enzyme in the reactive oxygen species (ROS) detoxification system of plants. The participation of MDHAR in ascorbate (AsA) recycling in the ascorbate-glutathione cycle is important in the acquired tolerance of crop plants to abiotic environmental stresses. Thus, MDHAR represents a strategic target protein for the improvement of crop yields. Although physiological studies have intensively characterized MDHAR, a structure-based functional analysis is not available. Here, a cytosolic MDHAR (OsMDHAR) derived from Oryza sativa L. japonica was expressed using Escherichia coli strain NiCo21 (DE3) and purified. The purified OsMDHAR showed specific enzyme activity (approximately 380 U per milligram of protein) and was crystallized using the hanging-drop vapour-diffusion method at pH 8.0 and 298 K. The crystal diffracted to 1.9 Å resolution and contained one molecule in the asymmetric unit (the Matthews coefficient VM is 1.98 Å(3) Da(-1), corresponding to a solvent content of 38.06%) in space group P41212 with unit-cell parameters a = b = 81.89, c = 120.4 Å. The phase of the OsMDHAR structure was resolved by the molecular-replacement method using a ferredoxin reductase from Acidovorax sp. strain KKS102 (PDB entry 4h4q) as a model.
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Hackwon
authors.initials: H
authors.lastname: Do
authors.affiliation: Department of Biology, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Il-Sup
authors.initials: IS
authors.lastname: Kim
authors.affiliation: Department of Biology, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Young-Saeng
authors.initials: YS
authors.lastname: Kim
authors.affiliation: Department of Biology, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Sun-Young
authors.initials: SY
authors.lastname: Shin
authors.affiliation: Department of Biology, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Jin-Ju
authors.initials: JJ
authors.lastname: Kim
authors.affiliation: Department of Biology, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Ji-Eun
authors.initials: JE
authors.lastname: Mok
authors.affiliation: Department of Biology, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Seong-Im
authors.initials: SI
authors.lastname: Park
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Ah Ram
authors.initials: AR
authors.lastname: Wi
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Hyun
authors.initials: H
authors.lastname: Park
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Department of Biology, Kyungpook National University, Daegu 702-701, Republic of Korea.
authors.forename: Ho-Sung
authors.initials: HS
authors.lastname: Yoon
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, Republic of Korea.
authors.forename: Han-Woo
authors.initials: HW
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D018360:Crystallography, X-Ray / Q000379:methods*; D017931:DNA Primers; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D012275:Oryza / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)
keywords: Oryza sativa L. japonica; abiotic stress; ascorbate regeneration; monodehydroascorbate reductase