Skip to main content
SpringerLink
Log in

Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi

  • Published:
Planta Aims and scope Submit manuscript

Abstract

The cyclitol 1d-4-O-methyl-myo-inositol (d-ononitol) is accumulated in certain legumes in response to abiotic stresses. S-Adenosyl-l-methionine:myo-inositol 6-O-methyltransferase (m6OMT), the enzyme which catalyses the synthesis of d-ononitol, was extracted from stems of Vigna umbellata Ohwi et Ohashi and purified to apparent homogeneity by a combination of conventional chromatographic techniques and by affinity chromatography on immobilized S-adenosyl-l-homocysteine (SAH). The purified m6OMT was photoaffinity labelled with S-adenosyl-l-[14C-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m6OMT for myo-inositol and S-adenosyl-l-methionine (SAM) were Michaelis-Menten type with K m values of 2.92 mM and 63 μM, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K i of 1.63 μM). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn2+, Zn2+ and Cu2+ and sulfhydryl group inhibitors. The purified m6OMT was found to be highly specific for the 6-hydroxyl group of myo-inositol and showed no activity on other naturally occurring isomeric inositols and inositol O-methyl-ethers. Neither d-ononitol, nor d-3-O-methyl-chiro-inositol, d-1-O-methyl-muco-inositol or d-chiro-inositol (end products of the biosynthetic pathway in which m6OMT catalyses the first step), inhibited the activity of the enzyme.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

DTT:

dithiothreitol

m6OMT:

myo-inositol 6-O-methyltransferase

SAH:

S-adenosyl-l-homocysteine

SAM:

S-adenosyl-l-methionine

References

  • Adams P, Thomas JC, Vernon DM, Bohnert HJ, Jensen RG (1992) Distinct cellular and organismic responses to salt stress. Plant Cell Physiol 33: 1215–1223

    Google Scholar 

  • Biffen M, Hanke DE (1991) Metabolic fate of myo-inositol in soybean callus cells. Plant Sci 75: 203–213

    Google Scholar 

  • Delia Ragione F, Cartení-Farina M, Porcelli M, Cacciapuoti V, Zappia V (1981) High-performance liquid chromatographic analysis of 5′-methylthioadenosine in rat tissues. J Chromatogr s226: 243–249

    Google Scholar 

  • Dittrich P, Brandl A (1987) Revision of the pathway of d-pinitol formation in leguminosae. Phytochemistry 26: 1925–1926

    Google Scholar 

  • Dittrich P, Gietl M, Kandler O (1971) d-1-O-Methyl-mucoinositol in higher plants. Phytochemistry 11: 245–250

    Google Scholar 

  • Edwards R, Dixon RA (1991) Isoflavone O-methyltransferase activities in elicitor-treated cell suspension cultures of Medicago sativa. Phytochemistry 30: 2597–2606

    Google Scholar 

  • Evans RD, Black RA, Loescher WH, Fellows RJ (1992) Osmotic relations of the drought-tolerant shrub Artemisia tridentata in response to water stress. Plant Cell Environ 15: 49–59

    Google Scholar 

  • Fazldeen H, Breitenbach M (1979) A new Sepharose derivative containing covalently bound myo-inositol: its structure and application. Mh Chem 110: 823–830

    Google Scholar 

  • Ford CW (1982) Accumulation of O-methyl-inositols in waterstressed Vigna species. Phytochemistry 21: 1149–1151

    Google Scholar 

  • Guranowski AB, Chiang PK, Cantoni GL (1981) 5′-Methylthioadenosine nucleosidase. Purification and characterisation of the enzyme from Lupinus luteus seeds. Eur J Biochem 114: 293–299

    Google Scholar 

  • Hipps PP, Sehgal RK, Holland WH, Sherman WR (1973) Identification and partial characterisation of inositol:NAD+ epimerase and inosose:NAD(P)H reductase from the fat body of the American cockroach, Periplaneta americana L. Biochemistry 12: 4705–4712

    Google Scholar 

  • Hoffmann-Ostenhof O, Pittner F (1982) The biosynthesis of myo-inositol and its isomers. Can J Chem 60: 1863–1871

    Google Scholar 

  • Hofmann H, Wagner I, Hoffmann-Ostenhof O (1969) Über ein lösliches Enzym aus Vinca rosea, das myo-Inosit zu l-Bornesit methyliert. Z Physiol Chem 350: 1465–1468

    Google Scholar 

  • Hurst JH, Billingsley ML, Lovenberg W (1984) Photoaffinity labelling of methyltransferase enzymes with S-adenosylmethionine: effects of methyl acceptor substrates. Biochem Biophys Res Commun 122: 499–508

    Google Scholar 

  • Keller F, Ludlow MM (1993) Carbohydrate metabolism in droughtstressed leaves of pigeonpea (Cajanus cajan). J Exp Bot 44: 1351–1359

    Google Scholar 

  • Khouri HE, De Luca V, Ibrahim RK (1988) Enzymatic synthesis of polymethylated flavonols in Chrysosplenium americanum. III. Purification and kinetic analysis of S-adenosyl-l-methionine:3-methylquercetin 7-O-methyltransferase. Arch Biochem Biophys 265: 1–7

    Google Scholar 

  • Kim S, Nochumson S, Chin W, Paik WK (1978) A rapid method for the purification of S-adenosylmethionine:protein-carboxyl O-methyltransferase by affinity chromatography. Anal Biochem 84: 415–422

    Google Scholar 

  • Kindl H, Hoffmann-Ostenhof O (1966) Cyclite: Biosynthese, Stoffwechsel und Vorkommen. Fortschr Chem Org Nat 24: 149–205

