title: Dihydroanatoxin-a Is Biosynthesized from Proline in Cylindrospermum stagnale PCC 7417: Isotopic Incorporation Experiments and Mass Spectrometry Analysis.
abstract: 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 stagnale PCC 7417 was definitively shown to produce dihydroanatoxin-a (1.2 mg/g dried cells). Oscillatoria sp. PCC 9107, Oscillatoria sp. PCC 6506, and C. stagnale PCC 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 ana cluster 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 ana gene cluster in C. stagnale PCC 7417. This is the first report of a cyanobacterium producing dihydroanatoxin-a, suggesting that other producers are present in the environment.
authors.affiliation: LIED, UMR 8236 CNRS, Université Paris Diderot , 75205 Paris Cedex 13, France.
authors.forename: Annick
authors.initials: A
authors.lastname: Méjean
authors.affiliation: LIED, UMR 8236 CNRS, Université Paris Diderot , 75205 Paris Cedex 13, France.
authors.forename: Klervi
authors.initials: K
authors.lastname: Dalle
authors.affiliation: LIED, UMR 8236 CNRS, Université Paris Diderot , 75205 Paris Cedex 13, France.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Paci
authors.affiliation: LCM, UMR 9168, École Polytechnique, CNRS , 91128 Palaiseau Cedex, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Bouchonnet
authors.affiliation: MCAM, UMR 7245, Sorbonne Université, Muséum National d'Histoire Naturelle, CNRS , 75005 Paris, France.
authors.forename: Stéphane
authors.initials: S
authors.lastname: Mann
authors.affiliation: LSABM CBI, UMR 8231 ESPCI ParisTech, CNRS, PSL Research University , 75005 Paris, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Pichon
authors.affiliation: LIED, UMR 8236 CNRS, Université Paris Diderot , 75205 Paris Cedex 13, France.|Chimie ParisTech, ENSCP , 75005 Paris, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Ploux
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry / Q000302:isolation & purification; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry / Q000302:isolation & purification; D000458:Cyanobacteria / Q000737:chemistry; D000087522:Cyanobacteria Toxins; D008401:Gas Chromatography-Mass Spectrometry; D015394:Molecular Structure; D005810:Multigene Family; D054300:Oscillatoria / Q000737:chemistry; D011392:Proline / Q000031:analogs & derivatives* / Q000737:chemistry / Q000302:isolation & purification; D014326:Tropanes / Q000737:chemistry / Q000302:isolation & purification
chemical_list: D001427:Bacterial Toxins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D000087522:Cyanobacteria Toxins; D014326:Tropanes; C000618148:dihydroanatoxin-A; C524097:homoanatoxin-a; C509783:anatoxin a; D011392:Proline