Metabolic inhibitor induces dynamic changes in saxitoxin biosynthesis and metabolism in the dinoflagellate Alexandrium pacificum (Group IV) under in vivo labeling condition

Abstract

In paralytic shellfish toxin-producing dinoflagellates, intracellular levels of saxitoxin and its analogues (STXs) are controlled by a balance between degradation and biosynthesis in response to marine environmental fluctuations and stresses. The purpose of this study was to demonstrate the utility of statistical analysis of in vivo labeling data for the dynamic analysis of variations in toxin production under stress. A toxic strain of the dinoflagellate Alexandrium pacificum (Group IV) was cultured in colchicine-containing 15N-labeled sodium nitrate-medium and metabolite levels were analyzed over time by liquid chromatography-mass spectrometry. Quantitative values of all isotopomers of precursor amino acids, biosynthetic intermediates, and major STXs were subjected to statistical analysis. The decrease of the nitrogen incorporation rates for all compounds suggested that colchicine decreased nitrate assimilation upstream of glutamate biosynthesis. In colchicine-treated cultures, the per-cell content of total STX analogues did not change significantly over time; however, the production rate of each pathway varied greatly. De novo STX biosynthesis was decreased by colchicine until Day 3, while the salvage pathway was not. Subsequently, biosynthesis by both pathways was enhanced. This analysis of dynamic metabolism provides new insights into the complex mechanisms regulating STX metabolism in dinoflagellates.