N-methylputrescine oxidase from tobacco roots

Mizusaki, Shigenobu; Tanabe, Yoko; Noguchi, Masayoshi; Tamaki, Einosuke

doi:10.1016/s0031-9422(00)86509-7

Cited by 69 publications

(27 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…At pH 7.5 potassium phosphate buffer gave activity that was about 50% higher than that with Tris-HCl buffer. The optimum pH with MP as the substrate is similar to that reported for tobacco DAO (22).…”

Section: Results and Discussion Distribution Of Dao In Hyoscyamus Spesupporting

confidence: 81%

“…2; Table III) clearly demonstrate that the H. niger enzyme has a much greater affinity for N-methylated than for primary diamines, unlike pea, pig, and, probably, most other DAOs. The tobacco DAO that functions in the early steps of nicotine biosynthesis has also been reported to be more active with MP than with putrescine (22). Frydman et al (8) reported that N-ethyl-, N-propyl-, and N-butylputrescines at 0.83 mM were oxidized at rates of 25 to 76% of that of putrescine by the DAOs from pea seedling and pig kidney.…”

Section: Substrate Specificitymentioning

confidence: 99%

See 1 more Smart Citation

Diamine Oxidase from Cultured Roots of Hyoscyamus niger

Hashimoto¹,

Mitani²,

Yamada³

1990

Plant Physiol.

View full text Add to dashboard Cite

Diamine oxidase was partially purified from cultured roots of Hyoscyamus niger L. that produce considerable amounts of tropane alkaloids, and then characterized. N-Methylated amines inhibited the activity of the enzyme more strongly than the corresponding primary amines. N-Methylputrescine was the best substrate of those studied, the respective Km values for it and for putrescine and cadaverine being 0.33, 2.85, and 6.25 millimolar. The specificity constants Vmax/Km for putrescine and cadaverine were 11 and 1% of the constant for N-methylputrescine. Marked specificity for the N-methylated diamine would enable the Hyoscyamus enzyme to function specifically in tropane alkaloid biosynthesis.The roots of several solanaceous plants synthesize anticholinergic alkaloids such as hyoscyamine and scopolamine. The tropine moiety of these alkaloids originates from ornithine and/or arginine. In Hyoscyamus albus, the symmetric diamine putrescine derived from these amino acids is first converted to MP' by putrescine N-methyltransferase (EC 2.1.1.53) and then deaminated oxidatively to 4-methylaminobutanal which spontaneously cyclizes to give the N-methylpyrrolinium cation ( Fig. 1) (12,14,15). This oxidative deamination is catalyzed by a DAO (EC 1.4.3.6 Alkaloid-producing, cultured roots of Hyoscyamus albus contain both free putrescine and free MP in their cells (14). But to function specifically in alkaloid biosynthesis, the DAO in Hyoscyamus roots must oxidize MP more efficiently than putrescine and other diamines present. Or, these diamines 'Abbreviations: MP, N-methylputrescine; DAO, diamine oxidase. must be localized in different metabolic compartments in the cells. We here characterize the DAO from cultured Hyoscyamus roots with emphasis on its substrate specificity. MATERIALS AND METHODS Plant MaterialsRoots of Hyoscyamus niger L. have been maintained in vitro in our laboratory since 1984 as described elsewhere (13). Prior to the experiments reported here, samples of these roots were transferred to 300-mL flasks containing 75 mL of auxinfree B5 medium (9) supplemented with 3% (w/v) sucrose, and were then cultured for 6 d. After being harvested with a suction filter, the roots were frozen immediately with liquid nitrogen and then homogenized in a Waring Blendor. The frozen homogenate was kept at -20°C until use.Pea seeds (Pisum sativum cv Alaska) were surface-sterilized and then germinated in moistened vermiculite in the dark for 8 d. The epicotyls of the etiolated pea seedlings were excised and frozen in liquid nitrogen after which they were processed in the same way as Hyoscyamus roots.All the other root, shoot and cell-suspension cultures used had been maintained in our laboratory for several years. The conditions for their culture are described elsewhere ( 14 To study substrate specificity, the ammonia produced by the DAO reaction was measured enzymatically with glutamate dehydrogenase (from beef liver; Oriental Yeast Co., Tokyo) by monitoring the decrease in NADH at 339 nm (18).When purified pea DAO preparati...

show abstract

Section: Results and Discussion Distribution Of Dao In Hyoscyamus Spesupporting

confidence: 81%

Section: Substrate Specificitymentioning

confidence: 99%

Diamine Oxidase from Cultured Roots of Hyoscyamus niger

Hashimoto¹,

Mitani²,

Yamada³

1990

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…There is convincing evidence for the presence of bound Cu2+ as cofactor in the enzymes from pig kidney (Mondovi et al, 1967), pea seedling (Hill & Mann, 1964), tobaoco root (Mizusaki et al, 1972), pig plasma (BIuffoni & Blaschko, 1964;Buffoni etaL., 1968), bovine plasma (Yamada & Yasunobu, 1962), Aspergillus niger (Yamada et al, 1968) and chick aorta (Siegal et al, 1970). It has also been found that all amine oxidases, except that from chick aorta, fail to oxidize lysyl peptides or collagen precursors; this observation has been confirmed in the present study with the diamine oxidase from pig kidney.…”

