PURIFICATION AND CHARACTERIZATION OF MYOINOSITOL 6-O-METHYLTRANSFERASE FROM VIGNA-UMBELLATA OHWI ET OHASHI

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 (m60MT), 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 chromatogra phy on immobilized S-adenosyl-L-homocysteine (SAH). The purified m60MT was photoaffinity labelled with S-adenosyl-L-[C-14-methyl]methionine. The native molecular weight was determined to be 106 kDa, with a subunit molecular weight of 40 kDa. Substrate-saturation kinetics of m60MT for myo-inositol and S-adenosyl-L-methionine (SAM) were Michaelis-Menten type with K-m values of 2.99 mM and 63 mu M, respectively. The SAH competitively inhibited the enzyme with respect to SAM (K-i of 1.63 mu M). The enzyme did not require divalent cations for activity, but was strongly inhibited by Mn2+, Zn2+ and CU2+ and sulfhydryl group inhibitors. The purified m60MT was found to be highly specific for the 6-hydroxyl group of mfo-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 m60MT catalyses the first step), inhibited the activity of the enzyme.