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PubMed2000

Characterization and partial purification of microsomal NAD(P)H:quinone oxidoreductases.

Quinone oxidoreductases are flavoproteins that catalyze two-electron reduction and detoxification of quinones. This leads to the protection of cells against toxicity, mutagenicity, and cancer due to exposure to environmental and synthetic quinones and its precursors. Two cytosolic forms of quinone oxidoreductases [NAD(P)H:quinone oxidoreductase 1 (NQO1) and NRH:quinone oxidoreductase 2 (NQO2)] were previously identified, purified, and cloned. A role of cytosolic NQO1 in protection of cells from oxidative stress, cytotoxicity, and mutagenicity of quinones was established. Currently, we have characterized and partially purified the NQO activity from rat liver microsomes. This activity was designated as microsomal NQO (mNQO). The mNQO activity showed significantly higher affinity for NADH than NADPH as electron donors and catalyzed reduction of 2,6-dichlorophenolindophenol and menadione. The mNQO activity was insensitive to dicoumarol, a potent inhibitor of cytosolic NQO1. Western analysis of microsomal proteins revealed 29- and 18-kDa bands that cross-reacted with polyclonal antibodies raised against cytosolic NQO1. The mNQO activity was partially purified by solubilization of microsomes with detergent Chaps, ammonium sulfate fractionation, and DEAE-Sephacel column chromatography. The microsomal mNQO proteins are expected to provide additional protection after cytosolic NQOs against quinone toxicity and mutagenicity.

2,6-Dichloroindophenol↗
PubMed1992

High levels of xanthine oxidoreductase in rat endothelial, epithelial and connective tissue cells. A relation between localization and function?

The localization of xanthine oxidoreductase activity was investigated in unfixed cryostat sections of various rat tissues by an enzyme histochemical method which specifically demonstrates both the dehydrogenase and oxidase forms of xanthine oxidoreductase. High activity was found in epithelial cells from skin, vagina, uterus, penis, liver, oral and nasal cavities, tongue, esophagus, fore-stomach and small intestine. In addition activity was demonstrated in sinusoidal cells of liver and adrenal cortex, endothelial cells in various organs and connective tissue fibroblasts. Xanthine oxidoreductase produces urate which is a scavenger of oxygen-derived radicals. Because the enzyme is found in epithelial and endothelial cells which are subject to relatively high oxidant stress, it is postulated that in these cells xanthine oxidoreductase is involved in the antioxidant enzyme defense system. In addition, a possible role for the enzyme in proliferation and differentiation processes is discussed.

Adrenal Cortex↗
PubMed2003

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1.

The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as "hydrophilic" and "hydrophobic" (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372-1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H(2)O(2)-Fe(2+). Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme.

Animals↗
PubMed1990

New evidence for the dimeric nature of NADH:Q oxidoreductase in bovine-heart submitochondrial particles.

The initial velocity of NADH oxidation by bovine-heart submitochondrial particles was measured at pH 8.0 after pretreatment of these particles with different amounts of the inhibitor piericidine A together with 0.035 mM NADH. The amount of piericidine A required to fully inhibit the NADH oxidation activity extrapolated to exactly 1.0 per Fe-S cluster 2 of NADH:Q oxidoreductase. When no reducing equivalents from NADH were present during the pretreatment, this ratio was 1.2. The difference is explained by assuming that NADH:Q oxidoreductase binds piericidine A more effectively in the reduced state than in the oxidized state. It was also found that after Q10-extraction and reincorporation of submitochondrial particles, the amount of piericidine A required to fully inhibit the NADH oxidation activity of the particles increased with the amount of Q10 present during reincorporation. This is explained by assuming that binding of piericidine A, to the inhibitory site of NADH:Q oxidoreductase requires Q10. When 0.035 mM NADPH instead of NADH was present during the pretreatment of submitochondrial particles with piericidine A, the amount of inhibitor per cluster 2 required to fully inhibit the initial NADH-oxidation activity extrapolated to 0.5. This result strongly suggests that NADH:Q oxidoreductase is a functional dimer.

Animals↗
PubMed1992

The PSI-E subunit of photosystem I binds ferredoxin:NADP+ oxidoreductase.

