PURIFICATION AND CHARACTERIZATION OF A SULFOTRANSFERASE SPECIFIC TO N‐21 OF SAXITOXIN AND GONYAUTOXIN 2+3 FROM THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

Abstract

A sulfotransferase (ST) specific to N‐21 of saxitoxin (STX) and gonyautoxin 2+3 (GTX2+3) designated as N‐ST was purified to homogeneity from the cytosolic fraction of clonal‐axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum Graham GC21V, which causes paralytic shellfish poisoning. The enzyme transferred a sulfate group from 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to N‐21 in the carbamoyl group of STX and GTX2+3 to produce GTX5 and C1+2, respectively. The molecular mass of the purified enzyme was determined by SDS‐PAGE to be 59 kDa. Gel filtration chromatography showed a native molecular mass of 65 kDa, indicating that the N‐ST is a monomeric enzyme. The N‐ST was specific to only N‐21 of STX and GTX2+3, and O‐22 sulfation was not observed. Moreover, the N‐ST was not active toward neo STX and GTX1+4, which differed from STX and GTX2+3, respectively, in only N‐1 hydroxylation. When various compounds previously reported to be substrates for STs in other organisms and paralytic shellfish poisoning toxins other than STX and GTX2+3 were added to the reaction mixture, N‐ST activity was not decreased. The enzyme required PAPS as the sole source of sulfate. The enzyme was optimally active at pH 6.0 and 25° C, and its activity was enhanced by Mg2+ and Co2+. The Km values of the N‐ST for STX and GTX2+3 were 16.1 μM and 29.8 μM, respectively.

Biosynthetic Intermediate Analysis and Functional Homology Reveal a Saxitoxin Gene Cluster in Cyanobacteria

“…circinalis AWQC131C and C. raciborskii T3 also produce N-and O-sulfated analogues of STX [GTX-5, C-2/C-3, (dc)GTX-3/GTX-4]. The activity of two 3Ј-phosphate 5Ј-phosphosulfate (PAPS)-dependent sulfotransferases, which were specific for the N-21 of STX and GTX-3/GTX-2 and the O-22 of 11-hydroxy STX, respectively, has been described previously in studies of the PSP toxin-producing dinoflagellate Gymnodinium catenatum (44,62). The sxt gene cluster from C. raciborskii T3 encodes SxtN, a putative sulfotransferase.…”

“…Although STX biosynthesis seems complex and unique, organisms from two kingdoms, including certain species of marine dinoflagellates and freshwater cyanobacteria, are capable of producing these toxins, apparently by the same biosynthetic route (47). In spite of considerable efforts, none of the enzymes or genes involved in the biosynthesis and modification of STX have been previously identified (15,39,40,44,62). Here, based on previously published knowledge regarding the possible steps in STX biosynthesis (47), together with information from our recent in vitro biosynthesis of STX (22), we used an approach that employed reverse genetics to identify the candidate STX biosynthetic gene cluster (sxt) in the cyanobacterium Cylindrospermopsis raciborskii T3 (23).…”

Reduction in the toxicity of the dinoflagellate Gymnodinium catenatum when fed on by the heterotrophic dinoflagellate Polykrikos kofoidii

“…G. catenatum has many types of toxins (i.e. STX, neoSTX, GTX1-6, C1-4, deSTX, deneoSTX, deGTX1-4, 11-α-hydroxySTX, 11-β-hydroxySTX) and one type can change to another under certain conditions (Sako et al 2001, Holmes et al 2002. Therefore, to investigate whether P. kofoidii has the ability to reduce the toxicity produced by G. catenatum and, if so, to quantify the reduction rate of the toxicity, we measured total toxicity in a population of P. kofoidii originally fed G. catenatum and then starved as a function of elapsed starvation time using the mouse bioassay.…”

Relationship of proteomic variation and toxin synthesis in the dinoflagellate Alexandrium tamarense CI01 under phosphorus and inorganic nitrogen limitation

“…Our findings further support the hypothesis that the PSTs biosynthesis is related to methionine and arginine. (Taroncher-Oldenburg and Anderson 2000; Sako et al 2001;Pomati et al 2006;Kellmann and Neilan 2007). S-Adenosyl-homocysteine hydrolase, arginine deiminase, sulfotransferase in A. fundyense might bear to STX (a kind of PSTs) biosynthesis (Taroncher-Oldenburg and Anderson 2000).…”