Synthesis and Identification of decarbamoyloxySaxitoxins in Toxic Microalgae and their Reactions with the Oxygenase, SxtT, Reveal Saxitoxin Biosynthesis.

Saxitoxin (STX, 1) is a representative compound of paralytic shellfish toxins (PSTs) that are produced by marine dinoflagellates and freshwater cyanobacteria. Although several pathways have been proposed for the biosynthesis of STX, the order of ring and side chain hydroxylation, and formation of the tricyclic skeleton have not been well established. In this study, 12,12-dideoxy-decarbamoyloxySTX (dd-doSTX, 2), the most reduced STX analogue having the tricyclic skeleton, and its analogues, 12ß-deoxy-doSTX (12ß-d-doSTX, 3), 12α-deoxy-doSTX (12α-d-doSTX, 4), and doSTX (5), were synthesized, and these compounds were screened in the toxic microalgae using high-resolution LCMSMS. dd-doSTX (2) and 12ß-d-doSTX (3) were identified in the PSTs-producing dinoflagellates (Alexandrium catenella, A. pacificum, and/or Gymnodinium catenatum) and in the cyanobacterium Dolichospermum circinale (TA04). doSTX (5), previously isolated from the dinoflagellate G. catenatum, was also identified in D. circinale (TA04). Furthermore, the conversion of 2 to 3, and 4 to 5, by SxtT with VanB, a reported Rieske oxygenase and its redox partner in STX biosynthesis, was confirmed. These results support that 2 is a possible biosynthetic precursor of STX, and that ring and side-chain hydroxylations proceed after cyclization.

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

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.

First Identification of 12ß-Deoxygonyautoxin 5 (12α-Gonyautoxinol 5) in the Cyanobacterium Dolichospermum circinale (TA04) and 12ß-Deoxysaxitoxin (12α-Saxitoxinol) in D. circinale (TA04) and the Dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC).

Saxitoxin and its analogues, paralytic shellfish toxins (PSTs), are potent and specific voltage-gated sodium channel blockers. These toxins are produced by some species of freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates of PSTs, as well as new analogues, from such organisms and proposed the biosynthetic and metabolic pathways of PSTs. In this study, 12ß-deoxygonyautoxin 5 (12α-gonyautoxinol 5 = gonyautoxin 5-12(R)-ol) was identified in the freshwater cyanobacterium, Dolichospermum circinale (TA04), and 12ß-deoxysaxitoxin (12α-saxitoxinol = saxitoxin-12(R)-ol) was identified in the same cyanobacterium and in the marine dinoflagellate Alexandrium pacificum (Group IV) (120518KureAC) for the first time from natural sources. The authentic standards of these compounds and 12α-deoxygonyautoxin 5 (12ß-gonyautoxinol 5 = gonyautoxin 5-12(S)-ol) were prepared by chemical derivatization from the major PSTs, C1/C2, produced in D. circinale (TA04). These standards were used to identify the deoxy analogues by comparing the retention times and MS/MS spectra using high-resolution LC-MS/MS. Biosynthetic or metabolic pathways for these analogues have also been proposed based on their structures. The identification of these compounds supports the α-oriented stereoselective oxidation at C12 in the biosynthetic pathway towards PSTs.

SxtA localizes to chloroplasts and changes to its 3'UTR may reduce toxin biosynthesis in non-toxic Alexandrium catenella (Group I)✰.

SxtA is the enzyme that catalyses the first step of saxitoxin biosynthesis. We developed an immunofluorescent method to detect SxtA using antibodies against SxtA peptides. Confocal microscopy revealed the presence of abundant, sub-cellularly localized signal in cells of toxic species and its absence in non-toxic species. Co-localization of SxtA with Rubisco II and ultra-structural observation by transmission electron microscopy strongly suggested the association of SxtA with chloroplasts. We also characterized a non-toxic sub-clone of Alexandrium catenella (Group I) to elucidate the mutation responsible for its loss of toxicity. Although sxtA4 gene copy number was indistinguishable in toxic and non-toxic sub-clones, mRNA and protein expression were significantly reduced in the non-toxic sub-clone and we uncovered sequence variation at the 3' untranslated region (3'UTR) of sxtA4 mRNA. We propose that differences in the sxtA4 mRNA 3'UTR lead to down-regulation of STX biosynthesis post-transcriptionally, thereby explaining the differences in toxicity amongst different A. catenella (Group I) sub-clones.

