Publications

• Monodehydroascorbate reductase from cucumber is a flavin adenine dinucleotide enzyme.

  Hossain M.A., Asada K.

  Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduce ... >> More

  Monodehydroascorbate reductase (EC 1.6.5.4) was purified from cucumber fruit to a homogeneous state as judged by polyacrylamide gel electrophoresis. The cucumber monodehydroascorbate reductase was a monomer with a molecular weight of 47,000. It contained 1 mol of FAD/mol of enzyme which was reduced by NAD(P)H and reoxidized by monodehydroascorbate. The enzyme had an exposed thiol group whose blockage with thiol reagents inhibited the electron transfer from NAD(P)H to the enzyme FAD. Both NADH and NADPH served as electron donors with Km values of 4.6 and 23 microM, respectively, and Vmax of 200 mol of NADH and 150 mol of NADPH oxidized mol of enzyme-1 s-1. The Km for monodehydroascorbate was 1.4 microM. The amino acid composition of the enzyme is presented. In addition to monodehydroascorbate, the enzyme catalyzed the reduction of ferricyanide and 2,6-dichloroindophenol but showed little reactivity with calf liver cytochrome b5 and horse heart cytochrome c. The kinetic data suggested a ping-pong mechanism for the monodehydroascorbate reductase-catalyzed reaction. Cucumber monodehydroascorbate reductase occurs in soluble form and can be distinguished from NADPH dehydrogenase, NADH dehydrogenase, DT diaphorase, microsome-bound NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase by its molecular weight, amino acid composition, and specificity of electron acceptors and donors. << Less

  J. Biol. Chem. 260:12920-12926(1985) [PubMed] [EuropePMC]

• Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, Physcomitrella patens and characterization of the expressed enzymes.

  Drew D.P., Lunde C., Lahnstein J., Fincher G.B.

  Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in t ... >> More

  Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The Km and kcat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 microM and 120-130 s(-1), respectively, using NADH as the electron donor. The Km values were at least an order of magnitude higher for NADPH. The Km values for the MDHA radical were approximately 0.5-1.0 microM for each of the purified enzymes, and further kinetic analyses indicated that PpMDHARs follow a 'ping-pong' kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated that the conserved cysteine residue is not directly involved in the reduction of MDHA. << Less

  Planta 225:945-954(2007) [PubMed] [EuropePMC]

• Tobacco nectarin III is a bifunctional enzyme with monodehydroascorbate reductase and carbonic anhydrase activities.

  Carter C.J., Thornburg R.W.

  Tobacco plants secrete a limited array of proteins (nectarins) into their floral nectar. N-terminal sequencing of the Nectarin II ( NEC2; 35kD) and the Nectarin III ( NEC3; 40kD) proteins revealed that they both share identity with dioscorin, the major soluble protein of yam tubers. These sequence ... >> More

  Tobacco plants secrete a limited array of proteins (nectarins) into their floral nectar. N-terminal sequencing of the Nectarin II ( NEC2; 35kD) and the Nectarin III ( NEC3; 40kD) proteins revealed that they both share identity with dioscorin, the major soluble protein of yam tubers. These sequences also revealed that NEC2 is a breakdown product of NEC3. Using these N-terminal peptide sequences, degenerate oligonucleotides were designed that permitted the isolation of a partial NEC3 cDNA. This cDNA was then used to probe a nectary specific cDNA library and a full-length NEC3 cDNA clone was isolated. Complete sequence analysis confirmed the identity of NEC3 as a dioscorin-like protein. MALDI-TOF mass spectrometric fingerprinting of tryptic peptides derived from the purified NEC3 confirmed that this protein was encoded by the isolated cDNA. NEC3 was shown to possess both carbonic anhydrase and monodehydroascorbate reductase activities. RT-PCR based expression analyses demonstrated that NEC3 transcript is expressed throughout nectary development as well as in other floral organs. A proposed function in the maintenance of pH and oxidative balance in nectar is discussed. << Less

  Plant Mol. Biol. 54:415-425(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Ascorbate free radical reductase mRNA levels are induced by wounding.

  Grantz A.A., Brummell D.A., Bennett A.B.

  A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide ... >> More

  A cDNA clone encoding ascorbate free radical (AFR) reductase (EC 1.6.5.4) was isolated from tomato (Lycopersicon esculentum Mill.) and its mRNA levels were analyzed. The cDNA encoded a deduced protein of 433 amino acids and possessed amino acid domains characteristic of flavin adenine dinucleotide- and NAD(P)H-binding proteins but did not possess typical eukaryotic targeting sequences, suggesting that it encodes a cytosolic form of AFR reductase. Low-stringency genomic DNA gel blot analysis indicated that a single nuclear gene encoded this enzyme. Total ascorbate contents were greatest in leaves, with decreasing amounts in stems and roots and relatively constant levels in all stages of fruit. AFR reductase activity was inversely correlated with total ascorbate content, whereas the relative abundance of AFR reductase mRNA was directly correlated with enzyme activity in tissues examined. AFR reductase mRNA abundance increased dramatically in response to wounding, a treatment that is known to also induce ascorbate-dependent prolyl hydroxylation required for the accumulation of hydroxyproline-rich glycoproteins. In addition, AFR reductase may contribute to maintaining levels of ascorbic acid for protection against wound-induced free radical-mediated damage. Collectively, the results suggest that AFR reductase activity is regulated at the level of mRNA abundance by low ascorbate contents or by factors that promote ascorbate utilization. << Less

  Plant Physiol. 108:411-418(1995) [PubMed] [EuropePMC]

• A direct demonstration of the catalytic action of monodehydroascorbate reductase by pulse radiolysis.