A photosystem I complex containing the polypeptides PSI-A to PSI-L, light-harvesting complex I and ferredoxin:NADP+ oxidoreductase has been isolated from barley using the non-ionic detergent n-decyl-beta-D-maltopyranoside. The ratio between bound ferredoxin:NADP+ oxidoreductase and P700 is 0.4 +/- 0.2. The complex is highly active in catalyzing light-induced transfer of electrons from plastocyanin to NADP+ at rates of 280 +/- 150 and 1800 +/- 800 mumol NADPH/(mg chl.h), without and in the presence of saturating amounts of exogenously added ferredoxin:NADP+ oxidoreductase, respectively. Endogenously bound ferredoxin:NADP+ oxidoreductase interacts with the PSI-E subunit as demonstrated by cross-linking experiments using two different types of cross-linkers and identification of the products by Western blotting and the use of monospecific antibodies.

Cross-Linking Reagents↗
PubMed2006

FCD1 encoding protein homologous to cellobiose: quinone oxidoreductase in Fusarium oxysporum.

We had cloned and characterized a gene from Fusarium oxysporum designated FCD1, encoding a putative cellobiose: quinone oxidoreductase (CBQ) which is a member of the extracellular redox enzyme family and also a member of glucose-methanol-choline (GMC) oxidoreductases. CBQ is known to be a free flavin domain of a cellobiose dehydrogenase (CDH) generated by proteolysis, but FCD1 gene encodes CBQ directly. In a phylogenetic tree of amino acid sequences of FCD1, GMC oxidoreductases and hypothetical GMC oxidoreductases, FCD1 clustered together with flavin domains (CBQs) of CDHs and putative proteins with unknown function of ascomycetes. FCD1-disruptants showed no reduction in virulence toward tomato and no obvious morphological effects such as production of conidia and mycelial growth as compared to the wild type strain, suggesting that FCD1 is not essential for virulence and vigor in F. oxysporum.

Amino Acid Sequence↗
PubMed2004

Influence of the oxidoreductase ER57 on the folding of an antibody fab fragment.

Oxidation and folding of secretory proteins in the endoplasmic reticulum (ER) depends on the presence of chaperones and oxidoreductases. Two of the oxidoreductases present in the ER of mammalian cells are protein disulfide isomerase (PDI) and ERp57. In this study, we investigated the influence of ERp57 on the in vitro reoxidation and refolding of an antibody Fab fragment. Our results show that ERp57 shares functional properties with PDI and that both are clearly different from other oxidoreductases. The reactivation of the denatured and reduced Fab fragment was enhanced significantly in the presence of ERp57 with kinetics and redox dependence of the reactivation reaction comparable to those obtained for PDI. These properties were not influenced by the presence of calnexin. Furthermore, whereas PDI cooperates with the immunoglobulin heavy chain binding protein (BiP), no synergistic effect could be observed for BiP and ERp57. These results indicate that the cooperation of the two oxidoreductases with different partner proteins may explain their different roles in the folding of proteins in the ER.

Enzyme-Linked Immunosorbent Assay↗
PubMed1989

The pathway of electron transfer in NADH:Q oxidoreductase.

The pre-steady-state reduction by NADPH of NADH:Q oxidoreductase, as present in submitochondrial particles, has been further investigated with the rapid-mixing, rapid-freezing technique. It was found that trypsin treatment, that had previously been used to inactivate the transhydrogenase activity (Bakker, P.T.A. and Albracht, S.P.J. (1986) Biochim. Biophys. Acta 850, 413-422), considerably affected the stability at pH 6.2 of the NAD(P)H oxidation activity of submitochondrial particles. Use of the inhibitor butadione circumvented this problem, thus allowing a more careful investigation of the kinetics at pH 6.2. In the presence of the inhibitor rotenone it was found that 50% of the Fe-S clusters 3 and all of the Fe-S clusters 2 and 4 could be reduced by NADPH within 30 ms at pH 6.2. The remainder of the Fe-S clusters 3 and all of the Fe-S clusters 1 were reduced slowly (complete reduction only after more than 60 s). It was concluded that these latter Fe-S clusters play no role in the NADPH oxidation activity. In the absence of rotenone at pH 6.2 only 50% of the Fe-S clusters 2-4 could be reduced within 30 ms, while Fe-S cluster 1 was again not reduced. This difference was attributed to the fast reoxidation of part of the Fe-S clusters 2 and 4 by ubiquinone. At pH 8.0, where the NADPH oxidation activity is almost zero, 50% of the Fe-S clusters 2-4 could still be reduced by NADPH within 30 ms, while Fe-S cluster 1 was not reduced. The presence of rotenone had no effect on this reduction. From these observations it is concluded that the Fe-S clusters 2 and 4, which were rapidly reduced by NADPH and reoxidised by ubiquinone at pH 6.2, could not be reduced by NADPH at 8.0. This provides an explanation why NADH:Q oxidoreductase was not able to oxidise NADPH at pH 8.0, while part of the Fe-S clusters were still rapidly reduced. As a working hypothesis a dimeric structure for NADH:Q oxidoreductase is proposed. One protomer (B) contains FMN and Fe-S clusters 1-4 in equal amounts; the other protomer (A) is identical except for the absence of Fe-S cluster 1. NADH is able to react with both protomers, while NADPH only reacts with protomer A. A pH-dependent electron transfer from protomer A to protomer B is proposed, which would allow the reduction of Fe-S clusters 2 and 4 of protomer B by NADPH at pH 6.2, which is required for NADPH:Q oxidoreductase activity.

