A new pathway for the biosynthesis of spermidine.

The only pathway known for the biosynthesis of sperrnidine is from putrescine and S-adenosylmethionine. S-Adenosylmethionine is decarboxylated, and the aminopropyl group of the product is transferred to putrescine to form spermidine. The enzymes of this pathway, S-adenosylmethionine decarboxylase (EC 4.1.1 S O ) and aminopropyltransferase (EC 2.5.1.16), have been found in a number of bacteria, yeasts and mammalian cells (Tabor & Tabor, 1972; Poso et al., 1976). In this report it is shown that Micrococcus denitriJcans and Rhodopseudomonas spheroides do not form spermidine in this way. They form it from putrescine and L-aspartic P-semialdehyde. M . denitrifcans synthesizes and excretes three siderochromes, two of which contain spermidine, when grown in media deficient in iron (Tait, 1975). In preliminary experiments the siderochromes containing spermidine were labelled after adding to cultures L-[U. ''C]arginine or ~~-[5-'~C]ornithine, known precursors of putrescine or [l ,4-14C] putrescine itself, but not after adding ~-[U-'~C]methionine. S-Adenosylmethionine decarboxylase activity could not be detected in cell-free extracts. These results suggested that M. denitrifcans might make spermidine by a novel route. M. denitrifcans was grown anaerobically in a defined nitrate medium deficient in iron (Tait, 1975), and R. spheroides was grown semi-anaerobically in the light as described by Neuberger et al. (1973). Cells were harvested by centrifugation, resuspended in 0.05~KH2P04/NaOH buffer, pH 7.5, radioactive and unlabelled compounds were added, and suspensions were incubated at 30°C. Suspensions of R. spheroides were illuminated. Concentrated suspensions of cells were disrupted by ultrasonication and centrifuged at 105000g for 90min. The supernatants were used as enzyme, either before or after dialysis. After incubation, cells and enzyme extracts were extracted with 10% (w/v) trichloroacetic acid. These extracts were saturated with Na2S04, adjusted to pH 12-1 3 with NaOH, and the amines were extracted into butan-1-01. The amines were separated by electrophoresis or chromatography on paper (Herbst et al., 1958). Quantitative determination of the amines was done as described by Russell et al. (1970), and their radioactivity was measured by liquid-scintillation spectrometry. M. denitrifcans and R. spheroides both contain about five times more spermidine than putrescine. Suspensions of these organisms formed radioactive spermidine from ~-[U-'~C]arginine, ~~-[5-'~C]ornithine a d [l ,4-'4C]putrescine, but not when incubated with ~-[U'~C]methionine and unlabelled putrescine. Enzyme extracts did not form radioactive spermidine from [I ,4-'4C]putrescine and S-adenosylmethionine. Of a number of labelled compounds incubated with cell suspensions in the presence of putrescine, ~-[U-'~C]aspartate and 14C-labelled fumarate gave the highest incorporations of radioactivity into spermidine; [2,3-'4C]fumarate was twice as effective as [1,4-'*C]fumarate. It was concluded that a metabolic product of L-aspartate, possibly L-aspartic 8-semialdehyde, might be the donor of the aminopropyl group of spermidine. In bacteria L-aspartic 8-semialdehyde is an intermediate in the biosynthesis of L-homoserine (Fig. 1) and a number of other amino acids (cf. Greenberg, 1969). It is known (Gibson et al., 1962) that R. spheroides contains L-aspartate kinase (EC 2.7.2.4), an NADP+-dependent L-aspartic b-semialdehyde dehydrogenase (EC 1.2.1.1 l), and an NADP+-dependent L-homoserine dehydrogenase (EC 1.1.1.3), and that extracts can make L-aspartic 8semialdehyde from L-aspartate or from L-homoserine (Fig. 1). Labelled spermidine was formed on incubating enzyme extracts from M. denitrifcaas and from R . spheroides with [I ,4-'4C]putrescine and L-aspartate (or putrescine and L[U-'4C]aspartate) plus ATP, Mg2+, NADPH and dithiothreitol, or with [1,4-"C]-