Search

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 1H-1H, 1H-13C, and 1H-15N as well as 2D 1H-1H 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 Oscillatoriasp. PCC 6506 were overproduced in Escherichia coliand 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: KM= 0.21 ± 0.05 μM and kcat= 0.19 ± 0.02 min–1. CyrG contained 0.55 mol of zinc per mol of monomer but could be activated by FeIIor CoII. 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.

7-Deoxy-desulfo-cylindrospermopsin was purified at small-scale from the supernatant of a culture of the cyanobacterium Oscillatoriasp. 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 Oscillatoriasp. PCC 10702. Isotopic incorporation studies using [2-13C,15N]glycine, a cylindrospermopsin precursor, and Oscillatoriasp. PCC 10702 cells showed that glycine was incorporated into 7-deoxy-desulfo-cylindrospermopsin, 7-deoxy-cylindrospermopsin, 7-epi-cylindrospermopsin, and cylindrospermopsin. The isotopic incorporation rate was consistent with the following metabolic flux: 7-deoxy-desulfo-cylindrospermopsin → 7-deoxy-cylindrospermopsin → 7-epi-cylindrospermopsin and cylindrospermopsin. We have cloned the cyrJgene into an expression vector and overproduced the putative sulfotransferase CyrJ in Escherichia coli. The purified protein CyrJ catalyzed, in vitro, 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: KM app.(PAPS) = 0.12 μM, Vmax app.= 20 nM/min, KM app.(7-deoxy-desulfo-cylindrospermopsin) = 0.12 μM, and KI app.(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.

Anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a are potent cyanobacterial neurotoxins. They are biosynthesized in cyanobacteria from proline and acetate by a pathway involving three polyketide synthases. We report the identification of carboxy-anatoxin-a, carboxy-homoanatoxin-a, and carboxy-dihydroanatoxin-a in acidic extracts of Cuspidothrix issatschenkoiCHARLIE-1, Oscillatoriasp. PCC 6506, and Cylindrospermum stagnalePCC 7417, respectively, using liquid chromatography coupled to mass spectrometry. The structure of these carboxy derivatives was confirmed by mass spectrometry and by isotopic incorporation experiments using labeled proline and acetate. Each of these three cyanobacteria only produce one carboxy-anatoxin, suggesting that these metabolites are the product of the hydrolysis by AnaA, the type II thioesterase, of the thioesters bound to AnaG, the last polyketide synthase of the pathway. By measuring the rate of isotopic incorporation of labeled proline into carboxy-homoanatoxin-a and homoanatoxin-a produced by Oscillatoriasp. PCC 6506, we show that carboxy-homoanatoxin-a is the intracellular precursor of homoanatoxin-a, and that homoanatoxin-a is then excreted into the extracellular medium. The transformation of carboxy-homoanatoxin-a into homoanatoxin-a is a very slow two-step process, with accumulation of carboxy-homoanatoxin-a, suggesting that the decarboxylation is spontaneous and not enzymatically catalyzed. However, an unidentified and extracellular catalyst accelerates the decarboxylation when the cell extracts are prepared at neutral pH.

LC-MS and GC-MS analytical conditions have been developed to detect the cis- and trans-epimers (relative configuration of the carbon bearing the acetyl or propionyl group) of dihydroanatoxin-a and dihydrohomoanatoxin-a, in biological samples. These compounds epimerize under acidic conditions, yielding a major species that was tentatively assigned as the cis-epimer. Cylindrospermum stagnalePCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoriasp. PCC 9107, Oscillatoriasp. PCC 6506, and C. stagnalePCC 7417, which produce anatoxin-a, homoanatoxin-a, and dihydroanatoxin-a, respectively, were cultivated in the presence of isotopically labeled proline, and the toxins were extracted. Interpretation of the GC-MS electron ionization mass spectra of these labeled anatoxins showed that they are all biosynthesized from proline and that the positions of the labels in these molecules are identical. These data and the fact that the anacluster of genes is conserved in these cyanobacteria suggest that dihydroanatoxin-a is formed by the reduction of either anatoxin-a or its precursor in a specific step involving AnaK, an F420-dependent oxido-reductase whose gene is found in the anagene cluster in C. stagnalePCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment.

The mechanism of action of amiclenomycin (1a), a naturally occuring inhibitor of diaminopelargonic acid aminotransferase, has been established. The enzyme catalyzes the formation of an aromatic adduct between the inhibitor and pyridoxal-5'-phosphate. The structure of the adduct, determined by mass spectrometry, is in agreement with the reported X-ray crystal structure. Kinetic parameters, characteristic of kcat inhibitors, have been observed, with a KI value of 2 μM and a kinact value of 0.4 min⁻¹. The irreversibility of the inactivation observed, in spite of the absence of covalent bond between the inhibitor and the protein, reveals the high affinity of the adduct for the active site. Two other cis-1-amino-4-substituted-cyclohexa-2,5-dienes, 3a and 4a, were also found to efficiently inhibit the enzyme. The trans-isomers were either much less potent (1b) or inactive (3b and 4b). The aminocyclohexadiene moiety, which is, apparently, responsible for the inhibition, could constitute an original pharmacophore for the design of new herbicides.

Biotin synthase catalyses the last step of the biosynthesis of biotin in microorganisms and plants. The active protein isolated from Bacillus sphaericusand Escherichia colicontains an iron‐sulphur (FeS) cluster. The native enzymes were depleted of their iron and inorganic sulphide and the resulting apoenzymes were chemically reconstituted with FeCl3and Na2[34S] to give labelled (Fe34S) enzymes. These enzymes were functional and when assayed in vitro produced labelled biotin containing about 65% of 34S. These data strongly support the hypothesis that the sulphur of biotin is derived from the (FeS) centre of the enzyme.

For Abstract see ChemInform Abstract in Full Text.