Animals↗
PubMed1998

Reduction of the plastoquinone pool by exogenous NADH and NADPH in higher plant chloroplasts. Characterization of a NAD(P)H-plastoquinone oxidoreductase activity

Chlorophyll fluorescence measurements were performed on osmotically lysed potato chloroplasts in order to characterize the reactions involved in the dark reduction of photosynthetic inter-system chain electron carriers. Addition of NADH or NADPH to lysed chloroplasts increased the chlorophyll fluorescence level measured in the presence of a non-actinic light until reaching Fmax, thus indicating an increase in the redox state of the plastoquinone (PQ) pool. The fluorescence increase was more pronounced when the experiment was carried out under anaerobic conditions and was about 50% higher when NADH rather than NADPH was used as an electron donor. The NAD(P)H-PQ oxidoreductase reaction was inhibited by diphenylene iodonium, N-ethylmaleimide and dicoumarol, but insensitive to rotenone, antimycin A and piericidin A. By comparing the substrate specificity and the inhibitor sensitivity of this reaction to the properties of spinach ferredoxin-NADP+-reductase (FNR), we infer that FNR is not involved in the NAD(P)H-PQ oxidoreductase activity and conclude to the participation of rotenone-insensitive NAD(P)H-PQ oxidoreductase. By measuring light-dependent oxygen uptake in the presence of DCMU, methyl viologen and NADH or NADPH as an electron donors, the electron flow rate through the NAD(P)H-PQ oxidoreductase is estimated to about 160 nmol O2 min-1 mg-1 chlorophyll. The nature of this enzyme is discussed in relation to the existence of a thylakoidal NADH dehydrogenase complex encoded by plastidial ndh genes. Copyright 1998 Elsevier Science B.V.

Journal Article↗
PubMed2004

Congenital adrenal hyperplasia caused by mutant P450 oxidoreductase and human androgen synthesis: analytical study.

BACKGROUND: Congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency is associated with accumulation of steroid metabolites, indicating impaired activity of 17alpha-hydroxylase and 21-hydroxylase. However, no mutations have been reported in the CYP17 and CYP21 genes, which encode these P450 enzymes. Affected girls are born with ambiguous genitalia, but their circulating androgens are low, and virilisation does not progress. We aimed to investigate the underlying molecular basis of congenital adrenal hyperplasia with apparent combined P450C17 and P450C21 deficiency in affected children. METHODS: We did sequence analysis of the human gene encoding P450 oxidoreductase, an enzyme that is important in electron transfer from NADPH to P450C17 and P450C21. We studied two unrelated families with a total of three affected children and 100 healthy controls. Wild-type and mutant P450 oxidoreductase proteins were bacterially expressed, purified, and assayed for cytochrome c reductase activity. FINDINGS: We identified four mutations encoding single aminoacid changes in P450 oxidoreductase. All patients were compound heterozygotes, whereas their parents and an unaffected sibling harboured a mutation in only one allele. By contrast, no mutations were noted in the controls. Bacterial expression of recombinant mutant proteins revealed deficient or reduced enzyme activity. INTERPRETATION: Molecular pathogenesis of this form of congenital adrenal hyperplasia is caused by mutations in the gene encoding P450 oxidoreductase. Deficiency of this enzyme could suggest an alternative pathway in human androgen synthesis, present only in fetal life, which explains the combination of antenatal androgen excess and postnatal androgen deficiency.

