Molecular cloning and characterization of a cDNA encoding pea monodehydroascorbate reductase.
Monodehydroascorbate radicals are generated in plant cells enzymatically by the hydrogen peroxide scavenging enzyme, ascorbate peroxidase, and nonenzymatically via the univalent oxidation of ascorbate by superoxide, hydroxyl, and various organic radicals. Regeneration of ascorbate is achieved by monodehydroascorbate reductase (EC 1.6.5.4) using NAD(P)H as an electron donor or, alternatively, by a set of two coupled reactions requiring dehydroascorbate reductase, glutathione reductase, glutathione, and NAD(P)H. As monodehydroascorbate reductase is a key enzyme in maintaining reduced pools of ascorbate, an important antioxidant, we undertook this study to learn more about its structure, function, and regulation. Herein we report the molecular cloning and characterization of a cDNA encoding monodehydroascorbate reductase of pea (Pisum sativum L.). The cDNA encodes a 433-amino acid polypeptide that shows, respectively, 73 and 87% identity with peptide fragments from soybean and cucumber monodehydroascorbate reductase. Monodehydroascorbate reductase contains the NAD(P)H and FAD binding domains of other flavin oxidoreductases. The cloned enzyme lacks a transit peptide, but the sequence of the carboxyl terminus is Ser-Lys-Ile, similar to the targeting motif found in peroxisomal proteins. When expressed in Escherichia coli fused to maltose-binding protein, monodehydroascorbate reductase has enzymatic properties comparable with purified soybean and cucumber monodehydroascorbate reductase. Northern blot analysis shows that the monodehydroascorbate reductase transcript is 1.6 kilobase in size and is expressed at relatively low levels in all plant tissues examined.
Murthy
S S
SS
Department of Plant Science, Cook College, Rutgers University, New Brunswick, New Jersey 08903-0231.
Zilinskas
B A
BA
D018528:ATP-Binding Cassette Transporters; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D018532:Pisum sativum / Q000201:enzymology*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D018076:DNA, Complementary; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; D009247:NADH, NADPH Oxidoreductases; C023927:monodehydroascorbate reductase (NADH)