BIOSYNTHESIS OF ANATOXIN-A BY ANABAENA-FLOS-AQUAE - EXAMINATION OF PRIMARY ENZYMATIC STEPS

L-[U-C-14]Arginine, L-[U-C-14]ornithine and [1,4-C-14]putrescine were incorporated into anatoxin-a by Anabnena flos-aquae IC-1. To test the hypothesis that arginine and ornithine give rise to putrescine, which is oxidized to Delta(1)-pyrroline, and that this, in turn, is a precursor of anatoxin-a, cell-free extracts were incubated with [2,5-C-14]Delta(1)-pyrroline. Subsequent chromatography demonstrated the disappearance of Delta(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 anatoxina-a also metabolized the C-14-labelled Delta(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 Delta(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 CO2 release gave rates up to 7 x those determined by amine production. With each of the two decarboxylases, chromatography on DEAE-cellulose gave two major peaks of CO2- 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 dlfluoromethylarginine 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 (NH4)(2)SO4, 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 get-filtration chromatography on Sephadex G-200. A 55-fold purification of the oxidase was attained. It has a K-m of 2.5 mu M and an apparent M(r) of 21-22 x 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.