Evolution of the key alkaloid enzyme putrescine N-methyltransferase from spermidine synthase

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.

Physiology and proteomics research on the leaves of ancient Platycladus orientalis (L.) during winter

“…Spermidine synthases (SPDSs) are ubiquitous enzymes that function in primary metabolism in plants. This enzymes use the substrate putrescine to transfer aminopropyl from decarboxylated S-adenosyl-L-methionine (SAM), forming spermidine [99]. The cellular polyamines putrescine, spermidine, and spermine, which are ubiquitous, have been implicated in a wide range of growth and developmental processes [100].…”

Genomic Insights into the Evolution of the Nicotine Biosynthesis Pathway in Tobacco

“…S1; Stenzel et al, 2006). Whereas ODC2, which acts in parallel with ODC1, has retained its original catalytic activity, PMT and MPO1, which have been derived from SPDS and DAO respectively, have acquired novel activities through neofunctionalization (Junker et al, 2013;Naconsie et al, 2014), thus contributing to the innovation of the pyrrolidine-forming extension (Fig. 1).…”

“…1). It has been proposed that PMT and MPO have evolved from spermidine synthase (SPDS) and diamine oxidase (DAO), two homologous enzymes with different catalytic activities, both of which accept putrescine as a substrate and thus are involved in polyamine metabolism (Hibi et al, 1994;Hashimoto et al, 1998;Junker et al, 2013;Naconsie et al, 2014). Two orphan oxidoreductases of different families, A622 (Hibi et al, 1994;DeBoer et al, 2009;Kajikawa et al, 2009) and berberine bridge enzyme-like (BBL) proteins (Kajikawa et al, 2011), are required for later steps including coupling of the two rings ( Fig.…”

Something old, something new: Conserved enzymes and the evolution of novelty in plant specialized metabolism

“…2C). Site-directed mutagenesis experiments suggest that a single amino acid change can convert the SPDS to PMT activity (Junker et al, 2013; Fig. 1D), although the actual evolutionary path taken by the duplicate of SPDS is not known.…”