  Kobayashi K., Tagawa S., Sano S., Asada K.

  To elucidate the catalytic mechanism of monodehydroascorbate (MDA) reductase from cucumber, its interaction with MDA radical was investigated by the use of pulse radiolysis. When approximately equimolar MDA radical to the fully reduced MDA reductase was generated, the fully reduced enzyme reacted ... >> More

  To elucidate the catalytic mechanism of monodehydroascorbate (MDA) reductase from cucumber, its interaction with MDA radical was investigated by the use of pulse radiolysis. When approximately equimolar MDA radical to the fully reduced MDA reductase was generated, the fully reduced enzyme reacted first with MDA radical to form the red semiquinone, and the semiquinone further reacted with MDA radical to form the oxidized enzyme. At a low ratio (< 20) of MDA radical to enzyme concentration, the fully reduced enzyme reacted quantitatively with MDA radical to form the semiquinone with a second-order rate constant of 2.6 x 10(8) M-1 s-1 at pH 7.4. At excess MDA radical to enzyme concentration, a similar rate constant was obtained from the decay of MDA radical. These results suggest that the reaction of the semiquinone with MDA radical occurs at the same rate or rate-limiting step of the oxidation of the fully reduced enzyme by MDA radical. The rate constants decreased with an increase in NaCl concentration, suggesting that the localization of cationic groups of amino acid residue near the active site may provide electrostatic guidance to the anionic substrate of MDA radical. << Less

  J Biol Chem 270:27551-27554(1995) [PubMed] [EuropePMC]

• Molecular cloning and characterization of a cDNA encoding pea monodehydroascorbate reductase.

  Murthy S.S., Zilinskas B.A.

  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 m ... >> More

  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. << Less

  J. Biol. Chem. 269:31129-31133(1994) [PubMed] [EuropePMC]

• Purification and characterization of monodehydroascorbate reductase from soybean root nodules.

  Dalton D.A., Langeberg L., Robbins M.

  Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silv ... >> More

  Soybean (Glycine max (L.) Merr.) root nodules contain the enzymes of the ascorbate-glutathione cycle as an important defense against activated forms of oxygen. A key enzyme in this cycle--monodehydroascorbate reductase (MR)--was purified 646-fold and appeared as a single band on SDS-PAGE with silver or Coomassie blue staining. Purified MR contained 0.7 mol FAD/mol enzyme and had a specific activity of 288 mumol NADH oxidized.min-1.mg protein-1. The enzyme was a single subunit occurring as two isozymes (MR I and MR II) with Mr values of 39,000 and 40,000. Isoelectric focusing revealed that each isozyme consisted of two forms with pl values of 4.6 to 4.7. Ferricyanide and 2,6-dichlorophenol-indophenol were effective as electron acceptors. The purified enzyme did not possess leghemoglobin reductase activity. Inhibition by p-chloromercuribenzoate indicated the involvement of a thiol group in MR activity. The Km values were 5.6, 150, and 7 microM for NADH, NADPH, and monodehydroascorbate, respectively. The pH optimum was 8 to 9. The N-terminal sequence of 10 amino acids of MR II had little homology to known protein sequences. << Less

  Arch. Biochem. Biophys. 292:281-286(1992) [PubMed] [EuropePMC]

• Crystallization and preliminary crystallographic analysis of monodehydroascorbate radical reductase from cucumber.

  Sano S., Kang Y.N., Shigemizu H., Morishita N., Yoon H.J., Saito K., Asada K., Mikami B.

  Monodehydroascorbate (MDA) radical reductase (EC 1.6.5.4) is an FAD enzyme that catalyzes the univalent reduction of MDA radical to ascorbate using NAD(P)H as an electron donor. The recombinant MDA reductase from cucumber was crystallized using polyethylene glycol 6000 as a precipitant. The crysta ... >> More

  Monodehydroascorbate (MDA) radical reductase (EC 1.6.5.4) is an FAD enzyme that catalyzes the univalent reduction of MDA radical to ascorbate using NAD(P)H as an electron donor. The recombinant MDA reductase from cucumber was crystallized using polyethylene glycol 6000 as a precipitant. The crystals belong to space group P2(1), with unit-cell parameters a = 60.8, b = 138.6, c = 61.7 A, beta = 114.5 degrees, and contained two molecules per asymmetric unit. The Matthews coefficient (VM) and the solvent content are 2.46 A3 Da(-1) and 50.0%, respectively. Diffraction data were collected to a resolution of 2.4 A at 100 K using Cu Kalpha radiation with a multi-wire area detector and gave a data set with an overall Rsym of 10.0% and a completeness of 92.5%. << Less

  Acta Crystallogr D Biol Crystallogr 60:1498-1499(2004) [PubMed] [EuropePMC]