Adolescent↗
PubMed1993

DNA sequencing of the seven remaining structural genes of the gene cluster encoding the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans.

In our previous papers, seven structural genes (NQO1-7) of the energy-transducing NADH-quinone (Q) oxidoreductase of Paracoccus denitrificans were characterized [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991a) Biochemistry 30, 8678-8684; (1991b) Biochemistry 30, 6422-6428; (1992a) Biochemistry 31, 6925-6932; (1992b) Arch. Biochem. Biophys. 296, 40-48]. This paper reports the identification, cloning, and sequencing of seven additional structural genes in the same gene cluster (P. denitrificans enzyme complex). These seven genes, designated NQO8-14, are composed of 1038, 492, 603, 306, 2112, 1542, and 1500 base pairs, respectively. The polypeptides encoded by the NQO8-14 genes are homologous, respectively, to the ND1 product, the 23-kDa polypeptide, and the ND6, ND4L, ND5, ND4, and ND2 products of the bovine NADH-Q oxidoreductase. The order of the 14 structural genes of the Paracoccus energy-transducing NADH-Q oxidoreductase in the gene cluster is NQ07, NQO6, NQO5, NQO2, NQO1, NQO3, NQO8, NQO9, NQO10, NQO11, NQO12, NQO13, and NQO14. Downstream from the NQO14 gene an open reading frame (designated URF240) was detected which encodes a predicted polypeptide homologous to the biotin [acetyl-CoA-carboxylase] ligase of Escherichia coli. In addition, a putative terminal sequence motif was observed downstream of the NQO14 gene, suggesting that the structural gene NQO14 is the 3'-terminal gene of the Paracoccus NADH-Q oxidoreductase gene cluster. Nucleotide sequencing of the entire gene cluster revealed the presence of three unidentified reading frames: one between the NQO3 and NQO8 genes and other two between the NQO9 and NQO10 genes. These are designated URF4, URF5, and URF6 and are composed of 768, 393, and 405 base pairs, respectively. The possible functions of the putative proteins encoded by URF5 and URF6 are discussed.

Amino Acid Sequence↗
PubMed1992

Gene cluster of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans: characterization of four structural gene products.

In previous reports from our laboratory, the three structural genes (NQO1, NQO2, and NQO3) of the energy-transducing NADH-quinone oxidoreductase of Paracoccus denitrificans were characterized [Xu, X., Matsuno-Yagi, A., & Yagi, T. (1991) Biochemistry 30, 6422-6428; (1991) Biochemistry 30, 8678-8684; (1992) Arch. Biochem. Biophys. 296, 40-48]. In this report, the four structural genes NQO4, NQO5, NQO6, and NQO7 of the same Paracoccus denitrificans oxidoreductase were cloned and sequenced. On the basis of sequence homology and immunological cross-reactivity, these genes encode counterparts of the 49-, 30-, and 20-kDa polypeptides and the mitochondrial DNA ND3 polypeptides of bovine mitochondrial complex I. These seven structural genes were found to be located in the same gene cluster. The order of the seven structural genes of the Paracoccus NADH-quinone oxidoreductase in the gene cluster is NQO7, NQO6, NQO5, NQO4, NQO2, NQO1, and NQO3. Upstream of the NQO7 gene, an open reading frame encoding a predicted polypeptide homologous to the UV repair enzyme A of Escherichia coli and Micrococcus lysodeikticus was detected. The 5'-terminus of the gene cluster carrying the Paracoccus NADH-quinone oxidoreductase was studied, and the possible promoter region is discussed. The NQO4 and NQO5 genes appear to code for the M(r) 48,000 and 21,000 polypeptides of the isolated Paracoccus NADH dehydrogenase complex [Yagi, T. (1986) Arch. Biochem. Biophys. 250, 302-311] on the basis of amino acid analyses and N-terminal protein sequence analyses. The antisera to the bovine complex I 49- and 30-kDa polypeptides cross-reacted with the Paracoccus 48- and 21-kDa subunits, respectively.

Amino Acid Sequence↗
PubMed1986

NADPH-cytochrome P-450 oxidoreductase: flavin mononucleotide and flavin adenine dinucleotide domains evolved from different flavoproteins.