    Google Scholar 

  • Koller F, Hoffmann-Ostenhof O (1976) Purification of myo-inositol 3-methyltransferase from Pisum sativum and of myo-inositol 1-methyltransferase from Vinca minor to homogeneity by affinity chromatography. Z Physiol Chem 357: 1465–1468

    Google Scholar 

  • Loewus FA, Loewus MW (1983) myo-Inositol: its biosynthesis and metabolism. Annu Rev Plant Physiol 34: 137–161

    Google Scholar 

  • Maxwell CA, Edwards R, Dixon RA (1992) Identification, purification, and characterisation of S-adenosyl-l-methionine:isoliquiritigenin 2′-O-methyltransferase from alfalfa (Medicago sativa L.). Arch Biochem Biophys 293: 158–166

    Google Scholar 

  • Pakusch AE, Matern U, Schiltz E (1991) Elicitor-inducible caffeoylcoenzyme A 3-O-methyltransferase from Petroselinum crispum cell suspensions. Plant Physiol 95: 137–143

    Google Scholar 

  • Peterson GL (1977) A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem 83: 346–356

    CAS  PubMed  Google Scholar 

  • Plouvier V (1990) Alditols et cyclitols: répartition et taxonomie chez les plantes supérieures. Les recherches publiées depuis 1962. Bull Mus Nat Hist Nat (Paris) 12: 209–223

    Google Scholar 

  • Popp M, Smirnoff N (1995) Polyol accumulation and metabolism during water deficit. In: Smirnoff N (ed) Environment and plant metabolisms ßios, Oxford UK, pp 199–215

  • Poulton JE (1981) Transmethylation and demethylation reactions in the metabolism of secondary plant products. In: Conn EE (ed) The biochemistry of plants vol. 7 Academic Press, New York pp 667–723

    Google Scholar 

  • Preisig CL, Matthews DE, VanEtten HD (1989) Purification and characterisation of S-adenosyl-l-methionine:6a-hydroxymaackiain 3-O-methyltransferase from Pisum sativum. Plant Physiol 91: 559–566

    Google Scholar 

  • Richter A (1992) Viscumitol, a dimethyl-ether of muco-inositol from Viscum album. Phytochemistry 31: 3925–3927

    Google Scholar 

  • Richter A, Popp M (1992) The physiological importance of accumulation of cyclitols in Viscum album L. New Phytol 121: 431–438

    Google Scholar 

  • Richter A, Thonke B, Popp M (1990) 1d-1-O-methyl-muco-inositol in Viscum album and members of the Rhizophoraceae. Phytochemistry 29: 1785–1786

    Google Scholar 

  • Scholda R, Billek G, Hoffmann-Ostenhof, O (1964) Die Bildung von Scyllit in Calycanthus occidentalis. Z Physiol Chem 33: 28–32

    Google Scholar 

  • Sharma SK, Brown SA (1978) Affinity chromatography on immobilized S-adenosyl-l-homocysteine. Purification of a furanocoumarin O-methyltransferase from cell culture of Ruta graveolens L. J Chromatogr 157: 427–431

    Google Scholar 

  • Sharma SK, Brown SA (1979) Affinity chromatography of Ruta graveolens L. O-methyltransferases. Studies demonstrating the potential of the technique in the mechanistic investigation of O-methyltransferases. Can J Biochem 57: 986–994

    Google Scholar 

  • Sommer C, Thonke B, Popp M (1990) The compatibility of d-pinitol and 1d-1-O-methyl-muco-inositol with malate dehydrogenase activity. Bot Acta 103: 270–273

    Google Scholar 

  • Timasheff SN (1992) A physicochemical basis for the selection of osmolytes by nature. In: Somero GN, Osmond CB, Bolis CL (eds) Water and Life. Springer, Berlin Heidelberg New York, pp 70–84

    Google Scholar 

  • Ueno Y, Hasegawa A, Tsuchiya T (1973) Isolation of O-methyl-scyllo-inositol from mung bean seeds. Carbohydr Res 29: 520–521

    Google Scholar 

  • Vernon DM, Bohnert HJ (1992a) A novel methyltransferase induced by osmotic stress in the facultative halophyte Mesembryanthemum crystallinum. EMBO J 11: 2077–2085

    Google Scholar 

  • Vernon DM, Bohnert HJ (1992b) Increased expression of a myo-inositol methyltransferase in Mesembryanthemum crystallinum is part of stress response distinct from Crassulaceaen acid metabolism induction. Plant Physiol 99: 1695–1698

    Google Scholar 

  • Wagner I, Hofmann H, Hoffmann-Ostenhof O (1969) Über ein lösliches Enyzm aus Erbsenkeimlingen, das myo-Inosit zu d-Bornesit methyliert. Z Physiol Chem 350: 1460–1464

    Google Scholar 

  • Walton NJ, Peerles ACJ, Robins RJ, Rhodes MJC, Boswell HD, Robins DJ (1994) Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L. Planta 193: 9–15

    Google Scholar 

  • Wanek W, Richter A (1993) l-Iditol:NAD+ 5-oxidoreductase in Viscum album: utilization of host-derived sorbitol. Plant Physiol Biochem 31: 205–211

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Plant Physiology, University of Vienna, PF 285, A-1091, Vienna, Austria

    Wolfgang Wanek & Andreas Richter

Authors
  1. Wolfgang Wanek
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Andreas Richter
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andreas Richter.

Additional information

We are greatful to Professor M. Popp (University of Vienna) for helpful discussion and comment. This work was supported by Grant P09595-BIO from the Austrian Science Foundation (FWF).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wanek, W., Richter, A. Purification and characterization of myo-inositol 6-O-methyltransferase from Vigna umbellata Ohwi et Ohashi. Planta 197, 427–434 (1995). https://doi.org/10.1007/BF00196663

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00196663

Key words

Search

Navigation