RESUMO
Putrescine N-methyltransferase (PMT) is a key enzyme of plant secondary metabolism at the start of the specific biosynthesis of nicotine, of tropane alkaloids, and of calystegines that are glycosidase inhibitors with nortropane structure. PMT is assumed to have developed from spermidine synthases (SPDS) participating in ubiquitous polyamine metabolism. In this study decisive differences between both enzyme families are elucidated. PMT sequences were known from four Solanaceae genera only, therefore additional eight PMT cDNA sequences were cloned from five Solanaceae and a Convolvulaceae. The encoded polypeptides displayed between 76% and 97% identity and typical amino acids different from plant spermidine synthase protein sequences. Heterologous expression of all enzymes proved catalytic activity exclusively as PMT and K (cat) values between 0.16 s(-1) and 0.39 s(-1). The active site of PMT was initially inferred from a protein structure of spermidine synthase obtained by protein crystallisation. Those amino acids of the active site that were continuously different between PMTs and SPDS were mutated in one of the PMT sequences with the idea of changing PMT activity into spermidine synthase. Mutagenesis of active site residues unexpectedly resulted in a complete loss of catalytic activity. A protein model of PMT was based on the crystal structure of SPDS and suggests that overall protein folds are comparable. The respective cosubstrates S-adenosylmethionine and decarboxylated S-adenosylmethionine, however, appear to bind differentially to the active sites of both enzymes, and the substrate putrescine adopts a different position.
Assuntos
Perfilação da Expressão Gênica , Metiltransferases/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Sítios de Ligação , Northern Blotting , Clonagem Molecular , Simulação por Computador , Convolvulaceae/enzimologia , Convolvulaceae/genética , DNA Complementar/química , DNA Complementar/genética , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Estrutura Terciária de Proteína , Putrescina/química , Putrescina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Solanaceae/enzimologia , Solanaceae/genética , Espermidina Sintase/química , Espermidina Sintase/genética , Espermidina Sintase/metabolismoRESUMO
Nicotine or nornicotine enriched with stable isotopes in either the N'-methyl group or the pyrrolidine-N were fed to Nicotiana plumbaginifolia suspension cell cultures that do not form endogenous nicotine. The metabolism of these compounds was investigated by analysing the incorporation of isotope into other alkaloids using gas chromatography-mass spectroscopy (GC-MS). Nicotine metabolism primarily resulted in the accumulation of nornicotine, the N'-demethylation product. In addition, six minor metabolites appeared during the course of nicotine metabolism, four of which were identified as cotinine, myosmine, N'-formylnornicotine and N'-carboethoxynornicotine. While cotinine was formed from [(13)C,(2)H(3)-methyl]nicotine without dilution of label, N'-formylnornicotine was labelled at only about 6% of the level of nicotine and N'-carboethoxynornicotine was unlabelled. Feeding with [1'-(15)N]nornicotine resulted in incorporation without dilution of label into both N'-formylnornicotine and N'-carboethoxynornicotine. This pattern strongly indicates that, while nornicotine and cotinine are derived directly from nicotine, N'-formylnornicotine and N'-carboethoxynornicotine are metabolites of nornicotine. Thus, it is directly demonstrated that N'-formylnornicotine is not an intermediate in nicotine demethylation.