Cloning of a tropane alkaloid biosynthesis gene and analysis of its expression

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Biosynthesis of tropane alkaloids is thought to proceed by way of the diamine putrescine,followed by its methylation by putrescine N-methyltransferase(PMT). High PMT activities were found in branch roots and/or cultured roots of several solanaceous plants. PMT was partially purified and characterized from cultured roots of Hyoscyamus albus. Initial velocity studies and product inhibition patterns of PMT are consistent with an ordered bi bi mechanism,in which the Km values for putrescine and S-adenosyl-L-methionine are 277 muM and 203 muM,respectively,and the Ki value for S-adenosyl-L-homocysteine is 110 muM. PMT efficiently N- methylated amines that have at least two amino groups separated by three or four methylene groups. Monoamines were good competitive inhibitors of PMT,among which n-butylamine,cyclohexylamine and exo- 2-aminonorbornane were most inhibitory, with respective Ki values of 11.0,9.1 and 10.0 muM. When n- butylamine was fed to root cultures of H. albus, the alkamine intermediates (tropinone, tropine and pseudotropine) drastically decreased at 1 mM of the exogenous monoamine, and the hyoscyamine content decreased by 52% at6 mM. The n-butylamine treatment caused a large increase in the putrescine content (especially in the conjugated pool),and the spermine content also slightly increased, whereas the spermidine content slightly decreased. The increase in the putrescine pool size (approximately 40 nmol/mg dry weight) was large enough to account for the decrease in the total alkaloid pool size. These studies further support the role of PMT as the first committed enzyme specific to alkaloid biosynthesis.