Poster: Enzymology
Abs #
246: Characterization of recombinant b-alanine N-methyltransferase, a novel enzyme involved in osmoprotectant synthesis in Limonium latifolium.
b-alanine N-methyltransferase (NMTase) is a S-adenosyl-L-methonine dependent methyltransferase involved in the synthesis of b-alanine betaine, an osmoprotectant found in most members of the Plumbaginaceae. b-alanine betaine’s potential role in plant tolerance to saline and hypoxic conditions makes its synthetic pathway an interesting target for metabolic engineering. Toward this goal, we recently cloned and characterized the full-length cDNA for NMTase (1428 bp) from Limonium latifolium, a member of the Plumbaginaceae. Sequence analysis revealed that NMTase is an unusual N-methyltransferase in having phylogenetic relations to O-methyltransferases. In a previous study, this methyltransferase was highly purified from L. latifolium leaves. However, the conventional protein purification techniques used, negatively affected enzyme stability, and hence, kinetic characterization was limited to partially purified fractions. Here, we report on the successful expression of NMTase cDNA in baker’s yeast under the galactose inducible promoter, as a fusion protein with a hexa-histidinyl tag at the N-terminus. The expression was monitored by activity assays, SDS-PAGE and immuno blots probed with anti (His6) antibody. Following purification using nickel-chelating affinity chromatography, the tag sequence was removed by enterokinase digestion. The over-expressed NMTase had a molecular weight of 43 KD comparable to the theoretical value of 41.3 KD. The recombinant NMTase was trifunctional using b-alanine, N- methyl b-alanine and N, N dimethyl b-alanine as methyl acceptor substrates. Kinetic parameters, substrate and inhibitor interactions are discussed.
