Purification and characterization of sulfotransferase specific to O-22 of 11-hydroxy saxitoxin from the toxic dinoflagellate Gymnodinium catenatum (dinophyceae)

Abstract

A novel sulfotransferase (O‐ST), which transferred the sulfate group of 3′‐phosphoadenosine 5′‐phosphosulfate (PAPS) to O‐22 of 11‐α,β‐hydroxy saxitoxin (STX) and produced GTX2 + 3, was purified to homogeneity from the cytosolic fraction of clonal‐axenic vegetative cells of the toxic dinoflagellate Gymnodinium catenatum GC21V. After four purification steps, including affinity chromatography and anion exchange chromatography, the enzyme was purified 500‐fold and the yield was 4%. On affinity chromatography with a PAP‐agarose column, O‐ST was observed in the bound fraction, and N‐ST specific to N‐21 of STX and GTX2 + 3 was found in the unbound fraction. The molecular mass of the purified enzyme was determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to be 65 kDa. Gel filtration chromatography showed a native molecular mass of 67 kDa, indicating that O‐ST is a monomeric enzyme. The enzyme was optimally active at pH 6.0 and 35°C. O‐ST did not require metal cations for its activity. O‐ST required PAPS as the sole source of sulfate. O‐ST transferred a sulfate group from PAPS to only O‐22 of 11‐α,β‐hydroxy STX and not to N‐21 of these toxins. These observations suggested that two ST, N‐ST and O‐ST, participate in the sulfation of PSP toxins.

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).…”

Bioconcentration, Bioaccumulation, and Metabolism of Pesticides in Aquatic Organisms

“…The range of these pH and temperature optima profiles indicate the presence of isozymes, which has been demonstrated by Yoshida et al (2002). Using successive column chromatography, these authors purified two isozymes that catalyze the sulfation of 11-hydroxy saxitoxin from cytosolic fractions of dinoflagellates.…”

“…Because the amount of endogeneous PAPS is less than that of UDPG(A), a larger amount of xenobiotic may limit the extent of sulfate conjugation. Sulfotransferase enzyme activity has been demonstrated to be mainly located in cytosolic fractions; tissues rich in this enzyme include mid-gut gland of clam (Kobayashi et al 1987), digestive gland and gonad of mussel (Lavado et al 2006), digestive gland of gumboot chiton (De Busk et al 2000), antennal glands and hepatopancreas of lobster (Elmamlouk and Gessner 1978;Li and James 1993;Schell and James 1989), digestive tube of sea urchin (Janer et al 2005a), and in dinoflagellates (Yoshida et al 2002). Phenols, either intact or produced via phase-I metabolism such as hydroxylation, demethylation, or ester cleavage, are the main substrates.…”

“…The optima for pH and temperature of this enzyme are approximately 6-9 and ≥35 • C, respectively (Elmamlouk and Gessner 1978;Lavado et al 2006;Li and James 1993;Schell and James 1989;Yoshida et al 2002), and are highly speciesdependent. The range of these pH and temperature optima profiles indicate the presence of isozymes, which has been demonstrated by Yoshida et al (2002).…”