PURIFICATION AND CHARACTERIZATION OF A NOVEL METHYLTRANSFERASE RESPONSIBLE FOR BIOSYNTHESIS OF HALOMETHANES AND METHANETHIOL IN BRASSICA-OLERACEA

A novel S-adenosyl-L-methionine:halide/bisulfide methyltransferase (EC 2.1.1.-) was purified approximately 1000-fold to apparent homogeneity from leaves of Brassica oleracea, The enzyme catalyzed the S adenosyl -L-methionine-dependent methylation of the halides iodide, bromide, an d chloride to monohalomethanes and of bisulfide to methanethiol. The d ual function of the enzyme was demonstrated through co-purification of the halide- and bisulfide-methylating activities in the same ratio an d by studies of competition between the alternative substrates iodide and bisulfide, The purification procedure included gel filtration, ani on exchange chromatography, and affinity chromatography on adenosine-a garose. Elution of the protein from a chromate-focusing column indicat ed a pI value of 4.8. The pH optimum of halide methylation (5.5-7.0) w as different from that of bisulfide methylation (7.0-8.0). The molecul ar mass values for the native and denatured protein were 29.5 and 28 k Da, respectively, suggesting that the active enzyme is a monomer, The enzyme had the highest specificity constant for iodide and the next hi ghest for bisulfide, Substrate interaction kinetics and product inhibi tion patterns were consistent with an Ordered Bi Bi mechanism.