Purification and Properties of S-Adenosyl-L-methionine: L-Methionine S-Methyltransferase from Wollastonia biflora Leaves

Abstract

The plant enzyme S-adenosylmethionine:methionine S-methyltransferase (EC 2.1.1.12, MMT) catalyzes the synthesis of S-methylmethionine. MMT was purified 620-fold to apparent homogeneity from leaves of Wollastonia biflora. The four-step purification included fractionation with polyethylene glycol, affinity chromatography on adenosine-agarose, anion exchange chromatography, and gel filtration. Protein yield was about 180 g/kg of leaves. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native gel filtration chromatography were, respectively, 115 and 450 kDa, suggesting a tetramer of 115-kDa subunits. The 115-kDa subunit was photoaffinity labeled by S-adenosyl[ 3 H]methionine. Antibodies raised against W. biflora MMT recognized a 115-kDa polypeptide in partially purified MMT preparations from leaves of lettuce, cabbage, clover, and maize.The pH optimum of W. biflora MMT was 7.2. Kinetic analysis of substrate interaction and product inhibition patterns indicated an Ordered Bi Bi mechanism, with S-adenosylmethionine the first reactant to bind and Sadenosylhomocysteine the last product to be released. The enzyme catalyzed methylation of selenomethionine and ethionine, but not of S-methylcysteine, homocysteine, cysteine, or peptidylmethionine. Tests with other substrate analogs indicated that a free carboxyl group was required for enzyme activity, and that a free amino group was not.

Evidence That the Pathway of Dimethylsulfoniopropionate Biosynthesis Begins in the Cytosol and Ends in the Chloroplast

“…The SDS-PAGE, electrotransfer, and immunodetection procedures were as given by James et al (1995a), except that the polyacrylamide concentration for DDH separations was 8% and the transfer buffers were those of Svoboda et al (1985). Methods for immunohistochemical localization of MMT were as described by Nolte and Koch (1993), except that 7-pm sections were used and the antiserum dilution was 1:250.…”

“…[35S]Met (44 GBq pmol-', NEN-DuPont) and Met were mixed to give the desired specific activity; basic contaminants were removed by shaking with 0.1 volume of Dowex-50 (NH,+) (Bio-Rad) for 1 min. [35S]SMM was synthesized enzymatically using W. biflora MMT purified by PEG precipitation and adenosine-agarose chromatography (James et al, 1995a). The reaction mixture contained 0.2 pmol of [35S]Met (56 MBq pmol-'), 0.3 pmol of AdoMet, and 30 pg of enzyme protein in 0.1 mL of Tris-Mes-acetate buffer (James et al, 1995a).…”

“…[35S]SMM was synthesized enzymatically using W. biflora MMT purified by PEG precipitation and adenosine-agarose chromatography (James et al, 1995a). The reaction mixture contained 0.2 pmol of [35S]Met (56 MBq pmol-'), 0.3 pmol of AdoMet, and 30 pg of enzyme protein in 0.1 mL of Tris-Mes-acetate buffer (James et al, 1995a). Incubation was for 12 h at 20°C.…”

“…Antisera raised previously against W. bifova MMT (James et al, 1995a) and spinach BADH (Weretilnyk and Hanson, 1989) were used. For immunotitration of DDH, 50-pL mixtures containing 10 mM BisTris-HCI, pH 7.0, stromal extract, and various volumes of serum were incubated for 15 min at 21°C.…”

“…Only one enzyme of DMSP biosynthesis has been described: MMT, the methyltransferase catalyzing the conversion of Met to SMM. MMT has been purified and characterized from W. bifora (James et al, 1995a); it has also been detected in various other plants that synthesize SMM but not DMSP (Giovanelli et al, 1980;Mudd and Datko, 1990;Gessler et al, 1991). The subcellular location of MMT has not been established.…”