Identification of a Novel Saxitoxin Analogue, 12ß-Deoxygonyautoxin 3, in the Cyanobacterium, Anabaena circinalis (TA04).

Saxitoxin (STX) and its analogues, the potent voltage-gated sodium channel blockers, are biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified several biosynthetic intermediates in the extract of the cyanobacterium, Anabaena circinalis (TA04), that are primarily produced during the early and middle stages in the biosynthetic pathway to produce STX. These findings allowed us to propose a putative biosynthetic pathway responsible for STX production based on the structures of these intermediates. In the present study, we identified 12ß-deoxygonyautoxin 3 (12ß-deoxyGTX3), a novel STX analogue produced by A. circinalis (TA04), by comparing the retention time and MS/MS fragmentation pattern with those of synthetic standards using LC-MS. The presence of this compound in A. circinalis (TA04) is consistent with stereoselective enzymatic oxidations at C11 and C12, and 11-O-sulfation, during the late stage of STX biosynthesis, as proposed in previous studies.

Metabolomic study of saxitoxin analogues and biosynthetic intermediates in dinoflagellates using 15N-labelled sodium nitrate as a nitrogen source.

A stable-isotope-labelling method using 15N-labelled sodium nitrate as a nitrogen source was developed for the toxic dinoflagellate Alexandrium catenella. The labelled saxitoxin analogues (STXs), their precursor, and the biosynthetic intermediates were analyzed by column-switching high-resolution hydrophilic interaction liquid chromatography with mass spectrometry. The low contents on Day 0, high 15N incorporation % of Int-C'2 and Int-E' suggested that their turn-over rates are high and that the sizes of the pool of these compounds are smaller than those of the other intermediates. The experimentally determined isotopomer distributions showed that arginine, Int-C'2, 11-hydroxy-Int-C'2, Int-E', GTX5, GTX4, C1, and C2, each existed as a combination of three populations that consisted of the non-labelled molecules and the labelled isotopomers representing molecules newly synthesized by incorporation of 15N assimilated from the medium with two different incorporation rates. The order of 15N incorporation % values of the labelled populations predicted by this model largely agreed with the proposed biosynthetic route. The stable-isotope-labelling method will be useful for understanding the complex mechanism of nitrogen flux in STX-producing dinoflagellates.

Synthesis and Identification of Key Biosynthetic Intermediates for the Formation of the Tricyclic Skeleton of Saxitoxin.

Saxitoxin (STX) and its analogues are potent voltage-gated sodium channel blockers biosynthesized by freshwater cyanobacteria and marine dinoflagellates. We previously identified genetically predicted biosynthetic intermediates of STX at early stages, Int-A' and Int-C'2, in these microorganisms. However, the mechanism to form the tricyclic skeleton of STX was unknown. To solve this problem, we screened for unidentified intermediates by analyzing the results from previous incorporation experiments with 15 N-labeled Int-C'2. The presence of monohydroxy-Int-C'2 and possibly Int-E' was suggested, and 11-hydroxy-Int-C'2 and Int-E' were identified from synthesized standards and LC-MS. Furthermore, we observed that the hydroxy group at C11 of 11-hydroxy-Int-C'2 was slowly replaced by CD3 O in CD3 OD. Based on this characteristic reactivity, we propose a possible mechanism to form the tricyclic skeleton of STX via a bicyclic intermediate from 11-hydroxy-Int-C'2.

Column switching combined with hydrophilic interaction chromatography-tandem mass spectrometry for the analysis of saxitoxin analogues, and their biosynthetic intermediates in dinoflagellates.

Hydrophilic-interaction chromatography (HILIC) is reportedly useful for the analysis of saxitoxin (STX) analogues, collectively known as paralytic shellfish toxins. Column switching and two-step gradient elution using HILIC combined with mass spectrometry enabled the simultaneous analysis of the 15 primary STX analogues and their biosynthetic intermediates, arginine, Int-A', and Int-C'2, and the shunt product, Cyclic-C'. Crude extracts of toxin-producing dinoflagellates can be injected without any treatment except filtration. Enrichment of the compounds using this method was highly reproducible with respect to retention times (% RSD was under 1%) and highly sensitive (limits of detection (LODs) were in the range 0.9 (Int-C'2) - 116 (C3) µM) in terms of avoiding matrix effects associated with co-eluting substances. Validation studies demonstrated acceptable performance of this method for specificity, repeatability, linearity and recovery. A comparison of the quantitative results for STX analogues in Alexandrium tamarense using HPLC with post-column fluorescent derivatization and the column-switching HILIC-MS method revealed good agreement. The presence of Int-A', Int-C'2, and Cyclic-C' in toxic dinoflagellate species with different toxin profiles was confirmed using this method. Our data support the hypothesis that the early stages of the STX biosynthesis and shunt pathways are the same in dinoflagellates and cyanobacteria.

