PURIFICATION AND PROPERTIES OF PUTRESCINE N-METHYLTRANSFERASE FROM TRANSFORMED ROOTS OF DATURA-STRAMONIUM L

Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrro lidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight es timated by gel-permeation chromatography on Superose-6 of 40 kDa; sodi um dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fra ctions from Superose-6 chromatography revealed a band of 36 kDa molecu lar weight. Kinetic studies of the purified enzyme gave K-m values for putrescine and S-adenosyl-L-methionine of 0.31 mM and 0.10 mM, respec tively, and K-i values for S-adenosyl-L-homocysteine and N-methylputre scine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2 -hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was ob served with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for subs trate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhib itor of the enzyme, with a K-i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discu ssed.