Abstract
Putrescine N-methyltransferases (PMTs) are the first specific enzymes of the biosynthesis of nicotine and tropane alkaloids. PMTs transfer a methyl group onto the diamine putrescine from S-adenosyl-l-methionine (SAM) as coenzyme. PMT proteins have presumably evolved from spermidine synthases (SPDSs), which are ubiquitous enzymes of polyamine metabolism. SPDSs use decarboxylated SAM as coenzyme to transfer an aminopropyl group onto putrescine. In an attempt to identify possible and necessary steps in the evolution of PMT from SPDS, homology based modeling of Datura stramonium SPDS1 and PMT was employed to gain deeper insight in the preferred binding positions and conformations of the substrate and the alternative coenzymes. Based on predictions of amino acids responsible for the change of enzyme specificities, sites of mutagenesis were derived. PMT activity was generated in D. stramonium SPDS1 after few amino acid exchanges. Concordantly, Arabidopsis thaliana SPDS1 was mutated and yielded enzymes with both, PMT and SPDS activities. Kinetic parameters were measured for enzymatic characterization. The switch from aminopropyl to methyl transfer depends on conformational changes of the methionine part of the coenzyme in the binding cavity of the enzyme. The rapid generation of PMT activity in SPDS proteins and the wide-spread occurrence of putative products of N-methylputrescine suggest that PMT activity is present frequently in the plant kingdom.
Highlights
Plants contain an unparalleled variety of chemical structures, and many of the compounds are utilized as aromas, dyes and medicines
The chiral sulfur of SAM must take an orientation different from that in decarboxylated SAM (dcSAM), or the substrate putrescine must change places. This consideration motivated the earlier hypothesis of a binding location for putrescine in Putrescine N-methyltransferases (PMTs) different from spermidine synthases (SPDSs) (Biastoff et al, 2009b)
A different configuration at the sulfur or different conformation of SAM may enable methyl transfer to putrescine, which stays at the position that it takes in SPDS
Summary
Plants contain an unparalleled variety of chemical structures, and many of the compounds are utilized as aromas, dyes and medicines. Putrescine N-methyltransferase (PMT, E.C. 2.1.1.53) is the first specific enzyme of tropane, nortropane, and nicotine biosynthesis in Solanaceae and Convolvulaceae (Biastoff et al, 2009a). Alkaloids of this large group are essential sympatholytic drugs used clinically like atropine and scopolamine. These alkaloids confer advantages to the plants that produce them (Berenbaum, 1995). PMT as key enzyme for those alkaloids transfers a methyl group from S-adenosyl-L-methionine (SAM) to the ubiquitous diamine putrescine, forming N-methylputrescine (Figure 1). SPDSs are ubiquitous enzymes of primary metabolism and use the same substrate putrescine for an aminopropyl transfer from decarboxylated SAM (dcSAM) forming spermidine, an essential polyamine in all eukaryotic organisms
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