Identification of 7-Deoxy-desulfo-argino-cylindrospermopsin, the Missing Piece in Cylindrospermopsin Biosynthesis.

Cylindrospermopsin, a major cyanotoxin, is produced by freshwater cyanobacteria. Its biosynthesis starts from arginine and glycine and involves five polyketide synthases and several tailoring enzymes. We report the identification of 7-deoxy-desulfo-argino-cylindrospermopsin in several cylindrospermopsin-producing cyanobacteria using mass spectrometry experiments. We have purified this new metabolite and established its structure by 1D and 2D NMR spectroscopy using scalar-based ¹ H- ¹ H, ¹ H- ¹³ C, and ¹ H- ¹⁵ N as well as 2D ¹ H- ¹ H ROESY correlation experiments. Using labeled arginines in isotopic incorporation experiments, we have shown that arginine is fully incorporated into 7-deoxy-desulfo-argino-cylindrospermopsin and that the uracil ring of cylindrospermopsin originates from the guanidino moiety of arginine, thus solving a long-standing puzzling question. CyrG and CyrH from the cylindrospermopsin-producing Oscillatoria sp. PCC 6506 were overproduced in Escherichia coli and purified to homogeneity. We showed that CyrG is a zinc-dependent hydrolase, homologous to adenosine deaminases, that transforms 7-deoxy-desulfo-argino-cylindrospermopsin into 7-deoxy-desulfo-cylindrospermopsin and ornithine, with the following kinetic parameters: K _M = 0.21 ± 0.05 μM and k _cat = 0.19 ± 0.02 min ^-1 . CyrG contained 0.55 mol of zinc per mol of monomer but could be activated by Fe ^II or Co ^II . CyrH contained almost no metal and showed no such activity even in the presence of excess metal. Using structure-based alignments and secondary structure predictions, we propose that the fifth and last polyketide synthase CyrF in cylindrospermopsin biosynthesis contains an unprecedented C-terminal domain homologous to N -acetyltransferases. We suggest that this domain catalyzes the condensation of the CyrF product with arginine to give 7-deoxy-desulfo-argino-cylindrospermopsin. This would be an unprecedented termination step for a polyketide synthase.