Section: +H202+nh3 Ch2n(ch3)2mentioning

confidence: 99%

Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity

Crabbe

Waight

Bardsley

et al. 1976

Biochemical Journal

View full text Add to dashboard Cite

1. Isoelectric focusing studies on human placental diamine oxidase showed the pl value of the active enzyme to be 6.5. This infonnation was used in modifying the enzyme purification by incorporating column chromatography on DEAE-Sephadex with ionic strength and pH-gradient elution and this, together with affinity chromatography on concanavalin A-Sepharose, gave a highly purified preparation, with a specific activity of 7.0 units/mg. 2. The enzyme gave the expected stoicheiometry with p-dimethylaminomethylbenzylamine as substrate (Keq. 2700) and also oxidized [8-arginine]vasopressin, [8-lysine]vasopressin, collagen and tropocollagen. Polyacrylamide gel slices showed identical migration ofdiamine-oxidizing and [8-lysine]vasopressin-oxidizing activity. 3. The molecular weight, determined by ultrantrifugation, sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, variable polyacrylamide-gel electrophoresis and Sephadex G-200 column chromatography, was estimated to be approx. 70000. 4. E.s.r. spectroscopy showed that copper and manganese were present in the purified enzyme. This result was confirmed by atomic absorption spectroscopy, which indicated a stoicheiometry for copper and manganese of approx.

show abstract

“…Nicotine is synthesized in the roots (Dawson, 1941) by the condensation of two precursors, nicotinic acid and N-methylpyrrolinium cation, which are produced independently by the pyridine-nucleotide cycle and the methylpyrroline pathway, respectively (Wagner et al, 1986;Bush et al, 1999;Kajikawa et al, 2011). In the methylpyrroline pathway, the biosynthesis of N-methylputrescine from putrescine requires AdoMet as a cosubstrate (Mizusaki et al, 1972;Hibi et al, 1994). The synthesized nicotine is translocated through the xylem and transported to the leaves (Morita et al, 2009;Shoji et al, 2009;Hildreth et al, 2011), where it accumulates.…”

mentioning

confidence: 99%

“…It has been well established that the methyl group for nicotine biosynthesis is derived from the THF-mediated C1 metabolic pathway via AdoMet (Mizusaki et al, 1972;Hibi et al, 1994). In commercial tobacco plants, nicotine accounts for 90% of the total alkaloid content, which is about 2% to 5% of dry leaf weight (Saitoh et al, 1985).…”

mentioning

confidence: 99%

Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

et al. 2012

View full text Add to dashboard Cite

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme of the tetrahydrofolate (THF)-mediated one-carbon (C1) metabolic network. This enzyme catalyzes the reduction of 5,10-methylene-THF to 5-methyl-THF. The latter donates its methyl group to homocysteine, forming methionine, which is then used for the synthesis of S-adenosyl-methionine, a universal methyl donor for numerous methylation reactions, to produce primary and secondary metabolites. Here, we demonstrate that manipulating tobacco (Nicotiana tabacum) MTHFR gene (NtMTHFR1) expression dramatically alters the alkaloid profile in transgenic tobacco plants by negatively regulating the expression of a secondary metabolic pathway nicotine N-demethylase gene, CYP82E4. Quantitative real-time polymerase chain reaction and alkaloid analyses revealed that reducing NtMTHFR expression by RNA interference dramatically induced CYP82E4 expression, resulting in higher nicotine-tonornicotine conversion rates. Conversely, overexpressing NtMTHFR1 suppressed CYP82E4 expression, leading to lower nicotine-to-nornicotine conversion rates. However, the reduced expression of NtMTHFR did not affect the methionine and Sadenosyl-methionine levels in the knockdown lines. Our finding reveals a new regulatory role of NtMTHFR1 in nicotine Ndemethylation and suggests that the negative regulation of CYP82E4 expression may serve to recruit methyl groups from nicotine into the C1 pool under C1-deficient conditions. Tetrahydrofolate (THF)-mediated one-carbon (C1) metabolism generates and provides C1 units in different oxidation states for various anabolic pathways, including alkaloid biosynthesis in plants (Hanson et al., 2000). Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylene-THF into the most reduced C1 derivative, 5-methyl-THF (Guenther et al., 1999;Roje et al., 1999;Gelling et al., 2004). The latter then serves as the methyl group donor for Met synthesis from homocysteine (Guenther et al., 1999;Hanson et al., 2000). More than 80% of synthesized Met is further converted to S-adenosyl-methionine (AdoMet), which is the universal methyl group donor in reactions leading to modifications of various primary and secondary metabolites (Giovanelli et al., 1985;Hanson et al., 2000;Roje, 2006). In plants, MTHFRs have been cloned and characterized from Arabidopsis (Arabidopsis thaliana) and maize (Zea mays; Roje et al., 1999). In contrast to the mammalian enzymes, which are NADPH dependent and inhibited by AdoMet, plant MTHFRs are NADH dependent and AdoMet insensitive. In addition, the reaction that they catalyze is likely reversible in the cytosol (Roje et al., 1999). To 1 This work was supported by the Golden LEAF Foundation (a startup fund to the Biomanufacturing Research Institute & Technology Enterprise), the National Institute of General Medical Sciences (grant no. SC3GM088084 to J.X.), the National Science Foundation of China and the Yunnan Tobacco Company (grant nos. 31060046 and 2011YN04 to B.X.), and the National Science Foundation (grant nos. MCB-04...

show abstract

N-methylputrescine oxidase from tobacco roots

Cited by 69 publications

References 11 publications

Diamine Oxidase from Cultured Roots of Hyoscyamus niger

Diamine Oxidase from Cultured Roots of Hyoscyamus niger

Human placental diamine oxidase. Improved purification and characterization of a copper- and manganese-containing amine oxidase with novel substrate specificity

Alteration of the Alkaloid Profile in Genetically Modified Tobacco Reveals a Role of Methylenetetrahydrofolate Reductase in Nicotine N-Demethylation

Contact Info

Product

Resources

About