Biosynthetic route towards saxitoxin and shunt pathway.

Saxitoxin, the most potent voltage-gated sodium channel blocker, is one of the paralytic shellfish toxins (PSTs) produced by cyanobacteria and dinoflagellates. Recently, putative biosynthetic genes of PSTs were reported in these microorganisms. We previously synthesized genetically predicted biosynthetic intermediates, Int-A' and Int-C'2, and also Cyclic-C' which was not predicted based on gene, and identified them all in the toxin-producing cyanobacterium Anabaena circinalis (TA04) and the dinoflagellate Alexandrium tamarense (Axat-2). This study examined the incorporation of (15)N-labeled intermediates into PSTs (C1 and C2) in A. circinalis (TA04). Conversions from Int-A' to Int-C'2, from Int-C'2 to Cyclic-C', and from Int-A' and Int-C'2 to C1 and C2 were indicated using high resolution-LC/MS. However, Cyclic-C' was not converted to C1 and C2 and was detected primarily in the extracellular medium. These results suggest that Int-A' and Int-C'2 are genuine precursors of PSTs, but Int-C'2 converts partially to Cyclic-C' which is a shunt product excreted to outside the cells. This paper provides the first direct demonstration of the biosynthetic route towards saxitoxin and a shunt pathway.

Synthesis of a tricyclic bisguanidine compound structurally related to saxitoxin and its identification in paralytic shellfish toxin-producing microorganisms.

We recently reported the chemical synthesis and identification of the genetically predicted biosynthetic intermediates of saxitoxin (STX), including a 2-aminoimidazole-bearing monoguanidine compound (Int-C'2) in two paralytic shellfish toxin (PST)-producing microorganisms. In this study, we achieved the direct conversion of Int-C'2 into a tricyclic bisguanidine compound (called Cyclic-C'), which is structurally related to STX, through oxidative intramolecular guanidine transfer to 2-aminoimidazole catalyzed by Pd/C under basic conditions in air. By using HPLC-MS analysis, Cyclic-C' was also identified in the PST-producing microorganisms, suggesting that Cyclic-C' is either another biosynthetic intermediate or a shunt product of PSTs. In addition, a weak inhibitory activity of Cyclic-C' to the voltage-gated sodium channels was detected by using a cell-based assay.

Indirect quantitation of saxitoxin by HPLC with post-column oxidation and fluorometric detection.

The indirect identification and quantification of saxitoxin (STX) using other STX analogues by high-performance liquid chromatography with post-column oxidation and fluorescent detection (HPLC-FD) was investigated. Decarbamoylsaxitoxin (dcSTX) among the many STX analogues was selected as an external standard to identify and quantify STX. The retention time of STX in shellfish extracts by HPLC-FD was reproducibly estimated by using the retention time of dcSTX and the separation factor (α) between STX and dcSTX. Almost all of the columns tested to setup the method were useful to identify STX. Because a molar fluorescent coefficient of dcSTX was slightly different from that of STX, a factor used to correct the fluorescent coefficient in STX/dcSTX was determined to be 1.30. The indirect quantification of STX in scallop extracts by using the correction factor agreed to 80 - 100% precision with direct quantification using STX as an external standard.

Synthesis and identification of proposed biosynthetic intermediates of saxitoxin in the cyanobacterium Anabaena circinalis (TA04) and the dinoflagellate Alexandrium tamarense (Axat-2).

Here, we describe the synthesis of the genetically predicted biosynthetic intermediates of the neurotoxin saxitoxin (STX) (1), 2, 6 and 7, and identification of 2 and 6 in toxin-producing microorganisms. This is the first chemical evidence supporting the genetically predicted biosynthetic route toward 1.

Biogenic polyamines capture CO2 and accelerate extracellular bacterial CaCO3 formation.

