Biosynthesis of anatoxin- a by Anabaena flos-aquae: Examination of primary enzymic steps

Abstract

l-[U- 14C]Arginine, l-[U- 14C]ornithine and [1,4- 14C]putrescine were incorporated into anatoxin- a by Anabaena flos-aquae IC-1. To test the hypothesis that arginine and ornithine give rise to putrescine, which is oxidized to Δ 1-pyrroline, and that this, in turn, is a precursor of anatoxin- a, cell-free extracts were incubated with [2,5- 14C]Δ 1-pyrroline. Subsequent chromatography demonstrated the disappearance of Δ 1-pyrroline and production of a major radioactive metabolite identified as anatoxin- a. A strain of A. flos-aquae that had lost its ability to synthesize anatoxin- a also metabolized the 14C-labelled Δ 1-pyrroline but without yielding a major radioactive product. The arginine and ornithine decarboxylases required to produce putrescine, and a diamine oxidase to cyclize it to Δ 1-pyrroline, were shown to be active in A. flos-aquae IC-1. The decarboxylases were associated with a particulate fraction sedimenting at 31 300 g. Assay of these enzymes by measuring CO 2 release gave rates up to 7 × those determined by amine production. With each of the two decarboxylases, chromatography on DEAE-cellulose gave two major peaks of CO 2-releasing activity, only one of which coincided with amine-producing activity. Based on agmatine production, the K m for the arginine decarboxylase was 10 mM. The arginine and ornithine decarboxylases were both inhibited to a similar extent by difluoromethylarginine and difluoromethylornithine and it is suggested that this, together with their chromatographic coincidence, may indicate a single protein. The diamine oxidase was inhibited by endogenous low M r compounds, had a broad pH optimum of 8.5–9.5, and was not sedimented at 31 300 g. On fractional precipitation with (NH 4) 2SO 4, enzymic activity was found in the 60–80% saturation fraction. Chromatography of this fraction on DEAE-cellulose gave a single peak of activity which was not further resolved by gel-filtration chromatography on Sephadex G-200. A 55-fold purification of the oxidase was attained. It has a K m of 2.5 μM and an apparent M r of 21–22 ×10 3. The toxin-producing and non-producing strains of A. flos-aquae showed similar rates of amine oxidase and ornithine decarboxylase activity. In a previously active anatoxin- a producing strain which has now lost this synthetic capacity, the 10 kb plasmid, described earlier, was replaced by a 6.5 kb plasmid.