Biosynthesis of Cylindrospermopsin in Cyanobacteria: Characterization of CyrJ the Sulfotransferase.

7-Deoxy-desulfo-cylindrospermopsin was purified at small-scale from the supernatant of a culture of the cyanobacterium sp. PCC 10702. This metabolite was obtained in a pure form using a three-step chromatographic procedure, and its identity was confirmed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). LC-MS quantification showed that this metabolite was excreted in the culture medium of sp. PCC 10702. Isotopic incorporation studies using [2-C,N]glycine, a cylindrospermopsin precursor, and sp. PCC 10702 cells showed that glycine was incorporated into 7-deoxy-desulfo-cylindrospermopsin, 7-deoxy-cylindrospermopsin, 7--cylindrospermopsin, and cylindrospermopsin. The isotopic incorporation rate was consistent with the following metabolic flux: 7-deoxy-desulfo-cylindrospermopsin → 7-deoxy-cylindrospermopsin → 7--cylindrospermopsin and cylindrospermopsin. We have cloned the gene into an expression vector and overproduced the putative sulfotransferase CyrJ in . The purified protein CyrJ catalyzed, , the transfer of a sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to 7-deoxy-desulfo-cylindrospermopsin to give 7-deoxy-cylindrospermopsin. Kinetic analysis afforded the following apparent constants: (PAPS) = 0.12 μM, = 20 nM/min, (7-deoxy-desulfo-cylindrospermopsin) = 0.12 μM, and (7-deoxy-desulfo-cylindrospermopsin) = 4.1 μM. Preliminary data suggested that CyrJ catalyzed the reaction through a ternary-complex kinetic mechanism. All these data confirmed that CyrJ catalyzed a sulfotransfer during the penultimate step of the biosynthesis of cylindrospermopsin.