Plant Physiol, July 2001, Vol. 126, pp. 1241-1249

-Alanine Betaine Synthesis in the Plumbaginaceae. Purification and Characterization of a Trifunctional, S-Adenosyl-L-Methionine-Dependent N-Methyltransferase from Limonium latifolium Leaves¹

Horticultural Sciences Department, Plant Molecular and Cellular Biology Program, University of Florida, Gainesville, Florida 32611-0690

-Alanine (-Ala) betaine is an osmoprotective compound accumulated by most members of the highly stress-tolerant family Plumbaginaceae. Its potential role in plant tolerance to salinity and hypoxia makes its synthetic pathway an interesting target for metabolic engineering. In the Plumbaginaceae, -Ala betaine is synthesized by S-adenosyl-L-methionine-dependent N-methylation of -Ala via N-methyl -Ala and N,N-dimethyl -Ala. It was not known how many N-methyltransferases (NMTases) participate in the three N-methylations of -Ala. An NMTase was purified about 1,890-fold, from Limonium latifolium leaves, using a protocol consisting of polyethylene glycol precipitation, heat treatment, anion-exchange chromatography, gel filtration, native polyacrylamide gel electrophoresis, and two substrate affinity chromatography steps. The purified NMTase was trifunctional, methylating -Ala, N-methyl -Ala, and N,N-dimethyl -Ala. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses indicated that the native NMTase is a dimer of 43-kD subunits. The NMTase had an apparent K_m of 45 µM S-adenosyl-l-methionine and substrate inhibition was observed above 200 µM. The apparent K_m values for the methyl acceptor substrates were 5.3, 5.7, and 5.9 mM for -Ala, N-methyl -Ala, and N,N-dimethyl -Ala, respectively. The NMTase had an isoelectric point of 5.15 and was reversibly inhibited by the thiol reagent p-hydroxymercuribenzoic acid.