PARENT SESSION
Posters P1B Photo-oxidative stress, photoinhibition. Abstracts (394-443)

Crystallization and preliminary crystallographic analysis of monodehydroascorbate radical reductase from cucumber. Satoshi Sano^*,1, You-Na Kang², Hiroko Shigemizu¹, Nobuhiko Morishita¹, Hye-Jin Yoon³, Kazumi Saito¹, Kozi Asada⁴, Bunzo Mikami², ¹ Laboratory of Chemistry of Biological Function, Graduate School of Agriculture, Kyoto, Japan² Laboratory of Food Quality Design and Development, Uji, Kyoto, Japan³ Structural Proteomics Laboratory, Seoul, Korea⁴ Department of Biotechnology, Fukuyama, Hiroshima, Japan

ABSTRACT- Ascorbate (AsA) plays a central function to protect eukaryotes from oxidative stress. When AsA acts as an antioxidant in cells, in most cases, monodehydroascorbate (MDA) radical is produced as the primary oxidation product. In plants, the reaction catalyzed by AsA peroxidase is a major source of MDA radicals, which scavenges hydrogen peroxide. MDA is a "sink" of radicals generated in plant cells. To maintain the antioxidant activity of AsA, the regeneration of AsA from MDA radical is obviously indispensable. Monodehydroascorbate (MDA) radical reductase (EC 1.6.5.4.) is an FAD enzyme that catalyzes the univalent-reduction of MDA radical to AsA using NAD(P)H as an electron donor. It is the only enzyme known to use an organic radical as an enzyme substrate. The kinetic analysis suggests that this enzymatic reaction is facilitated by the electrostatic interaction between the enzyme and substrates. To understand the reaction mechanism of this characteristic flavoenzyme, its three dimensional structure is indispensable. The recombinant MDA reductase from cucumber produced in E. coli was crystallized using polyethylene glycol 6000 as a precipitant. The crystals belong to space group P2₁, with unit-cell parameters a = 60.8 , b = 138.6 , c = 61.7 , = 114.5°, and contained two molecules per asymmetric unit. The Matthews coefficient (V_M) and the solvent content are 2.46 ³ Da^-1 and 50.0%, respectively. The diffraction data were collected up to a resolution of 2.4 at 100 K using CuK radiation with a multi-wire area detector and gave a data set with an overall R_sym of 10.0% and a completeness of 92.5%. Structure determination of MDA reductase by heavy atom derivative and by molecular replacement methods is currently under way.

KEY WORDS: monodehydroascorbate radical reductase, ascorbate, crystallization, three dimensional structure