The FMN-binding domain of NADPH-cytochrome P-450 oxidoreductase, residues 77-228, is homologous with bacterial flavodoxins, while the FAD-binding domain, residues 267-678, shows a high degree of similarity to two FAD-containing proteins, ferredoxin-NADP+ reductase and NADH-cytochrome b5 reductase. Comparison of these proteins to glutathione reductase, a flavoprotein whose three-dimensional structure is known, has permitted tentative identification of FAD- and cofactor-binding residues in these proteins. The remarkable conservation of sequence between NADPH-cytochrome P-450 oxidoreductase and ferredoxin-NADP+ reductase, coupled with the homology of the FMN-binding domain of the oxidoreductase with the bacterial flavodoxins, implies that NADPH-cytochrome P-450 oxidoreductase arose as a result of fusion of the ancestral genes for these two functionally linked flavoproteins.

Amino Acid Sequence↗
PubMed1990

Nucleotide and deduced amino acid sequence of a human cDNA (NQO2) corresponding to a second member of the NAD(P)H:quinone oxidoreductase gene family. Extensive polymorphism at the NQO2 gene locus on chromosome 6.

NAD(P)H:quinone oxidoreductases (NQOs) are flavoproteins that catalyze the oxidation of NADH or NADPH by various quinones and oxidation-reduction dyes. We have previously described a complementary DNA that encodes a dioxin-inducible cytosolic form of human NAD(P)H:quinone oxidoreductase (NQO1). In the present report we describe the nucleotide sequence and deduced amino acid sequence for a cDNA clone that is likely to encode a second form of NAD(P)H:quinone oxidoreductase (NQO2) which was isolated by screening a human liver cDNA library by hybridization with a NQO1 cDNA probe. The NQO2 cDNA is 976 nucleotides long and encodes a protein of 231 amino acids (Mr = 25,956). The human NQO2 cDNA and protein are 54% and 49% similar to human liver cytosolic NQO1 cDNA and protein, respectively. COS1 cells transfected with NQO2 cDNA showed a 5-7-fold increase in NAD(P)H:quinone oxidoreductase activity as compared to nontransfected cells when either 2,6-dichlorophenolindophenol or menadione was used as substrate. Western blot analysis of the expressed NQO1 and NQO2 cDNA proteins showed cross-reactivity with rat NQO1 antiserum, indicating that NQO1 and NQO2 proteins are immunologically related. Northern blot analysis shows the presence of one NQO2 mRNA of 1.2 kb in control and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) treated human hepatoblastoma Hep-G2 cells and that TCDD treatment does not lead to enhanced levels of NQO2 mRNA as it does for NQO1 mRNA. Southern blot analysis of human genomic DNA suggests the presence of a single gene approximately 14-17 kb in length. The NQO2 gene locus is highly polymorphic as indicated by several restriction fragment length polymorphisms detected with five different restriction enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1970

Specificity and locale of the L-3-glycerophosphate-flavoprotein oxidoreductase of mitochondria isolated from the flight muscle of Sarcophaga barbata thoms.

1. The oxidation of l-3-glycerophosphate by flight-muscle mitochondria isolated from the flesh fly Sarcophaga barbata has been studied. Use of substrate analogues indicates that the catalytic and effector l-3-glycerophosphate binding sites on the allosteric l-3-glycerophosphate-flavoprotein oxidoreductase differ markedly in specificity. 2. The l-3-glycerophosphate-cyanoferrate oxidoreductase system in these mitochondria is antimycin-insensitive whereas the corresponding NADH-cyanoferrate oxidoreductase is extremely sensitive to this respiratory-chain inhibitor. Also no swelling is observed when these mitochondria are suspended in iso-osmotic solutions of ammonium glycerophosphate in contrast with the extensive swelling seen in similar solutions of ammonium pyruvate. These observations indicate that l-3-glycerophosphate does not penetrate the mitochondrial matrix whereas pyruvate does. 3. Submitochondrial particles catalyse the ATP-driven reduction of NAD(+) by l-3-glycerophosphate but at a far lower rate than that seen when succinate is the electron donor. These particles do not have an energy-linked pyridine nucleotide transhydrogenase activity. 4. We conclude that the l-3-glycerophosphate-flavoprotein oxidoreductase is located on the outer surface of the inner membrane of the flight-muscle mitochondria.

Adenosine Triphosphate↗
PubMed1988

NN'-dicyclohexylcarbodi-imide-sensitivity of bovine heart mitochondrial NADH: ubiquinone oxidoreductase. Inhibition of activity and binding to subunits.

