cDNAs for S-adenosyl-L-methionine decarboxylase from Catharanthus roseus, heterologous expression, identification of the proenzyme-processing site, evidence for the presence of both subunits in the active enzyme, and a conserved region in the 5' mRNA leader.
S-Adenosyl-L-methionine decarboxylases (AdoMetDC) are pyruvoyl-dependent enzymes producing the aminopropyl group for spermidine biosynthesis, and this reaction is the rate-limiting step in polyamine biosynthesis. We characterized cDNAs from Catharanthus roseus (Madagascar periwinkle) and investigated the enzyme after heterologous expression. The largest cDNA (1842 bp) had an 5' leader of 469 bp and encoded a protein of 357 residues and 30-35% identity with mammalian AdoMetDC. The proenzyme expressed in Escherichia coli was processed into active enzyme, and the processing site was identified by site-directed mutagenesis as the second Ser in the sequence Leu-Ser-Glu-Ser-Ser. The analysis of affinity-purified proteins indicated that the active enzyme contained both subunits. The Km for S-adenosyl-L-methionine was 35-40 microM, and the enzyme activity was not stimulated by putrescine. The 5' leader of the mRNA contained start and stop codons for a polypeptide of 51 amino acids, and this region was conserved in the 5' leaders of other plant AdoMetDC mRNAs. The putative polypeptide had no similarity with the hexapeptide responsible for modulation of AdoMetDC mRNA translation in mammals. The possibility is discussed that plants evolved a different type of translational regulation.
Schröder
G
G
Institut für Biologie II, Universität Freiburg, Germany.
Schröder
J
J
D012437:Adenosylmethionine Decarboxylase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000032:analysis*; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D010944:Plants / Q000201:enzymology*; D011700:Putrescine / Q000494:pharmacology; D012333:RNA, Messenger / Q000737:chemistry*; D011994:Recombinant Proteins / Q000096:biosynthesis
D018076:DNA, Complementary; D004792:Enzyme Precursors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012437:Adenosylmethionine Decarboxylase; D011700:Putrescine