Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in Escherichia coli.

The Journal of Biological Chemistry
Satoshi SanoKozi Asada

Abstract

Monodehydroascorbate radical (MDA) reductase, an FAD-enzyme, is the first enzyme to be identified whose substrate is an organic radical and catalyzes the reduction of MDA to ascorbate by NAD(P)H. Its cDNA has been cloned from cucumber seedlings (Sano, S., and Asada, K. (1994) Plant Cell Physiol. 35, 425-437), and a plasmid was constructed in the present study that allowed a high level expression in Escherichia coli of the cDNA-encoding MDA reductase using the T7 RNA polymerase expression system. The recombinant MDA reductase was purified to a crystalline state, with a yield of over 20 mg/liter of culture, and it exhibited spectroscopic properties of the FAD similar to those of the enzyme purified from cucumber fruits during redox reactions with NADH and MDA. The red semiquinone of the FAD of MDA reductase was generated by photoreduction. p-Chloromercuribenzoate inhibited the reduction of the enzyme-FAD by NADH, and dicumarol suppressed electron transfer from the reduced enzyme to MDA. The specificity of electron acceptors of the recombinant enzyme appeared to be similar to that of MDA reductase, even though the amino acid sequence encoded by the cDNA was somewhat different from that of the enzyme purified from cucumber fruits. ...Continue Reading

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Citations

Jul 7, 2005·Antioxidants & Redox Signaling·Ken'ichi Ogawa
Dec 29, 2000·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·K Asada
Mar 12, 2004·Annual Review of Plant Physiology and Plant Molecular Biology·Kozi Asada
Apr 26, 2005·Bioscience, Biotechnology, and Biochemistry·Satoshi SanoKazumi Saito
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