Putrescine N-methyltransferases--a structure-function analysis.
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.
Teuber
Michael
M
Institute of Pharmacy, Faculty of Science I, Martin-Luther University Halle-Wittenberg, Halle, Saale, Germany.
Azemi
Mohammad E
ME
Namjoyan
Foroogh
F
Meier
Anna-Carolin
AC
Wodak
Anja
A
Brandt
Wolfgang
W
Dräger
Birgit
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D003001:Cloning, Molecular; D003198:Computer Simulation; D027722:Convolvulaceae / Q000201:enzymology / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011700:Putrescine / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D019657:Solanaceae / Q000201:enzymology / Q000235:genetics; D011436:Spermidine Synthase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D018076:DNA, Complementary; D010940:Plant Proteins; D008780:Methyltransferases; C112598:putrescine N-methyltransferase; D011436:Spermidine Synthase; D011700:Putrescine