Molecular characterization of monodehydroascorbate radical reductase from cucumber highly expressed in Escherichia coli.
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. The Km values for NADH and NADPH of the recombinant enzyme indicated a high affinity of the enzyme for NADH. The reaction catalyzed by the enzyme did not exhibit saturation kinetics with MDA up to 3 microM. A second order rate constant for the reduction of the enzyme-FAD with NADH was 1.25 x 10(8) M-1 s-1, as determined by a stopped-flow method, and its value decreased with increases in ionic strength, an indication of the enhanced electrostatic guidance of NADH to the enzyme-FAD.
Sano
S
S
Research Institute for Food Science, Kyoto University, Japan.
Miyake
C
C
Mikami
B
B
Asada
K
K
D003001:Cloning, Molecular; D018553:Cucumis sativus / Q000201:enzymology*; D018076:DNA, Complementary; D001728:Dicumarol / Q000494:pharmacology; D004583:Electrons; D004926:Escherichia coli; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010957:Plasmids; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D001728:Dicumarol; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)