β-Alanine Betaine Synthesis in the Plumbaginaceae. Purification and Characterization of a Trifunctional,S-Adenosyl-l-Methionine-DependentN-Methyltransferase from Limonium latifoliumLeaves

Abstract

␤-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-lmethionine-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 m S-adenosyl-l-methionine and substrate inhibition was observed above 200 m. 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.

β-Alanine N-Methyltransferase of Limonium latifolium. cDNA Cloning and Functional Expression of a Novel N-Methyltransferase Implicated in the Synthesis of the Osmoprotectant β-Alanine Betaine

“…The specific activity toward N,N-dimethyl ␤-Ala was less than that found with ␤-Ala or N-methyl ␤-Ala as the methyl acceptors (Fig. 6), like what was found with the enzyme purified from L. latifolium leaves (Rathinasabapathi et al, 2001).…”

“…The deduced amino acid sequence (Fig. 2) resulted in a theoretical mass of 41,286 D and a pI of 5.84, closely resembling experimental determinations of 43,000 D and 5.15 for the NMTase (Rathinasabapathi et al, 2001). ␤-Ala NMTase had all three motifs conserved for Ado-Met binding (Joshi and Chiang, 1998).…”

“…␤-Ala methylation, catalyzed by a S-adenosyl l-Met (Ado-Met)-dependent N-methyltransferase (␤-Ala N-methyltransferase [NMTase]) was specific to ␤-Ala betaine-accumulating members of the Plumbaginaceae and was absent in species lacking this pathway . An 86-kD leaf protein, a dimer of 43-kD subunits, purified from L. latifolium was trifunctional, catalyzing all three methylations of ␤-Ala betaine synthesis (Rathinasabapathi et al, 2001). This suggested that ␤-Ala betaine synthesis could be engineered in transgenic plants by simply expressing a single gene.…”

“…Active ␤-Ala NMTase protein was purified from L. latifolium leaves using a previously described protocol (Rathinasabapathi et al, 2001). The 43-kD band was extracted from a polyacrylamide gel and subjected to degradation by lysylendo-peptidase (lysC), and several peptides were sequenced (Table I).…”

“…Accordingly, ␤-Ala betaine was distributed among species of the Plumbaginaceae, adapted to a wide range of adverse stress environments including saline and hypoxic conditions (Hanson et al, 1994). Although ␤-Ala betaine accumulation has been intensely studied for its role in osmoprotection (Hanson et al, 1994;Rathinasabapathi et al, 2000Rathinasabapathi et al, , 2001, early work on this compound in marine algae also suggested it to have a cholesterol-reducing effect in animal feeding experiments (Abe and Kaneda, 1973 Article, publication date, and citation information can be found at www.plantphysiol.org/cgi …”