Purification and Characterization of S-Adenosyl-l-methionine:6a-Hydroxymaackiain 3-O-Methyltransferase from Pisum sativum

The isoflavonoid phytoalexin pisatin is synthesized by Pisum sativum in response to microbial infection and certain other forms of stress. An enzyme which synthesizes pisatin by methylating the 3-hydroxyl of (+)6a-hydroxymaackiain (HMK) was extracted from CuCl₂-stressed pea seedlings. The enzyme was enriched 370-fold by (NH₄)₂SO₄ precipitation, DEAE chromatography, chromatofocusing, and hydrophobic interaction chromatography (HIC), to a specific activity of 8.2 microkatals per gram protein. Enzyme activity profiles from chromatofocusing and HIC columns suggested the presence of two isozymes, of pl 5.2 and 4.9. Nondenaturing gel filtration of the HIC-purified enzyme gave a single peak of activity at the same elution volume as BSA (66 kilodaltons); the active fractions showed two proteins upon SDS-PAGE, of M_r 66,000 and 43,000. The smaller protein was most abundant in chromatographic fractions containing peak enzyme activity throughout purification. In a partially purified preparation, this 43 kilodalton protein was the only one photoaffinity labelled by [³H]S-adenosyl-l-methionine. The purified enzyme preferred the (+) over the (−) stereoisomer of HMK and other pterocarpans; overall, (+)HMK was the best substrate. K_m values were 2.3 micromolar for (+)HMK and 35 micromolar for S-adenosyl-l-methionine. The methyltransferase had a pH optimum of 7.9 and no apparent divalent cation requirement.