Dicyclohexylcarbodi-imide (DCCD) inhibition of NADH: ubiquinone oxidoreductase was studied in submitochondrial particles and in the isolated form, together with the binding of the reagent to the enzyme. DCCD inhibited the isolated enzyme in a time- and concentration-dependent manner. Over the concentration range studied, a maximum inhibition of 85% was attained within 60 min. The time course for the binding of DCCD to the enzyme was similar to that of activity inhibition. The NADH:ubiquinone oxidoreductase activity of the submitochondrial particles was also sensitive to DCCD, and the locus of binding of the inhibitor was studied by subsequent resolution of the enzyme into subunit polypeptides. Only two subunits (molecular masses 13.7 and 21.5 kDa) were labelled by [14C]DCCD, whereas, when the enzyme in its isolated form was treated with [14C]DCCD, six subunits (13.7, 16.1, 21.5, 39, 43 and 53 kDa) were labelled. Comparison with the subunit labelling of F1F0-ATPase and ubiquinol:cytochrome c oxidoreductase indicated that the labelling pattern of NADH:ubiquinone oxidoreductase, and enzyme complex with a multitude of subunits, is unique and not due to contamination by other inner-membrane proteins. The correlation between the electron- and proton-transport functions and the DCCD-binding components remains to be established.

Animals↗
PubMed2001

Quinone oxidoreductase message levels are differentially regulated in parasitic and non-parasitic plants exposed to allelopathic quinones.

Allelopathic chemicals released by plants into the rhizosphere have effects on neighboring plants ranging from phytoxicity to inducing organogenesis. The allelopathic activity of naturally occurring quinones and phenols is primarily a function of reactive radicals generated during redox cycling between quinone and hydroquinone states. We isolated cDNAs encoding two distinct quinone oxidoreductases from roots of the parasitic plant Triphysaria treated with the allelopathic quinone 2,6-dimethoxybenzoquinone (DMBQ). TvQR1 is a member of the zeta-crystallin quinone oxidoreductase family that catalyzes one-electron quinone reductions, generating free radical semiquinones. TvQR2 belongs to a family of detoxifying quinone oxidoreductases that catalyze bivalent redox reactions which avoid the radical intermediate. TvQR1 and TvQR2 message levels are rapidly upregulated in Triphysaria roots as a primary response to treatment with various allelopathic quinones. Inhibition of quinone oxidoreductase enzymatic activity with dicumarol prior to quinone treatment resulted in increased transcript levels. While TvQR2 homologs were upregulated by DMBQ in roots of all plants examined, TvQR1 homologs were upregulated only in roots of parasitic plants. Phylogenetic trees constructed of TvQR1 and TvQR2 protein homologs in Archea, Eubacteria and Eukaryotes indicated that both gene families are ancient, yet the families have dissimilar evolutionary histories in angiosperms. We hypothesize that TvQR2-like proteins function to detoxify allelopathic quinones in the rhizosphere, while TvQR1 has specific functions associated with haustorium development in parasitic plants.

Alleles↗
PubMed1983

Activation of plant quinate:NAD 3-oxidoreductase by Ca and calmodulin.

Quinate:NAD(+) 3-oxidoreductase (EC 1.1.1.24) from carrot cell suspension cultures has previously been shown to be activated by phosphorylation and inactivated by dephosphorylation. Here it is shown that the reactivation of the inactivated quinate:NAD(+) oxidoreductase is an enzyme-mediated process that requires ATP and protein kinase activity. The reactivation is completely inhibited by EGTA and can be restored by the addition of Ca(2+). Cyclic AMP at concentrations up to 5 muM did not have any effect on the reactivation either with or without EGTA in the medium. Calmodulin-depleted fractions containing quinate:NAD(+) oxidoreductase were obtained by passage of the crude extracts through an affinity column of 2-chloro-10-(3-aminopropyl)phenothiazine coupled to Sepharose 4B. The enzyme in this calmodulin-deficient fraction could be inactivated but not reactivated even in the presence of ATP and Ca(2+). However, addition of bovine brain calmodulin completely restored the activity of the enzyme. Half-maximal activation occurred at 130 nM calmodulin. We conclude from these data that the quinate:NAD(+) oxidoreductase is activated by a Ca(2+) - and calmodulin-dependent plant protein kinase.

Journal Article↗