Bacteria, including cyanobacteria, as well as some fungi, are known to deposit calcium carbonate (CaCO(3)) extracellularly in calcium-containing artificial medium. Despite extensive investigation, the mechanisms involved in extracellular formation of CaCO(3) by bacteria have remained unclear. The ability of synthetic amines to remove carbon dioxide (CO(2)) from natural gas led us to examine the role of biogenic polyamines in CaCO(3) deposition by bacteria. Here, we demonstrated that biogenic polyamines such as putrescine, spermidine, and spermine were able to react with atmospheric CO(2) and the resultant carbamate anion was characterized by using nuclear magnetic resonance (NMR) analysis. Biogenic polyamines accelerated the formation of CaCO(3), and we artificially synthesized the dumbbell-shaped calcites, which had the same form as observed with bacterial CaCO3 precipitates, under nonbacterial conditions by using polyamines. The reaction rate of calcification increased with temperature with an optimum of around 40 °C. Our observation suggests a novel scheme for CO(2) dissipation that could be a potential tool in reducing atmospheric CO(2) levels and, therefore, global warming.

Preparation of calibration standards of N1-H paralytic shellfish toxin analogues by large-scale culture of cyanobacterium Anabaena circinalis (TA04).

Mouse bioassay is the official testing method to quantify paralytic shellfish toxins (PSTs) in bivalves. A number of alternative analytical methods have been reported. Some methods have been evaluated by a single laboratory validation. Among the different types of methods, chemical analyses are capable of identifying and quantifying the toxins, however a shortage of the necessary calibration standards hampers implementation of the chemical analyses in routine monitoring of PSTs in bivalves. In our present study, we studied preparation of major PST analogues as calibrants by large-scale cultivation of toxic freshwater cyanobacteria Anabaena circinalis TA04. The cells were steadily grown in 10 L bottle for 28 days. The primary N1-H toxins, C1/C2, were produced at a concentration of 1.3 ± 0.1 µmol/L. The intracellular and extracellular toxins occupied 80% and 20%, respectively. Over 220 µmol of the toxins was obtained from approximately 200 L of the culture over six months, demonstrating that it is sufficient to prepare saxitoxin analogues. The toxins were chemically converted to six N1-H analogues. Preparation of the analogues was carried out at relatively high yields (50-90%). The results indicate that our preparation method is useful to produce N1-H toxins. In our present study, detailed conditions for preparation of one of the rare N1-H analogues, gonyautoxin-5, were investigated.

Detection of Rap1A as a yessotoxin binding protein from blood cell membranes.

As is the case with other ladder-shaped polyether compounds, yessotoxin is produced by marine dinoflagellate, and possesses various biological activities beside potent toxicity. To gain a better understanding of the molecular mechanism for high affinity between these polyethers and their binding proteins, which accounts for their powerful biological activities, we searched for its binding proteins from human blood cells by using the biotin-conjugate of desulfated YTX as a ligand. By a protein pull-down protocol with use of streptavidin beads, a band of specifically binding proteins was detected in SDS-PAGE. HPLC-tandem mass spectrometry (MS/MS) indicated that Rap 1A, one of Ras superfamily proteins, binds to the YTX-linked resins. Western blotting and surface plasmon resonance experiments further confirmed that Rap1A specifically binds to YTX with the K(D) value around 4 µM.

Development of quantitative NMR method with internal standard for the standard solutions of paralytic shellfish toxins and characterisation of gonyautoxin-5 and gonyautoxin-6.

The chemical analysis of paralytic shellfish toxins (PSTs) requires standard solutions with accurate concentration. The mouse toxicity in each toxin is also essential knowledge for the introduction of chemical analysis as an alternative method to mouse bioassay (MBA) in routine monitoring of shellfish. In this study, we developed the quantitative analysis of PSTs by nuclear magnetic resonance (NMR), using tert-butanol as an internal standard. Only proton signals with longitudinal relaxation time (T(1)) of less than 2.5 s, including the internal standard, were used for quantitation of toxins. Our method showed good precision (<3%) and accuracy (slope: 1.0038, R(2): 1.0000). The limit of quantitation (LOQ) at 5% relative standard deviation (RSD) was calculated to be 0.16 mM, which corresponded to 67 microg/mL as Saxitoxin (STX) diacetate form, while the limit of detection (LOD) was 0.04 mM. Gonyautoxin-5 (GTX5) and gonyautoxin-6 (GTX6) isolated from mussels were quantified by our method, and the toxicities of GTX5 and GTX6 were obtained by the MBA in which mice were standardized by STX provided from FDA. The specific toxicities of GTX5 and GTX6 newly calculated by the MBA were 120 MU/micromol (29 microg STX equiv./micromol) and 105 MU/micromol (25 microg STX equiv./micromol), respectively. These results are useful to convert the amount of GTX5 and GTX6 into the mouse toxicity, especially in the areas where the dinoflagellate Gymnodinium catenatum predominantly produces both toxins.

