β-Peltatin 6- O-methyltransferase from suspension cultures of Linum nodiflorum

Abstract

S-Adenosyl- l-methionine:β-peltatin 6- O-methyltransferase was isolated and characterized from cell suspension cultures of Linum nodiflorum L. (Linaceae), a Linum species accumulating aryltetralin lignans such as 6-methoxypodophyllotoxin. The enzyme transfers a methyl group from S-adenosyl- l-methionine to the only free OH-group of β-peltatin in position 6 thus forming β-peltatin-A methylether. This reaction is a putative biosynthetic step in the biosynthesis of 6-methoxypodophyllotoxin from deoxypodophyllotoxin. The enzyme has a pH-optimum at pH 7.7 and a temperature optimum at 40 °C. The enzyme activity is strongly inhibited by MnSO 4, FeCl 3, FeSO 4 and ZnSO 4 as well as S-adenosyl-homocysteine. Mg 2+ and EDTA did not influence the methylation of β-peltatin. Substrate saturation curves were obtained for S-adenosyl-methionine and β-peltatin and apparent K m-values of 15 μM and 40 μM, respectively, were determined for these substrates. Substrate inhibition was observed for β-peltatin. No other lignan substrate tested nor caffeic acid were accepted. The suspension cell line of Linum nodiflorum was characterized with respect to growth, medium alterations and lignan production as well as activity of SAM:β-peltatin 6- O-methyltransferase. Highest specific activities of β-peltatin 6- O-methyltransferase were determined on day 7 of the culture period corresponding to the highest levels of 6-methoxypodophyllotoxin on days 7 to 12.