Cloning and expression of a cDNA encoding homospermidine synthase from Senecio vulgaris (Asteraceae) in Escherichia coli.
The enzyme homospermidine synthase catalyzes the NAD+-dependent conversion of 2 mol putrescine into homospermidine. Instead of putrescine, spermidine can substitute for the first putrescine moiety in plants, in which case diaminopropane instead of ammonia is released. The enzyme facilitates the formation of the 'uncommon' polyamine homospermidine which is an important precursor in the biosynthesis of pyrrolizidine alkaloids. The first plant homospermidine synthase was purified to apparent chemical homogenity from the root tissue culture Senecio vernalis (Asteraceae) (Böttcher et al. 1994, Can. J. Chem. 72, 80-85; Ober 1997, Dissertation). Four endopeptidase LysC fragments were sequenced from the purified protein. With the aid of degenerate primers against these peptides, a cDNA encoding homospermidine synthase was now cloned and characterized from Senecio vulgaris. The nucleotide sequence of the cloned cDNA revealed an open reading frame of 1155-base pairs containing 385 amino acids with a predicted Mr of 44500. GenBank research revealed that the deduced amino acid sequence shows 59% identity to human deoxyhypusine synthase. The homospermidine synthase encoding cDNA was subcloned into the expression vector pet15b and overexpressed in E. coli. The recombinant enzyme formed upon expression catalyzed homospermidine synthesis.
Kaiser
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Institute of Pharmaceutical Biology, Braunschweig, Germany. akaiser@parasit.meb.uni-bonn.de
D019883:Alkyl and Aryl Transferases / Q000235:genetics*; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D008401:Gas Chromatography-Mass Spectrometry; D015870:Gene Expression; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D011073:Polyamines / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012674:Senecio / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D011073:Polyamines; D011993:Recombinant Fusion Proteins; D019883:Alkyl and Aryl Transferases; C028376:homospermidine synthetase