Surface plasmon resonance-based detection of ladder-shaped polyethers by inhibition detection method.

Ladder-shaped polyether (LSP) compounds represented by brevetoxins and ciguatoxins were largely discovered in association with seafood poisoning. Thus, a quick quantification method for LSPs is potentially important. We examined a surface plasmon resonance method using desulfated-yessotoxin (dsYTX) immobilized on a sensor chip and phosphodiesterase PDEII in a inhibition detection mode. Yessotoxin, brevetoxin B and synthetic LSP derivatives showed clear inhibition against PDEII binding to the immobilized dsYTX, by which their half inhibitory concentrations were successfully estimated. This inhibition method appeared to be superior in specificity to direct binding assays where binding proteins to LSP was immobilized on a sensor chip.

Interaction of ladder-shaped polyethers with transmembrane alpha-helix of glycophorin A as evidenced by saturation transfer difference NMR and surface plasmon resonance.

Ladder-shaped polyether (LSP) compounds are thought to interact with transmembrane alpha-helices, but direct evidence has scarcely obtained for these interactions. We adopted a transmembrane alpha-helix of glycophorin A, and quantitatively evaluated its interaction with LSPs such as yessotoxin (YTX), desulfated YTX and artificial LSPs, using surface plasmon resonance and saturation transfer difference NMR. As a result, dissociation constants (K(D)) of YTX and desulfated YTX to a transmembrane domain peptide of glycophorin A were determined to be in the submillimolar range. Furthermore, in saturation transfer difference NMR, the signals at the polyene side chain and the angular methyl groups of YTX were significantly attenuated, which probably comprised an interacting interface of LSPs with a transmembrane alpha-helix. These results suggest that hydrophobic interaction plays an important role in molecular recognition of the alpha-helix peptide by LSPs.

Purification and characterization of paralytic shellfish toxin-transforming enzyme, sulfocarbamoylase I, from the Japanese bivalve Peronidia venulosa.

The Japanese bivalve Peronidia venulosa contains paralytic shellfish toxin (PST)-transforming enzymes that convert the weakly toxic C-toxins to the more potent decarbamoyl toxins. The enzyme was purified 154-fold with a yield of 0.26% and was named sulfocarbamoylase I. It was found to be a protein with an estimated molecular weight of 300 kDa by gel filtration column chromatography. Observation of a single band equivalent to 150 kDa on SDS-PAGE with or without reducing agents suggested it to be a homodimer with ionically bound subunits. The enzyme catalyzes the hydrolysis of the carboxyl bond in the N-sulfocarbamoyl moiety of PSP-toxins. The sulfonyl moiety in the carbamoyl side chain of substrates is essential for enzyme recognition. The N-terminal amino acid sequences of nine tryptic peptides were determined by the Edman degradation method. In a database search using the BLAST program, no protein that shows remarkable homology was retrieved. Several characteristics of the enzyme were also compared with those of another PST-transforming enzyme, carbamoylase I, which was previously isolated from the Japanese clam Mactra chinensis.

Studies of diarrhetic activity on pectenotoxin-6 in the mouse and rat.

Diarrhetic activity of pectenotoxin-6 (PTX6), a shellfish contaminant in Japanese scallops (Patinopecten yessoensis), was studied in vivo. Mice gavaged with 5mg/kg PTX6 did not show diarrhea or fluid secretion, and no prominent pathological changes were observed. There was no synergistic toxicity of PTX6 with okadaic acid (OA) or pectenotoxin-2 (PTX2) when toxins were given to mice by gavage. Synergistic activity of PTX6 with OA was also not confirmed under crude conditional simulation with oil. In contrast to the oral administration to mice, PTX6 at 500 microg/kg by i.p. was the lethal dose with bleeding in the liver, injuries at the gastric organs and the kidney. When rats were gavaged with PTX6 at a dose of 2 mg/kg, PTX6 did not have diarrhetic activity; however, the middle-lower small intestine (jejunum-ileum) was eroded at villi by edema. PTX6 is a potent toxin if administered by intraperitoneal injection to mice, or if administered orally to the rat. However, it is not clear if PTX6 passes through the intestinal barrier if given by the oral route.